Liz Harry is Professor of Biology and Director of the ithree institute (infection, immunology and innovation) at the University of Technology, Sydney (UTS).
Liz obtained her PhD in Biochemistry from the University of Sydney. She then went to Harvard University as a National Institutes of Health (NIH, USA) Postdoctoral Fellow. There she pioneered the development of fluorescence microscopy techniques for bacteria that enabled visualization of the subcellular proteins inside bacterial cells. These techniques have revolutionized our view of the internal organization of bacterial cells. They revealed that bacterial cells are highly organized, with proteins having specific cellular addresses that are exquisitely controlled in time and space.
Liz then returned to Australia to be an Australian Research Council (ARC) Postdoctoral Fellow and then an ARC QEII Fellow in the School of Molecular Biosciences at the University of Sydney. She was then head-hunted to take up a position as Associate Professor at the ithree institute (then known as the Institute for the Biotechnology of Infectious Diseases) with the aim of building a team of bacterial biologists. She was promoted to Professor of Biology in 2010, and has played a leading role in the recruitment of several eminent Australian and international researchers to the institute.
Professor Harry’s research on bacterial cell division has had a significant impact on our understanding of how bacterial cells multiply, and how they control this process to ensure equal partitioning of chromosomes vital for survival. Her research has often changed the direction of thinking in the field, and has afforded excellent opportunities in antibacterial discovery. Her cutting-edge cell biology techniques including super resolution microscopy have provided unique insights in the mechanism and spatiotemporal control of the division process in bacteria. She made the surprising discovery that the cytokinetic ring, which is a polymer of the tubulin-like protein, FtsZ, forms at the division site at midcell as a result of the remodeling of a cytoskeletal helical assembly of polymers. Her research has shown that this Z ring is linked to the progress of the initiation phase of DNA replication, and gave rise to a new definition for the role of long-known sptatial regulators of bacterial division.
With the move to the ithree institute, CI Harry shifted some of her fundamental research focus on bacterial cell division toward bacteria that cause infectious disease, such as Staphylococcus aureus and Acinetobacter baumannii, and antibacterial discovery. Professor Harry has worked with industry in antibacterial discovery research for the development of novel antibiotics that target this process in pathogens, and to examine how natural products function as effective therapeutics for infectious disease.
Liz was awarded the 2002 Australian Eureka Prize for Scientific Research, and recently won the 2008 ASM Frank Fenner Award, awarded by the Australian Society for Microbiology in recognition of her distinguished contributions to Australian research in microbiology. She has had several Plenary invitations to international conferences and served on the Executive of the Australian Society for Microbiology for several years. She is currently a member of the Australian Academy of Science, National Committee for Biomedical Sciences.
Can supervise: YES
Liz Harry’s primary research interest is how bacterial cells divide and how they regulate this process. Cell division is essential for survival, colonisation and infection. What are the cues that signal cells to divide at the right place and at the right time? How do cells ensure that when division occurs to produce two newborn cells, each one receives the correct amount of DNA? The answers to these questions are essential to understand how organisms reproduce and grow. But they remain unknown. Research in Liz’s laboratory addresses these questions in bacteria to gain an understanding of the regulation of this vital process both in culture and in the host during infection to facilitate the design of novel therapeutics that target it.
Liz has successfully partnered with industry in the areas of antimicrobial drug discovery and natural product therapeutics.
Subject Coordinator and Lecturer for Introductory Pharmacology and Microbiology to Bachelor of Nursing and Bachelor of Midwifery students at UTS.
Antibiotic Resistance Tutorial, 2nd year Bachelor of Biotechnology students at UTS.
Bottomley, AL, Peterson, E, Iosifidis, G, Yong, AMH, Hartley-Tassell, LE, Ansari, S, McKenzie, C, Burke, C, Duggin, IG, Kline, KA & Harry, EJ 2020, 'The novel E. coli cell division protein, YtfB, plays a role in eukaryotic cell adhesion', Scientific Reports, vol. 10, no. 1.View/Download from: Publisher's site
© 2020, The Author(s). Characterisation of protein function based solely on homology searches may overlook functions under specific environmental conditions, or the possibility of a protein having multiple roles. In this study we investigated the role of YtfB, a protein originally identified in a genome-wide screen to cause inhibition of cell division, and has demonstrated to localise to the Escherichia coli division site with some degree of glycan specificity. Interestingly, YtfB also shows homology to the virulence factor OapA from Haemophilus influenzae, which is important for adherence to epithelial cells, indicating the potential of additional function(s) for YtfB. Here we show that E. coli YtfB binds to N'acetylglucosamine and mannobiose glycans with high affinity. The loss of ytfB results in a reduction in the ability of the uropathogenic E. coli strain UTI89 to adhere to human kidney cells, but not to bladder cells, suggesting a specific role in the initial adherence stage of ascending urinary tract infections. Taken together, our results suggest a role for YtfB in adhesion to specific eukaryotic cells, which may be additional, or complementary, to its role in cell division. This study highlights the importance of understanding the possible multiple functions of proteins based on homology, which may be specific to different environmental conditions.
Bouzo, D, Cokcetin, NN, Li, L, Ballerin, G, Bottomley, AL, Lazenby, J, Whitchurch, CB, Paulsen, IT, Hassan, KA & Harry, EJ 2020, 'Characterizing the mechanism of action of an ancient antimicrobial, manuka honey, against pseudomonas aeruginosa using modern transcriptomics', mSystems, vol. 5, no. 3.View/Download from: Publisher's site
Copyright © 2020 Bouzo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Manuka honey has broad-spectrum antimicrobial activity, and unlike traditional antibiotics, resistance to its killing effects has not been reported. However, its mechanism of action remains unclear. Here, we investigated the mechanism of action of manuka honey and its key antibacterial components using a transcriptomic approach in a model organism, Pseudomonas aeruginosa. We show that no single component of honey can account for its total antimicrobial action, and that honey affects the expression of genes in the SOS response, oxidative damage, and quorum sensing. Manuka honey uniquely affects genes involved in the explosive cell lysis process and in maintaining the electron transport chain, causing protons to leak across membranes and collapsing the proton motive force, and it induces membrane depolarization and permeabilization in P. aeruginosa. These data indicate that the activity of manuka honey comes from multiple mechanisms of action that do not engender bacterial resistance. IMPORTANCE The threat of antimicrobial resistance to human health has prompted interest in complex, natural products with antimicrobial activity. Honey has been an effective topical wound treatment throughout history, predominantly due to its broad-spectrum antimicrobial activity. Unlike traditional antibiotics, honey-resistant bacteria have not been reported; however, honey remains underutilized in the clinic in part due to a lack of understanding of its mechanism of action. Here, we demonstrate that honey affects multiple processes in bacteria, and this is not explained by its major antibacterial components. Honey also uniquely affects bacterial membranes, and this can be exploited for combination therapy with antibiotics that are otherwise ineffective on their own. We argue that honey should be included as part of the current array ...
Phan, M-D, Bottomley, AL, Peters, KM, Harry, EJ & Schembri, MA 2020, 'Uncovering novel susceptibility targets to enhance the efficacy of third-generation cephalosporins against ESBL-producing uropathogenic Escherichia coli.', The Journal of antimicrobial chemotherapy, vol. 75, no. 6, pp. 1415-1423.View/Download from: Publisher's site
BACKGROUND:Uropathogenic Escherichia coli (UPEC) are a major cause of urinary tract infection (UTI), one of the most common infectious diseases in humans. UPEC are increasingly associated with resistance to multiple antibiotics. This includes resistance to third-generation cephalosporins, a common class of antibiotics frequently used to treat UTI. METHODS:We employed a high-throughput genome-wide screen using saturated transposon mutagenesis and transposon directed insertion-site sequencing (TraDIS) together with phenotypic resistance assessment to identify key genes required for survival of the MDR UPEC ST131 strain EC958 in the presence of the third-generation cephalosporin cefotaxime. RESULTS:We showed that blaCMY-23 is the major ESBL gene in EC958 responsible for mediating resistance to cefotaxime. Our screen also revealed that mutation of genes involved in cell division and the twin-arginine translocation pathway sensitized EC958 to cefotaxime. The role of these cell-division and protein-secretion genes in cefotaxime resistance was confirmed through the construction of mutants and phenotypic testing. Mutation of these genes also sensitized EC958 to other cephalosporins. CONCLUSIONS:This work provides an exemplar for the application of TraDIS to define molecular mechanisms of resistance to antibiotics. The identification of mutants that sensitize UPEC to cefotaxime, despite the presence of a cephalosporinase, provides a framework for the development of new approaches to treat infections caused by MDR pathogens.
Valentin, E, Bottomley, AL, Chilambi, GS, Harry, EJ, Amal, R, Sotiriou, GA, Rice, SA & Gunawan, C 2020, 'Heritable nanosilver resistance in priority pathogen: A unique genetic adaptation and comparison with ionic silver and antibiotics', Nanoscale, vol. 12, no. 4, pp. 2384-2392.View/Download from: Publisher's site
© 2020 The Royal Society of Chemistry. The past decade has seen the incorporation of antimicrobial nanosilver (NAg) into medical devices, and, increasingly, in everyday 'antibacterial' products. With the continued rise of antibiotic resistant bacteria, there are concerns that these priority pathogens will also develop resistance to the extensively commercialized nanoparticle antimicrobials. Herein, this work reports the emergence of stable resistance traits to NAg in the WHO-listed priority pathogen Staphylococcus aureus, which has previously been suggested to have no, or very low, capacity for silver resistance. With no native presence of genetically encoded silver defence mechanisms, the work showed that the bacterium is dependent on mutation of physiologically essential genes, including those involved in nucleotide synthesis and oxidative stress defence. While some mutations were uniquely associated with resistance to NAg, the study also found common mutations that could be protective against both NAg and ionic silver. This is consistent with the observation of NAg/ionic silver cross-resistance. These mutations were detected following withdrawal of the silver exposure, denoting heritable characteristics that allow for spread of the resistance traits even with discontinued silver use. Heritable silver resistance in priority pathogen cautions that these nanoparticle antimicrobials should only be used as needed, to preserve their efficacy for treating infections.
Hajduk, IV, Mann, R, Rodrigues, CDA & Harry, EJ 2019, 'The ParB homologs, Spo0J and Noc, together prevent premature midcell Z ring assembly when the early stages of replication are blocked in Bacillus subtilis.', Molecular microbiology, vol. 112, no. 3, pp. 766-784.View/Download from: Publisher's site
Precise cell division in coordination with DNA replication and segregation is of utmost importance for all organisms. The earliest stage of cell division is the assembly of a division protein FtsZ into a ring, known as the Z ring, at midcell. What still eludes us, however, is how bacteria precisely position the Z ring at midcell. Work in B. subtilis over the last two decades has identified a link between the early stages of DNA replication and cell division. A recent model proposed that the progression of the early stages of DNA replication leads to an increased ability for the Z ring to form at midcell. This model arose through studies examining Z ring position in mutants blocked at different steps of the early stages of DNA replication. Here, we show that this model is unlikely to be correct and the mutants previously studied generate nucleoids with different capacity for blocking midcell Z ring assembly. Importantly, our data suggest that two proteins of the widespread ParB family, Noc and Spo0J are required to prevent Z ring assembly over the bacterial nucleoid and help fine tune the assembly of the Z ring at midcell during the cell cycle.
Lu, J, Cokcetin, NN, Burke, CM, Turnbull, L, Liu, M, Carter, DA, Whitchurch, CB & Harry, EJ 2019, 'Honey can inhibit and eliminate biofilms produced by Pseudomonas aeruginosa.', Scientific reports, vol. 9, no. 1.View/Download from: Publisher's site
Chronic wound treatment is becoming increasingly difficult and costly, further exacerbated when wounds become infected. Bacterial biofilms cause most chronic wound infections and are notoriously resistant to antibiotic treatments. The need for new approaches to combat polymicrobial biofilms in chronic wounds combined with the growing antimicrobial resistance crisis means that honey is being revisited as a treatment option due to its broad-spectrum antimicrobial activity and low propensity for bacterial resistance. We assessed four well-characterised New Zealand honeys, quantified for their key antibacterial components, methylglyoxal, hydrogen peroxide and sugar, for their capacity to prevent and eradicate biofilms produced by the common wound pathogen Pseudomonas aeruginosa. We demonstrate that: (1) honey used at substantially lower concentrations compared to those found in honey-based wound dressings inhibited P. aeruginosa biofilm formation and significantly reduced established biofilms; (2) the anti-biofilm effect of honey was largely driven by its sugar component; (3) cells recovered from biofilms treated with sub-inhibitory honey concentrations had slightly increased tolerance to honey; and (4) honey used at clinically obtainable concentrations completely eradicated established P. aeruginosa biofilms. These results, together with their broad antimicrobial spectrum, demonstrate that manuka honey-based wound dressings are a promising treatment for infected chronic wounds, including those with P. aeruginosa biofilms.
Kusuma, KD, Griffith, R, Harry, EJ, Bottomley, AL & Ung, AT 2019, 'In silico Analysis of FtsZ Crystal Structures Towards a New Target for Antibiotics', Australian Journal of Chemistry, vol. 72, no. 3, pp. 184-196.View/Download from: Publisher's site
© 2018 CSIRO. The bacterial cell division protein FtsZ is conserved in most bacteria and essential for viability. There have been concerted efforts in developing inhibitors that target FtsZ as potential antibiotics. Key to this is an in-depth understanding of FtsZ structure at the molecular level across diverse bacterial species to ensure inhibitors have high affinity for the FtsZ target in a variety of clinically relevant pathogens. In this study, we show that FtsZ structures differ in three ways: (1) the H7 helix curvature; (2) the dimensions of the interdomain cleft; and (3) the opening/closing mechanism of the interdomain cleft, whereas no differences were observed in the dimensions of the nucleotide-binding pocket and T7 loop. Molecular dynamics simulation may suggest that there are two possible mechanisms for the process of opening and closing of the interdomain cleft on FtsZ structures. This discovery highlights significant differences between FtsZ structures at the molecular level and this knowledge is vital in assisting the design of potent FtsZ inhibitors.
Kusuma, KD, Payne, M, Ung, AT, Bottomley, AL & Harry, EJ 2019, 'FtsZ as an Antibacterial Target: Status and Guidelines for Progressing This Avenue.', ACS infectious diseases, vol. 5, no. 8, pp. 1279-1294.View/Download from: Publisher's site
The disturbing increase in the number of bacterial pathogens that are resistant to multiple, or sometimes all, current antibiotics highlights the desperate need to pursue the discovery and development of novel classes of antibacterials. The wealth of knowledge available about the bacterial cell division machinery has aided target-driven approaches to identify new inhibitor compounds. The main division target being pursued is the highly conserved and essential protein FtsZ. Despite very active research on FtsZ inhibitors for several years, this protein is not yet targeted by any commercial antibiotic. Here, we discuss the suitability of FtsZ as an antibacterial target for drug development and review progress achieved in this area. We use hindsight to highlight the gaps that have slowed progress in FtsZ inhibitor development and to suggest guidelines for concluding that FtsZ is actually the target of these molecules, a key missing link in several studies. In moving forward, a multidisciplinary, communicative, and collaborative process, with sharing of research expertise, is critical if we are to succeed.
Lehmann, DM, Krishnakumar, K, Batres, MA, Hakola-Parry, A, Cokcetin, N, Harry, E & Carter, DA 2019, 'A cost-effective colourimetric assay for quantifying hydrogen peroxide in honey', Access Microbiology, vol. 1, no. 10.View/Download from: Publisher's site
Honey is a natural product with many beneficial properties including antimicrobial action. Production of hydrogen peroxide (H2O2) in diluted honey is central to this action. Here, we describe an optimized method for measuring levels of H2O2 in honey. This method is based on established methods, with the level of dilution, the time between dilution and reading the assay, and aeration of the samples during the assay identified as critical points for ensuring reliability and reproducibility. The method is cost-effective and easy to perform using common laboratory equipment. Using this method, we quantified the hydrogen peroxide content of five different, unprocessed polyfloral honeys collected in NC, USA. Our results show that H2O2 production by these honeys varies greatly, with some samples producing negligible levels of H2O2. We assessed the effect of colour on the assay by measuring the recovery of spiked H2O2 from light and dark honey and from serially diluted dark corn syrup, and found the amount of H2O2 that could be detected was lower in dark corn syrup and darker honey samples.
Mediati, DG, Burke, CM, Ansari, S, Harry, EJ & Duggin, IG 2018, 'High-throughput sequencing of sorted expression libraries reveals inhibitors of bacterial cell division.', BMC genomics, vol. 19, no. 1.View/Download from: Publisher's site
BACKGROUND:Bacterial filamentation occurs when rod-shaped bacteria grow without dividing. To identify genetically encoded inhibitors of division that promote filamentation, we used cell sorting flow cytometry to enrich filamentous clones from an inducible expression library, and then identified the cloned DNA with high-throughput DNA sequencing. We applied the method to an expression library made from fragmented genomic DNA of uropathogenic E. coli UTI89, which undergoes extensive reversible filamentation in urinary tract infections and might encode additional regulators of division. RESULTS:We identified 55 genomic regions that reproducibly caused filamentation when expressed from the plasmid vector, and then further localized the cause of filamentation in several of these to specific genes or sub-fragments. Many of the identified genomic fragments encode genes that are known to participate in cell division or its regulation, and others may play previously-unknown roles. Some of the prophage genes identified were previously implicated in cell division arrest. A number of the other fragments encoded potential short transcripts or peptides. CONCLUSIONS:The results provided evidence of potential new links between cell division and distinct cellular processes including central carbon metabolism and gene regulation. Candidate regulators of the UTI-associated filamentation response or others were identified amongst the results. In addition, some genomic fragments that caused filamentation may not have evolved to control cell division, but may have applications as artificial inhibitors. Our approach offers the opportunity to carry out in depth surveys of diverse DNA libraries to identify new genes or sequences encoding the capacity to inhibit division and cause filamentation.
Liu, MY, Cokcetin, NN, Lu, J, Turnbull, L, Carter, DA, Whitchurch, CB & Harry, EJ 2018, 'Rifampicin-Manuka Honey Combinations Are Superior to Other Antibiotic-Manuka Honey Combinations in Eradicating Staphylococcus aureus Biofilms.', Frontiers in Microbiology, vol. 8, pp. 1-12.View/Download from: Publisher's site
Chronic wound infections are a major burden to both society and the health care industry. Bacterial biofilms are the major cause of chronic wound infections and are notoriously recalcitrant to treatments with antibiotics, making them difficult to eradicate. Thus, new approaches are required to combat biofilms in chronic wounds. One possible approach is to use drug combination therapies. Manuka honey has potent broad-spectrum antibacterial activity and has previously shown synergistic activity in combination with antibiotics against common wound pathogens, including Staphylococcus aureus. In addition, manuka honey exhibits anti-biofilm activity, thereby warranting the investigation of its potential as a combination therapy with antibiotics for the topical treatment of biofilm-related infections. Here we report the first use of MacSynergy II to investigate the response of established S. aureus (strain NCTC 8325) biofilms to treatment by combinations of Medihoney (medical grade manuka honey) and conventional antibiotics that are used for preventing or treating infections: rifampicin, oxacillin, fusidic acid, clindamycin, and gentamicin. Using checkerboard microdilution assays, viability assays and MacSynergy II analysis we show that the Medihoney-rifampicin combination was more effective than combinations using the other antibiotics against established staphylococcal biofilms. Medihoney and rifampicin were strongly synergistic in their ability to reduce both biofilm biomass and the viability of embedded S. aureus cells at a level that is likely to be significant in vivo. Other combinations of Medihoney and antibiotic produced an interesting array of effects: Medihoney-fusidic acid treatment showed minor synergistic activity, and Medihoney-clindamycin, -gentamicin, and -oxacillin combinations showed overall antagonistic effects when the honey was used at sub-inhibitory concentration, due to enhanced biofilm formation at these concentrations which could not be counter...
Faiz, MB, Amal, R, Marquis, CP, Harry, EJ, Sotiriou, GA, Rice, SA & Gunawan, C 2018, 'Nanosilver and the microbiological activity of the particulate solids versus the leached soluble silver.', Nanotoxicology, vol. 12, no. 3, pp. 263-273.View/Download from: Publisher's site
Nanosilver (Ag NPs) is currently one of the most commercialized antimicrobial nanoparticles with as yet, still unresolved cytotoxicity origins. To date, research efforts have mostly described the antimicrobial contribution from the leaching of soluble silver, while the undissolved solid Ag particulates are often considered as being microbiologically inert, serving only as source of the cytotoxic Ag ions. Here, we show the rapid stimulation of lethal cellular oxidative stress in bacteria by the presence of the undissolved Ag particulates. The cytotoxicity characteristics are distinct from those arising from the leached soluble Ag, the latter being locked in organic complexes. The work also highlights the unique oxidative stress-independent bacterial toxicity of silver salt. Taken together, the findings advocate that future enquiries on the antimicrobial potency and also importantly, the environmental and clinical impact of Ag NPs use, should pay attention to the potential bacterial toxicological responses to the undissolved Ag particulates, rather than just to the leaching of soluble silver. The findings also put into question the common use of silver salt as model material for evaluating bacterial toxicity of Ag NPs.
Gardiner, M, Vicaretti, M, Sparks, J, Bansal, S, Bush, S, Liu, M, Darling, A, Harry, E & Burke, CM 2017, 'A longitudinal study of the diabetic skin and wound microbiome.', PeerJ, vol. 5, pp. e3543-e3543.View/Download from: Publisher's site
BACKGROUND: Type II diabetes is a chronic health condition which is associated with skin conditions including chronic foot ulcers and an increased incidence of skin infections. The skin microbiome is thought to play important roles in skin defence and immune functioning. Diabetes affects the skin environment, and this may perturb skin microbiome with possible implications for skin infections and wound healing. This study examines the skin and wound microbiome in type II diabetes. METHODS: Eight type II diabetic subjects with chronic foot ulcers were followed over a time course of 10 weeks, sampling from both foot skin (swabs) and wounds (swabs and debrided tissue) every two weeks. A control group of eight control subjects was also followed over 10 weeks, and skin swabs collected from the foot skin every two weeks. Samples were processed for DNA and subject to 16S rRNA gene PCR and sequencing of the V4 region. RESULTS: The diabetic skin microbiome was significantly less diverse than control skin. Community composition was also significantly different between diabetic and control skin, however the most abundant taxa were similar between groups, with differences driven by very low abundant members of the skin communities. Chronic wounds tended to be dominated by the most abundant skin Staphylococcus, while other abundant wound taxa differed by patient. No significant correlations were found between wound duration or healing status and the abundance of any particular taxa. DISCUSSION: The major difference observed in this study of the skin microbiome associated with diabetes was a significant reduction in diversity. The long-term effects of reduced diversity are not yet well understood, but are often associated with disease conditions.
Mann, R, Mediati, DG, Duggin, IG, Harry, EJ & Bottomley, AL 2017, 'Metabolic Adaptations of Uropathogenic E. coli in the Urinary Tract.', Frontiers in Cellular and Infection Microbiology, vol. 7, pp. 1-15.View/Download from: Publisher's site
Escherichia coli ordinarily resides in the lower gastrointestinal tract in humans, but some strains, known as Uropathogenic E. coli (UPEC), are also adapted to the relatively harsh environment of the urinary tract. Infections of the urine, bladder and kidneys by UPEC may lead to potentially fatal bloodstream infections. To survive this range of conditions, UPEC strains must have broad and flexible metabolic capabilities and efficiently utilize scarce essential nutrients. Whole-organism (or "omics") methods have recently provided significant advances in our understanding of the importance of metabolic adaptation in the success of UPECs. Here we describe the nutritional and metabolic requirements for UPEC infection in these environments, and focus on particular metabolic responses and adaptations of UPEC that appear to be essential for survival in the urinary tract.
Productive bacterial cell division and survival of progeny requires tight coordination between chromosome segregation and cell division to ensure equal partitioning of DNA. Unlike rod-shaped bacteria that undergo division in one plane, the coccoid human pathogen Staphylococcus aureus divides in three successive orthogonal planes, which requires a different spatial control compared to rod-shaped cells. To gain a better understanding of how this coordination between chromosome segregation and cell division is regulated in S. aureus, we investigated proteins that associate with FtsZ and the divisome. We found that DnaK, a well-known chaperone, interacts with FtsZ, EzrA
and DivIVA, and is required for DivIVA stability. Unlike in several rod shaped organisms, DivIVA in S. aureus associates with several components of the divisome, as well as the chromosome segregation protein, SMC. This data, combined with phenotypic analysis of mutants, suggests a novel role for S. aureus DivIVA in ensuring cell division and
chromosome segregation are coordinated.
Gorle, AK, Bottomley, AL, Harry, EJ, Collins, JG, Keene, FR & Woodward, CE 2017, 'DNA condensation in live E. coli provides evidence for transertion.', Molecular BioSystems, vol. 13, no. 4, pp. 677-680.View/Download from: Publisher's site
Condensation studies of chromosomal DNA in E. coli with a tetranuclear ruthenium complex are carried out and images obtained with wide-field fluorescence microscopy. Remarkably different condensate morphologies resulted, depending upon the treatment protocol. The occurrence of condensed nucleoid spirals in live bacteria provides evidence for the transertion hypothesis.
Gunawan, C, Marquis, CP, Amal, R, Sotiriou, GA, Rice, SA & Harry, EJ 2017, 'Widespread and Indiscriminate Nanosilver Use: Genuine Potential for Microbial Resistance.', ACS Nano, vol. 11, no. 4, pp. 3438-3445.View/Download from: Publisher's site
In this era of increasing antibiotic resistance, the use of alternative antimicrobials such as silver has become more widespread. Superior antimicrobial activity has been provided through fabrication of silver nanoparticles or nanosilver (NAg), which imparts cytotoxic actions distinct from those of bulk silver. In the wake of the recent discoveries of bacterial resistance to NAg and its rising incorporation in medical and consumer goods such as wound dressings and dietary supplements, we argue that there is an urgent need to monitor the prevalence and spread of NAg microbial resistance. In this Perspective, we describe how the use of NAg in commercially available products facilitates prolonged microorganism exposure to bioavailable silver, which underpins the development of resistance. Furthermore, we advocate for a judicial approach toward NAg use in order to preserve its efficacy and to avoid environmental disruption.
Hajduk, IV, Rodrigues, CD & Harry, EJ 2016, 'Connecting the dots of the bacterial cell cycle: Coordinating chromosome replication and segregation with cell division.', Seminars in cell & developmental biology, vol. 53, pp. 2-9.View/Download from: Publisher's site
Proper division site selection is crucial for the survival of all organisms. What still eludes us is how bacteria position their division site with high precision, and in tight coordination with chromosome replication and segregation. Until recently, the general belief, at least in the model organisms Bacillus subtilis and Escherichia coli, was that spatial regulation of division comes about by the combined negative regulatory mechanisms of the Min system and nucleoid occlusion. However, as we review here, these two systems cannot be solely responsible for division site selection and we highlight additional regulatory mechanisms that are at play. In this review, we put forward evidence of how chromosome replication and segregation may have direct links with cell division in these bacteria and the benefit of recent advances in chromosome conformation capture techniques in providing important information about how these three processes mechanistically work together to achieve accurate generation of progenitor cells.
Fluctuations in nutrient availability are a fact of life for bacterial cells in the 'wild'. To survive and compete, bacteria must rapidly modulate cell-cycle processes to accommodate changing nutritional conditions and concomitant changes in cell growth. Our understanding of how this is achieved has been transformed in recent years, with cellular metabolism emerging as a central player. Several metabolic enzymes, in addition to their normal catalytic functions, have been shown to directly modulate cell-cycle processes in response to changing nutrient levels. Here we focus on cell division, the final event in the bacterial cell cycle, and discuss recent compelling evidence connecting division regulation to nutritional status and metabolic activity.
Carter, DA, Blair, SE, Cokcetin, NN, Bouzo, D, Brooks, P, Schothauer, R & Harry, EJ 2016, 'Therapeutic Manuka Honey: No Longer So Alternative', FRONTIERS IN MICROBIOLOGY, vol. 7.View/Download from: Publisher's site
Cokcetin, NN, Pappalardo, M, Campbell, LT, Brooks, P, Carter, DA, Blair, SE & Harry, EJ 2016, 'The Antibacterial Activity of Australian Leptospermum Honey Correlates with Methylglyoxal Levels', PLOS ONE, vol. 11, no. 12.View/Download from: Publisher's site
Liu, S, Gunawan, C, Barraud, N, Rice, SA, Harry, EJ & Amal, R 2016, 'Understanding, Monitoring, and Controlling Biofilm Growth inDrinking Water Distribution Systems', Environmental Science and Technology (Washington), vol. 50, pp. 8954-8976.View/Download from: Publisher's site
In drinking water distribution systems (DWDS), biofilms are the predominant mode of microbial growth, with the presence of extracellular polymeric substance (EPS) protecting the biomass from environmental and shear stresses. Biofilm formation poses a significant problem to the drinking water industry as a potential source of bacterial contamination, including pathogens, and, in many cases, also affecting the taste and odor of drinking water and promoting the corrosion of pipes. This article critically reviews important research findings on biofilm growth in DWDS, examining the factors affecting their formation and characteristics as well as the various technologies to characterize and monitor and, ultimately, to control their growth. Research indicates that temperature fluctuations potentially affect not only the initial bacteria-to-surface attachment but also the growth rates of biofilms. For the latter, the effect is unique for each type of biofilm-forming bacteria; ammonia-oxidizing bacteria, for example, grow more-developed biofilms at a typical summer temperature of 22 °C compared to 12 °C in fall, and the opposite occurs for the pathogenic Vibrio cholerae. Recent investigations have found the formation of thinner yet denser biofilms under high and turbulent flow regimes of drinking water, in comparison to the more porous and loosely attached biofilms at low flow rates. Furthermore, in addition to the rather well-known tendency of significant biofilm growth on corrosion-prone metal pipes, research efforts also found leaching of growth-promoting organic compounds from the increasingly popular use of polymer-based pipes. Knowledge of the unique microbial members of drinking water biofilms and, importantly, the influence of water characteristics and operational conditions on their growth can be applied to optimize various operational parameters to minimize biofilm accumulation. More-detailed characterizations of the biofilm population size and structure are now...
Campbell, LT, Padula, MP, Harry, E & Carter, DA 2015, 'You are what you secrete: extracellular proteins and virulence in Cryptococcus', Microbiology Australia, vol. 36, no. 2, pp. 93-95.View/Download from: Publisher's site
Fungal organisms secrete a wide range of biomolecules,
including degradative enzymes that are essential for nutrition,
toxins, effectors and secondary compounds that modulate
interactions with host animals and plants, and a variety
of signaling and stress-related proteins1
. As these are likely
to be key determinants of virulence and may also be useful
diagnostic and therapeutic targets, we investigated the
secretome of different strains of the fungal pathogen Cryptococcus.
Virulent strains secreted predominantly hydrolytic
and proteolytic enzymes, while the least virulent strain
secreted a range of additional non-degradative proteins
including many that lacked secretion signals, some that
appear to be 'moonlighting', and a number that are known
to be allergenic. It appears that in Cryptococcus, the secretome
may influence virulence both through the presence of
harmful enzymes and through the absence of proteins that
alert the host defence mechanisms.
Campbell, LT, Simonin, AR, Chen, C, Ferdous, J, Padula, MP, Harry, E, Hofer, M, Campbell, IL & Carter, DA 2015, 'Cryptococcus Strains with Different Pathogenic Potentials Have Diverse Protein Secretomes', EUKARYOTIC CELL, vol. 14, no. 6, pp. 554-563.View/Download from: Publisher's site
Duggin, IG, Aylett, CHS, Walsh, JC, Michie, KA, Wang, Q, Turnbull, L, Dawson, EM, Harry, EJ, Whitchurch, CB, Amos, LA & Loewe, J 2015, 'CetZ tubulin-like proteins control archaeal cell shape', NATURE, vol. 519, no. 7543, pp. 362-+.View/Download from: Publisher's site
Liu, M, Lu, J, Mueller, P, Turnbull, L, Burke, CM, Schlothauer, RC, Carter, DA, Whitchurch, CB & Harry, EJ 2015, 'Antibiotic-specific differences in the response of Staphylococcus aureus to treatment with antimicrobiala combined with manuka honey', Frontiers in Microbiology, vol. 5, pp. 1-9.View/Download from: Publisher's site
Skin infections caused by antibiotic resistant Staphylococcus aureus are a significant health problem worldwide; often associated with high treatment cost and mortality rate. Complex natural products like New Zealand (NZ) manuka honey have been revisited and studied extensively as an alternative to antibiotics due to their potent broad-spectrum antimicrobial activity, and the inability to isolate honey-resistant S. aureus. Previous studies showing synergistic effects between manuka-type honeys and antibiotics have been demonstrated against the growth of one methicillin-resistant S. aureus (MRSA) strain. We have previously demonstrated strong synergistic activity between NZ manuka-type honey and rifampicin against growth and biofilm formation of multiple S. arueus strains. Here, we have expanded our investigation using multiple S. aureus strains and four different antibiotics commonly used to treat S. aureus-related skin infections: rifampicin, oxacillin, gentamicin, and clindamycin. Using checkerboard microdilution and agar diffusion assays with S. aureus strains including clinical isolates and MRSA we demonstrate that manuka-type honey combined with these four antibiotics frequently produces a synergistic effect. In some cases when synergism was not observed, there was a significant enhancement in antibiotic susceptibility. Some strains that were highly resistant to an antibiotic when present alone become sensitive to clinically achievable concentrations when combined with honey. However, not all of the S. aureus strains tested responded in the same way to these combinational treatments. Our findings support the use of NZ manuka-type honeys in clinical treatment against S. aureus-related infections and extend their potential use as an antibiotic adjuvant in combinational therapy. Our data also suggest that manuka-type honeys may not work as antibiotic adjuvants for all strains of S. aureus, and this may help determine the mechanistic processes behind honey syner...
Cleverley, RM, Barrett, JR, Baslé, A, Bui, NK, Hewitt, L, Solovyova, A, Xu, Z-Q, Daniel, RA, Dixon, NE, Harry, EJ, Oakley, AJ, Vollmer, W & Lewis, RJ 2014, 'Structure and function of a spectrin-like regulator of bacterial cytokinesis', Nature Communications, vol. 5, pp. 1-10.View/Download from: Publisher's site
Bacterial cell division is facilitated by a molecular machine—the divisome—that assembles at mid-cell in dividing cells. The formation of the cytokinetic Z-ring by the tubulin homologue FtsZ is regulated by several factors, including the divisome component EzrA. Here we describe the structure of the 60-kDa cytoplasmic domain of EzrA, which comprises five linear repeats of an unusual triple helical bundle. The EzrA structure is bent into a semicircle, providing the protein with the potential to interact at both N- and C-termini with adjacent membrane-bound divisome components. We also identify at least two binding sites for FtsZ on EzrA and map regions of EzrA that are responsible for regulating FtsZ assembly. The individual repeats, and their linear organization, are homologous to the spectrin proteins that connect actin filaments to the membrane in eukaryotes, and we thus propose that EzrA is the founding member of the bacterial spectrin family.
Monahan, LG, Hajduk, IV, Blaber, SP, Charles, IG & Harry, EJ 2014, 'Coordinating Bacterial Cell Division with Nutrient Availability: a Role for Glycolysis', MBIO, vol. 5, no. 3.View/Download from: Publisher's site
Yin, Z, Wang, Y, Whittell, LR, Jergic, S, Liu, M, Harry, E, Dixon, NE, Kelso, MJ, Beck, JL & Oakley, AJ 2014, 'DNA replication is the target for the antibacterial effects of nonsteroidal anti-inflammatory drugs.', Chemistry & biology, vol. 21, no. 4, pp. 481-487.View/Download from: Publisher's site
Evidence suggests that some nonsteroidal anti-inflammatory drugs (NSAIDs) possess antibacterial properties with an unknown mechanism. We describe the in vitro antibacterial properties of the NSAIDs carprofen, bromfenac, and vedaprofen, and show that these NSAIDs inhibit the Escherichia coli DNA polymerase III β subunit, an essential interaction hub that acts as a mobile tether on DNA for many essential partner proteins in DNA replication and repair. Crystal structures show that the three NSAIDs bind to the sliding clamp at a common binding site required for partner binding. Inhibition of interaction of the clamp loader and/or the replicative polymerase α subunit with the sliding clamp is demonstrated using an in vitro DNA replication assay. NSAIDs thus present promising lead scaffolds for novel antibacterial agents targeting the sliding clamp.
Yin, Z, Whittell, LR, Wang, Y, Jergic, S, Liu, M, Harry, EJ, Dixon, NE, Beck, JL, Kelso, MJ & Oakley, AJ 2014, 'Discovery of Lead Compounds Targeting the Bacterial Sliding Clamp Using a Fragment-Based Approach', Journal of Medicinal Chemistry, vol. 57, no. 6, pp. 2799-2806.View/Download from: Publisher's site
Lu, J, Turnbull, L, Burke, CM, Liu, MY, Carter, DA, Schlothauer, RC, Whitchurch, CB & Harry, L 2014, 'Manuka-type honeys can eradicate biofilms produced by Staphylococcus aureus strains with different biofilm-forming abilities', PeerJ, vol. 2.View/Download from: Publisher's site
Chronic wounds are a major global health problem. Their management is difficult and costly, and the development of antibiotic resistance by both planktonic and biofilm-associated bacteria necessitates the use of alternative wound treatments. Honey is now being revisited as an alternative treatment due to its broad-spectrum antibacterial activity and the inability of bacteria to develop resistance to it. Many previous antibacterial studies have used honeys that are not well characterized, even in terms of quantifying the levels of the major antibacterial components present, making it difficult to build an evidence base for the efficacy of honey as an antibiofilm agent in chronic wound treatment. Here we show that a range of well-characterized New Zealand manuka-type honeys, in which two principle antibacterial components, methylglyoxal and hydrogen peroxide, were quantified, can eradicate biofilms of a range of Staphylococcus aureus strains that differ widely in their biofilm-forming abilities. Using crystal violet and viability assays, along with confocal laser scanning imaging, we demonstrate that in all S. aureus strains, including methicillin-resistant strains, the manuka-type honeys showed significantly higher anti-biofilm activity than clover honey and an isotonic sugar solution. We observed higher anti-biofilm activity as the proportion of manuka-derived honey, and thus methylglyoxal, in a honey blend increased. However, methylglyoxal on its own, or with sugar, was not able to effectively eradicate S. aureus biofilms. We also demonstrate that honey was able to penetrate through the biofilm matrix and kill the embedded cells in some cases. As has been reported for antibiotics, sub-inhibitory concentrations of honey improved biofilm formation by some S. aureus strains, however, biofilm cell suspensions recovered after honey treatment did not develop resistance towards manuka-type honeys. New Zealand manuka-type honeys, at the concentrations they can be applie...
Li, F, Harry, L, Bottomley, AL, Edstein, MD, Birrell, GW, Woodward, CE, Keene, FR & Collins, JG 2014, 'Dinuclear ruthenium(II) antimicrobial agents that selectively target polysomes in vivo', Chemical Science, vol. 5, pp. 685-693.View/Download from: Publisher's site
Wide-field fluorescence microscopy at high magnification was used to study the intracellular binding site of Rubb16 in Escherichia coli. Upon incubation of E. coli cells at the minimum inhibitory concentration, Rubb16 localised at ribosomes with no significant DNA binding observed. Furthermore, Rubb16 condensed the ribosomes when they existed as polysomes. It is postulated that the condensation of polysomes would halt protein production, and thereby inhibit bacterial growth. The results of this study indicate that the family of inert dinuclear ruthenium complexes Rubbn selectively target RNA over DNA in vivo. Selective RNA targeting could be advantageous for the development of therapeutic agents, and because of differences in ribosome structure between bacteria and eukaryotic cells, the Rubbn complexes could be selectively toxic to bacteria. In support of this hypothesis, the toxicity of Rubb16 was found to be significantly less to liver and kidney cell lines than against a range of bacteria.
Monahan, LG, Liew, AT, Bottomley, AL & Harry, L 2014, 'Division site positioning in bacteria: one size does not fit all', Frontiers in Microbiology, vol. 5, no. 19.View/Download from: Publisher's site
Spatial regulation of cell division in bacteria has been a focus of research for decades. It has been well studied in two model rod-shaped organisms, Escherichia coli and Bacillus subtilis, with the general belief that division site positioning occurs as a result of the combination of two negative regulatory systems, Min and nucleoid occlusion. These systems influence division by preventing the cytokinetic Z ring from forming anywhere other than midcell. However, evidence is accumulating for the existence of additional mechanisms that are involved in controlling Z ring positioning both in these organisms and in several other bacteria. In some cases the decision of where to divide is solved by variations on a common evolutionary theme, and in others completely different proteins and mechanisms are involved. Here we review the different ways bacteria solve the problem of finding the right place to divide. It appears that a one-size-fits-all model does not apply, and that individual species have adapted a division-site positioning mechanism that best suits their lifestyle, environmental niche and mode of growth to ensure equal partitioning of DNA for survival of the next generation.
Turnbull, L, Strauss, MP, Liew, ATF, Monahan, LG, Whitchurch, CB & Harry, EJ 2014, 'Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)', JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, no. 91.View/Download from: Publisher's site
Chan, C, Deadman, B, Manley-harris, M, Wilkins, A, Alber, DG & Harry, L 2013, 'Analysis of the flavonoid component of bioactive New Zealand manuka (Leptospermum scoparium) honey and the isolation, characterisation and synthesis of an unusual pyrrole', Food Chemistry, vol. 141, no. 3, pp. 1772-1781.View/Download from: Publisher's site
The flavonoid components of New Zealand manuka (Leptospermum scoparium) honey have been quantified in a series of 31 honeys of varying non-peroxide antibacterial activity to clarify discrepancies between previous studies reported in the literature. Total
Bacterial cell division begins with the polymerization of the FtsZ protein to form a Z ring at the division site. This ring subsequently recruits the division machinery to allow cytokinesis. How the Z ring is positioned correctly remains a challenging qu
Burke, CM, Liu, MY, Britton, WJ, Triccas, JA, Thomas, T, Smith, A, Allen, S, Salomon, R & Harry, L 2013, 'Harnessing Single Cell Sorting To Identify Cell Division Genes And Regulators In Bacteria', Plos One, vol. 8, no. 4, pp. 1-13.View/Download from: Publisher's site
Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development.
Lu, J, Carter, DA, Turnbull, L, Rosendale, D, Hedderley, D, Stephens, J, Gannabathula, S, Steinhorn, G, Schlothauer, RC, Whitchurch, CB & Harry, L 2013, 'The effect of New Zealand kanuka, manuka and clover honeys on bacterial growth dynamics and cellular morphology varies according to the species', PLoS One, vol. 8, no. 2, pp. e55898-e55898.View/Download from: Publisher's site
Treatment of chronic wounds is becoming increasingly difficult due to antibiotic resistance. Complex natural products with antimicrobial activity, such as honey, are now under the spotlight as alternative treatments to antibiotics. Several studies have shown honey to have broad-spectrum antibacterial activity at concentrations present in honey dressings, and resistance to honey has not been attainable in the laboratory. However not all honeys are the same and few studies have used honey that is well defined both in geographic and chemical terms. Here we have used a range of concentrations of clover honey and a suite of manuka and kanuka honeys from known geographical locations, and for which the floral source and concentration of methylglyoxal and hydrogen peroxide potential were defined, to determine their effect on growth and cellular morphology of four bacteria: Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. While the general trend in effectiveness of growth inhibition was manuka.manuka-kanuka blend.kanuka.clover, the honeys had varying and diverse effects on the growth and cellular morphology of each bacterium, and each organism had a unique response profile to these honeys. P. aeruginosa showed a markedly different pattern of growth inhibition to the other three organisms when treated with sub-inhibitory concentrations of honey, being equally sensitive to all honeys, including clover, and the least sensitive to honey overall. While hydrogen peroxide potential contributed to the antibacterial activity of the manuka and kanuka honeys, it was never essential for complete growth inhibition. Cell morphology analysis also showed a varied and diverse set of responses to the honeys that included cell length changes, cell lysis, and alterations to DNA appearance. These changes are likely to reflect the different regulatory circuits of the organisms that are activated by the stress of honey treatment.
Muller, P, Alber, DG, Turnbull, L, Schlothauer, RC, Carter, DA, Whitchurch, CB & Harry, L 2013, 'Synergism between medihoney and rifampicin against methicillin-resistant Staphylococcus aureus (MRSA)', PLoS One, vol. 8, no. 2, pp. e57679-e57679.View/Download from: Publisher's site
Skin and chronic wound infections caused by highly antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are an increasing and urgent health problem worldwide, particularly with sharp increases in obesity and diabetes. New Zealand manuka honey has potent broad-spectrum antimicrobial activity, has been shown to inhibit the growth of MRSA strains, and bacteria resistant to this honey have not been obtainable in the laboratory. Combinational treatment of chronic wounds with manuka honey and common antibiotics may offer a wide range of advantages including synergistic enhancement of the antibacterial activity, reduction of the effective dose of the antibiotic, and reduction of the risk of antibiotic resistance. The aim of this study was to investigate the effect of Medihoney in combination with the widely used antibiotic rifampicin on S. aureus. Using checkerboard microdilution assays, time-kill curve experiments and agar diffusion assays, we show a synergism between Medihoney and rifampicin against MRSA and clinical isolates of S. aureus. Furthermore, the Medihoney/rifampicin combination stopped the appearance of rifampicin-resistant S. aureus in vitro. Methylglyoxal (MGO), believed to be the major antibacterial compound in manuka honey, did not act synergistically with rifampicin and is therefore not the sole factor responsible for the synergistic effect of manuka honey with rifampicin. Our findings support the idea that a combination of honey and antibiotics may be an effective new antimicrobial therapy for chronic wound infections.
Rodrigues, CD & Harry, L 2012, 'The Min System And Nucleoid Occlusion Are Not Required For Identifying The Division Site In Bacillus Subtilis But Ensure Its Efficient Utilization', PLoS Genetics, vol. 8, no. 3, pp. 1-20.View/Download from: Publisher's site
Precise temporal and spatial control of cell division is essential for progeny survival. The current general view is that precise positioning of the division site at midcell in rod-shaped bacteria is a result of the combined action of the Min system and
Chong, H, Campbell, L, Padula, M, Hill, C, Harry, L, Li, SS, Wilkins, MR, Herbert, BR & Carter, DA 2012, 'Time-Course Proteome Analysis Reveals the Dynamic Response of Cryptococcus gattii Cells to Fluconazole', PLoS One, vol. 7, no. 8, pp. 1-10.View/Download from: Publisher's site
Cryptococcus gattii is an encapsulated fungus capable of causing fatal disease in immunocompetent humans and animals. As current antifungal therapies are few and limited in efficacy, and resistance is an emerging issue, the development of new treatment strategies is urgently required. The current study undertook a time-course analysis of the proteome of C. gattii during treatment with fluconazole (FLC), which is used widely in prophylactic and maintenance therapies. The aims were to analyze the overall cellular response to FLC, and to find fungal proteins involved in this response that might be useful targets in therapies that augment the antifungal activity of FLC. During FLC treatment, an increase in stress response, ATP synthesis and mitochondrial respiratory chain proteins, and a decrease in most ribosomal proteins was observed, suggesting that ATP-dependent efflux pumps had been initiated for survival and that the maintenance of ribosome synthesis was differentially expressed. Two proteins involved in fungal specific pathways were responsive to FLC. An integrative network analysis revealed co-ordinated processes involved in drug response, and highlighted hubs in the network representing essential proteins that are required for cell viability. This work demonstrates the dynamic cellular response of a typical susceptible isolate of C. gattii to FLC, and identified a number of proteins and pathways that could be targeted to augment the activity of FLC
Packer, JM, Irish, JL, Herbert, BR, Hill, C, Padula, M, Blair, S, Carter, D & Harry, L 2012, 'Specific Non-Peroxide Antibacterial Effect Of Manuka Honey On The Staphylococcus Aureus Proteome', International Journal Of Antimicrobial Agents, vol. 40, no. 1, pp. 43-50.View/Download from: Publisher's site
Manuka honey, derived from the New Zealand flowering plant Leptospermum scoparium, shows promise as a topical antibacterial agent and effective chronic wound dressing. The aim of this study was to determine the non-peroxide antibacterial effects of this
Strauss, M, Liew, AT, Turnbull, L, Whitchurch, CB, Monahan, LG & Harry, L 2012, '3D-SIM super resolution microscopy reveals a bead-like arrangement for FtsZ and the division machinery: implications for triggering cytokinesis', Plos Biology, vol. 10, no. 9, p. e1001389.View/Download from: Publisher's site
FtsZ is a tubulin-like GTPase that is the major cytoskeletal protein in bacterial cell division. It polymerizes into a ring, called the Z ring, at the division site and acts as a scaffold to recruit other division proteins to this site as well as providing a contractile force for cytokinesis. To understand how FtsZ performs these functions, the in vivo architecture of the Z ring needs to be established, as well as how this structure constricts to enable cytokinesis. Conventional wide-field fluorescence microscopy depicts the Z ring as a continuous structure of uniform density. Here we use a form of super resolution microscopy, known as 3D-structured illumination microscopy (3D-SIM), to examine the architecture of the Z ring in cells of two Gram-positive organisms that have different cell shapes: the rod-shaped Bacillus subtilis and the coccoid Staphylococcus aureus. We show that in both organisms the Z ring is composed of a heterogeneous distribution of FtsZ. In addition, gaps of fluorescence were evident, which suggest that it is a discontinuous structure. Time-lapse studies using an advanced form of fast live 3D-SIM (Blaze) support a model of FtsZ localization within the Z ring that is dynamic and remains distributed in a heterogeneous manner. However, FtsZ dynamics alone do not trigger the constriction of the Z ring to allow cytokinesis. Lastly, we visualize other components of the divisome and show that they also adopt a bead-like localization pattern at the future division site. Our data lead us to propose that FtsZ guides the divisome to adopt a similar localization pattern to ensure Z ring constriction only proceeds following the assembly of a mature divisome.
Liew, AT, Theis, T, Jensen, SO, Garcia-Lara, J, Foster, S, Firth, N, Lewis, PG & Harry, L 2011, 'A simple plasmid-based system that allows rapid generation of tightly controlled gene expression in Staphylococcus aureus', Microbiology, vol. 157, pp. 666-676.View/Download from: Publisher's site
We have established a plasmid-based system that enables tightly controlled gene expression and the generation of GFP fusion proteins in Staphylococcus aureus simply and rapidly. This system takes advantage of an Escherichia coli S. aureus shuttle vector
Peters, PC, Cox, G, Monahan, LG & Harry, L 2011, 'Super-resolution imaging of the bacterial cytokinetic protein FtsZ', Micron, vol. 42, no. 4 Special Issue, pp. 336-341.View/Download from: Publisher's site
The idea of a bacterial cytoskeleton arose just 10 years ago with the identification of the cell division protein, FtsZ, as a tubulin homolog. Fts,Z plays a pivotal role in bacterial division, and is present in virtually all prokaryotes and in some eukaryotic organelles. The earliest stage of bacterial cell division is the assembly of FtsZ into a Z ring at the -division site, which subsequently constricts during cytokinesis. FtsZ also assembles into dynamic helical structures along the bacterial ceil, which are thought to act as precursors to the Z fing via a cell-cycle-mediated FtsZ polymer remodelling. The fine structures of the FtsZ helix and ring are unknown but crucial for identifying the molecular details of Z ring assembly and its regulation. We now reveal, using STED microscopy that the FtsZ helical structure in cells of the gram positive bacterium, Bacillus subtilis, is a highly irregular and discontinuous helix of FtsZ; very different to the smooth cable-like appearance observed by conventional fluorescence optics. STED also identifies a novel FtsZ helical structure of smaJ!er pitch that is invisible to standard optical methods, identifying a possible third intermediate in the pathway to Z ring assembly, which commits bacterial cells to divide.
Brzoska, AJ, Withers, R, Turner, KM, Robinson, A, Dixon, N, Harry, L & Lewis, P 2010, 'Identification of novel drug targets using model organisms', Microbiology Australia, vol. May, pp. 95-97.
A review that describes the potential for inhibiting essential, fundamental processes in model pathogens to facilitate the development of new antibiotics.
Review in token-refereed journal on the bacterial cytoskeketon
Moriya, S, Rashid, RA, Rodrigues, CD & Harry, L 2010, 'Influence of the nucleoid and the early stages of DNA replication on positioning the division site in Bacillus subtilis', Molecular Microbiology, vol. 76, no. 3, pp. 634-647.View/Download from: Publisher's site
Although division site positioning in rod~shaped bac~ teria is generally believed to occur through the com~ bined effect of nucleoid occlusion and the Min system, several lines of evidence suggest the existence of additional mechanisms. Studies using outgrown spores of Bacillus subtilis have shown that inhi·biting ~ the early stages of DNA replication, leading up to . assembly of the replisome at orie, influences Z ring positioning. Here we examine whether Z ring formation at midcell under various conditions of DNA replication inhibition is solely the result of relief of nucleoid occlusion. We show that midcell Z rings form preferentially over unreplicated nucleoids that have a bilobed mor~ phology (lowering DNA concentration at midcell), whereas acentral Z rings form beside a single~lobed nucleoid. Remarkably however, when the Dna8 repli~ cation initiation protein is inactivated midcell Z rings never form over bilobed nucleoids. Relieving nucleoid occlusion by deleting noc increased midcell Z ring frequency for all situations of DNA replication inhibItion, however not to the same extent, with the DnaB~ inactivated strain having the lowest frequency of midcell Z rings. We propose an additional mechanism for Z ring positioning in which the division site becomes increasingly potentiated for Z ring formation as initiation of replication is progressively completed.
Robinson, A, Brzoska, AJ, Turner, KM, Withers, R, Harry, EJ, Lewis, PJ & Dixon, NE 2010, 'Essential Biological Processes of an Emerging Pathogen: DNA Replication, Transcription, and Cell Division in Acinetobacter spp.', MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, vol. 74, no. 2, pp. 273-+.View/Download from: Publisher's site
Monahan, LG, Robinson, A & Harry, L 2009, 'Lateral Ftsz Association And The Assembly Of The Cytokinetic Z Ring In Bacteria', Molecular Microbiology, vol. 74, no. 4, pp. 1004-1017.View/Download from: Publisher's site
Cell division in bacteria is facilitated by a polymeric ring structure, the Z ring, composed of tubulin-like FtsZ protofilaments. Recently it has been shown that in Bacillus subtilis, the Z ring forms through the cell cycle-mediated remodelling of a helical FtsZ polymer. To investigate how this occurs in vivo, we have exploited a unique temperature-sensitive strain of B. subtilis expressing the mutant protein FtsZ(Ts1). FtsZ(Ts1) is unable to complete Z ring assembly at 49°C, becoming trapped at an intermediate stage in the helix-to-ring progression. To determine why this is the case, we used a combination of methods to identify the specific defect of the FtsZ(Ts1) protein in vivo. Our results indicate that while FtsZ(Ts1) is able to polymerize normally into protofilaments, it is defective in the ability to support lateral associations between these filaments at high temperatures. This strongly suggests that lateral FtsZ association plays a crucial role in the polymer transitions that lead to the formation of the Z ring in the cell. In addition, we show that the FtsZ-binding protein ZapA, when overproduced, can rescue the FtsZ(Ts1) defect in vivo. This suggests that ZapA functions to promote the helix-to-ring transition of FtsZ by stimulating lateral FtsZ association.
Moriya, S, Kawai, Y, Kaji, S, Smith, AC, Harry, L & Errington, J 2009, 'Effects of oriC relocation on control of replication initiation in Bacillus subtilis', Microbiology-uk, vol. 155, pp. 3070-3082.View/Download from: Publisher's site
In bacteria, DNA replication initiation is tightly regulated in order to coordinate chromosome replication with cell growth. In Escherichia coli, positive factors and negative regulatory mechanisms playing important roles in the strict control of DNA replication initiation have been reported. However, it remains unclear how bacterial cells recognize the right time for replication initiation during the cell cycle. In the Gram-positive bacterium Bacillus subtilis, much less is known about the regulation of replication initiation, specifically, regarding negative control mechanisms which ensure replication initiation only once per cell cycle. Here we report that replication initiation was greatly enhanced in strains that had the origin of replication (oriC) relocated to various loci on the chromosome. When oriC was relocated to new loci further than 250 kb counterclockwise from the native locus, replication initiation became asynchronous and earlier than in the wild-type cells. In two oriC-relocated strains (oriC at argG or pnbA, 257 degrees or 300 degrees on the 360 degrees chromosome map, respectively), DnaA levels were higher than in the wild-type but not enough to cause earlier initiation of replication. Our results suggest that the initiation capacity of replication is accumulated well before the actual time of initiation, and its release may be suppressed by a unique DNA structure formed near the native oriC locus.
Blair, S, Cokcetin, N, Harry, L & Carter, D 2009, 'The unusual antibacterial activity of medical-grade Leptospermum honey: antibacterial spectrum, resistance and transcriptome analysis', European Journal of Clinical Microbiology & Infecti..., vol. 28, no. 10, pp. 1199-1208.View/Download from: Publisher's site
There is an urgent need for new, effective agents in topical wound care, and selected honeys show potential in this regard. Using a medical-grade honey, eight species of problematic wound pathogens, including those with high levels of innate or acquired antibiotic resistance, were killed by 4.0-14.8% honey, which is a concentration that can be maintained in the wound environment. Resistance to honey could not be induced under conditions that rapidly induced resistance to antibiotics. Escherichia coli macroarrays were used to determine the response of bacterial cells to a sub-lethal dose of honey. The pattern of gene expression differed to that reported for other antimicrobial agents, indicating that honey acts in a unique and multifactorial way; 78 (2%) genes were upregulated and 46 (1%) genes were downregulated more than two-fold upon exposure to the medical-grade honey. Most of the upregulated genes clustered into distinct functional regulatory groups, with many involved in stress responses, and the majority of downregulated genes encoded for products involved in protein synthesis. Taken together, these data indicate that honey is an effective topical antimicrobial agent that could help reduce some of the current pressures that are promoting antibiotic resistance.
The growing problem of antibiotic resistance has been exacerbated by the use of new drugs that are merely variants of older overused antibiotics. While it is naive to expect to restrain the spread of resistance without controlling antibacterial usage, the desperate need for drugs with novel targets has been recognized by health organizations, industry and academia alike. The wealth of knowledge available about the bacterial cell-division pathway has aided target-driven approaches to identify novel inhibitors. Here, we discuss the therapeutic potential of inhibiting bacterial cell division, and review the progress made in this exciting new area of antibacterial discovery.
The growing problem of antibiotic resistance has been exacerbated by the use of new drugs that are merely variants of older overused antibiotics. While it is naive to expect to restrain the spread of resistance without controlling antibacterial usage, the desperate need for drugs with novel targets has been recognized by health organizations, industry and academia alike. The wealth of knowledge available about the bacterial cell-division pathway has aided target-driven approaches to identify novel inhibitors. Here, we discuss the therapeutic potential of inhibiting bacterial cell division, and review the progress made in this exciting new area of antibacterial discovery.
Wadsworth, KD, Rowland, SL, Harry, L & King, GF 2008, 'The divisomal protein DivIB contains multiple epitopes that mediate its recruitment to incipient division sites', Molecular Microbiology, vol. 67, no. 5, pp. 1143-1155.View/Download from: Publisher's site
Bacterial cytokinesis is orchestrated by an assembly of essential cell division proteins that form a supramolecular structure known as the divisome. DivIB and its orthologue FtsQ are essential members of the divisome in Gram-positive and Gram-negative ba
Peters, PC, Migocki, M, Thoni, C & Harry, L 2007, 'A new assembly pathway for the cytokinetic Z ring from a dynamic helical structure in vegetatively growing cells of Bacillus subtilis', Molecular Microbiology, vol. 64, no. 2, pp. 487-499.View/Download from: Publisher's site
The earliest event in bacterial cell division is the formation of a Z ring, composed of the tubulin-like FtsZ protein, at the division site at midcell. This ring then recruits several other division proteins and together they drive the formation of a division of septum between two replicated chromosomes. Here we show that, in addition to forming a cytokinetic ring, FtsZ localises in a helical-like pattern in vegetatively growing cells of Bacillus subtilis. FtsZ moves rapudly within this helix-like structure. Examination of FtsZ localisation in individual live cells undergoing a single cell cycle suggests a new assembly mechanism for Z ring formation tha involves a cell cycle-mediated multistep remodelling of FtsZ polymers. Our observations suggest that initially FtsZ localises in a helical pattern, with movement of FtsZ within this sturcture occurring along the entire length of the cell. Next, movement of FtsZ in a helical-like pattern is restricted to a central region of the cell. Finally the FtsZ ring forms precisely at midcell. We further show that another division protein, FtsA, shown to interact with FtsZ prior to Z ring formation in B. subtilis, also localises to similar helical patterns in vegetatively growing cells.
Herbert, BR, Grinyer, J, Mccarthy, J, Isaacs, M, Harry, L, Nevalainen, HK, Traini, M, Hunt, S, Schulz, B, Laver, M, Goodall, AR, Packer, JM, Harry, JL & Williams, K 2006, 'Improved 2-DE of microorganisms after acidic extraction', Electrophoresis, vol. 27, no. 8, pp. 1630-1640.View/Download from: Publisher's site
2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extrac
Michie, KA, Monahan, LG, Beech, PL & Harry, L 2006, 'Trapping of a spiral-like intermediate of the bacterial cytokinetic protein FtsZ', Journal Of Bacteriology, vol. 188, no. 5, pp. 1680-1690.View/Download from: Publisher's site
The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at m
Peters, PC, Thoni, C & Harry, L 2006, 'Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation', Biotechnic and Histochemistry, vol. 81, no. 2-3, pp. 105-106.View/Download from: Publisher's site
A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when us
Thompson, L & Harry, L 2006, 'Alternative Sigma factors: the master regulators', Microbiology Australia, vol. 27, no. 3, pp. 118-120.
Thompson, L, Beech, PL, Real, G, Henriques, AO & Harry, L 2006, 'Requirement for the cell division protein DivIB in polar cell division and engulfment during sporulation in Bacillus subtilis', Journal Of Bacteriology, vol. 188, no. 21, pp. 7677-7685.View/Download from: Publisher's site
During spore formation in Bacillus subtilis, cell division occurs at the cell pole and is believed to require essentially the same division machinery as vegetative division. Intriguingly, although the cell division protein DivIB is not required for veget
Hayashi, M, Ogura, Y, Harry, L, Ogasawara, N & Moriya, S 2005, 'Bacillus subtilis YabA is involved in determining the timing and synchrony of replication initiation', FEMS Microbiology Letters, vol. 247, no. 1, pp. 73-79.View/Download from: Publisher's site
It is shown here by flow cytometry that Bacillus subtilis YabA negatively regulates the timing of replication initiation. When the level of YabA was reduced, replication began at a decreased cell mass and when the level was increased, initiation was dela
Jensen, SO, Thompson, L & Harry, L 2005, 'Cell division in bacillus subtilis: FtsZ and FtsA association is Z-ring independent, and FtsA is required for efficient midcell Z-ring assembly', Journal Of Bacteriology, vol. 187, no. 18, pp. 6536-6544.View/Download from: Publisher's site
The earliest stage in cell division in bacteria is the assembly of a Z ring at the division site at midcell. Other division proteins are also recruited to this site to orchestrate the septation process. FtsA is a cytosolic division protein that interacts
Jensen, SO, Thompson, LS & Harry, EJ 2005, 'Cell division in bacillus subtilis: FtsZ and FtsA association is Z-ring independent, and FtsA is required for efficient midcell Z-ring assembly', JOURNAL OF BACTERIOLOGY, vol. 187, no. 18, pp. 6536-6544.View/Download from: Publisher's site
Real, G, Autret, S, Harry, L, Errington, J & Henriques, AO 2005, 'Cell division protein DivIB influences the Spo0J/Soj system of chromosome segregation in Bacillus subtilis', Molecular Microbiology, vol. 55, no. 2, pp. 349-367.View/Download from: Publisher's site
The initiation of the developmental process of sporulation in the rod-shaped bacterium Bacillus subtilis involves the activation of the Spo0A response regulator. Spo0A then drives the switch in the site of division septum formation from midcell to a polar position. Activated Spo0A is required for the transcription of key sporulation loci such as spoIIG, which are negatively regulated by the Soj protein. The transcriptional repressing activity of Soj is antagonized by Spo0J, and both proteins belong to the well-conserved Par family of partitioning proteins. Soj has been shown to jump from nucleoid to nucleoid via the cell pole. The dynamic behaviour of Soj is somehow controlled by Spo0J, which prevents the static association of Soj with the nucleoid, and presumably its transcriptional repression activity. Soj in turn is required for the proper condensation of Spo0J foci around the oriC region. The asymmetric partitioning of the sporangial cell requires DivIB and other proteins involved in vegetative (medial) division. We describe an allele of the cell division gene divIB (divIB80) that reduces the cellular levels of DivIB, and affects nucleoid structure and segregation in growing cells, yet has no major impact on cell division. In divIB80 cells Spo0J foci are not correctly condensed and Soj associates statically with the nucleoid. The divIB80 allele prevents transcription of spoIIG, and arrests sporulation prior to the formation of the asymmetric division septum. The defect in Spo0A-dependent gene expression, and the Spo- phenotype can be suppressed by expression of divIB in trans or by deletion of the soj-spo0J locus. However, deletion of the spo0J-soj region does not restore the normal cellular levels of DivIB. Therefore, the reduced levels of DivIB in the divIB80 mutant are sufficient for efficient cell division, but not to sustain a second, earlier function of DivIB related to the activity of the Spo0J/Soj system of chromosome segregation.
Proteomics has rapidly become an important tool for life science research, allowing the integrated analysis of global protein expression from a single experiment. To accommodate the complexity and dynamic nature of any proteome, researchers must use a co
Migocki, M, Lewis, PJ, Wake, G & Harry, L 2004, 'The midcell replication factory in Bacillus subtilis is highly mobile: implications for coordinating chromosome replication with other cell cycle events', Molecular Microbiology, vol. 54, no. 2, pp. 452-463.View/Download from: Publisher's site
During vegetative growth, rod-shaped bacterial cells such as Escherichia coli and Bacillus subtilis divide precisely at midcell. It is the Z ring that defines the position of the division site. We previously demonstrated that the early stages of chromosome replication are linked to midcell Z ring assembly in B. subtilis and proposed a direct role for the centrally located replication factory in masking and subsequently unmasking the midcell site for Z ring assembly. We now show that the replication factory is significantly more scattered about the cell centre than the Z ring in both vegetative cells and outgrown spores of B. subtilis. This finding is inconsistent with the midcell replication factory acting as a direct physical block to Z ring assembly. Time-lapse experiments demonstrated that the lower precision of replication factory positioning results from its high mobility around the cell centre. Various aspects of this mobility are presented and the results are discussed in the light of current views on the determinants of positional information required for accurate chromosome segregation and cell division
Harry, L 2003, 'The precision of bacterial cell division', Today's Life Science, vol. 15, no. 2, pp. 34-37.
Harry, L & Lewis, PJ 2003, 'Early Targeting of Min Proteins to the Cell Poles in Germinated Spores of Bacillus subtilis: Evidence for Division Apparatus-Independent Recruitment of Min Proteins to the Division Site', Molecular Microbiology, vol. 47, no. 1, pp. 37-48.View/Download from: Publisher's site
The earliest event in bacterial cell division is the assembly of a tubulin-like protein, FtsZ, at mid-cell to form a ring. In rod-shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell. The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions. All three proteins localize to the division site at mid-cell and to cell poles. Their recruitment to the division site is dependent on localization of both `early' and `late' division proteins. We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B. subtilis spores. We show that, although the FtsZ ring assembles at mid-cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z-ring constriction and completion of division. We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid-cell. Furthermore, these proteins were found to be present in mature, dormant spores. These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal-sized daughter cells are produced upon cell division
Migocki, M & Harry, L 2003, 'Splitting up: a time and place for it in bacteria', Australian Biochemist, vol. April.
Ogura, Y, Ogasawara, N, Harry, L & Moriya, S 2003, 'Increasing the ratio of Soj to SpoOJ levels promotes replication initiation in Bacillus subtilis', Journal Of Bacteriology, vol. 185, no. 21, pp. 6316-6324.View/Download from: Publisher's site
The ParA and ParB protein families are well conserved in bacteria. However, their functions are still unclear. In Bacillus subtilis, Soj and Spo0J are members of these two protein families, respectively. A previous report revealed that replication initiated early and asynchronously in spo0J null mutant cells, as determined by flow cytometry. In this study, we examined the cause of this promotion of replication initiation. Deletion of both the soj and spo0J genes restored the frequency of replication initiation to almost the wild-type level, suggesting that production of Soj in the absence of Spo0J leads to early and asynchronous initiation of replication. Consistent with this suggestion, overproduction of Soj in wild-type cells had the same effect on replication initiation as in the spo0J null mutant, and overproduction of both Soj and Spo0J did not. These results indicate that when the ratio of Soj to Spo0J increases, Soj interferes with tight control of replication initiation and causes early and asynchronous initiation. Whereas replication initiation also occurred significantly earlier in the two spo0J mutants, spo0J14 and spo0J17, it occurred only slightly early in the sojK16Q mutant and was delayed in the sojG12V mutant. Although Soj localized to nucleoids in the spo0J mutants, the two Soj mutant proteins were distributed throughout the cell or localized to cell poles. Thus, interestingly, the promotion of replication initiation seems to correlate with localization of Soj to nucleoids. This may suggest that Soj inhibits transcription of some cell cycle genes and leads to early and asynchronous initiation of replication. In wild-type cells Spo0J counteracts this Soj function.
Migocki, M, Freeman, M, Wake, G & Harry, L 2002, 'The Min system is not required for precise placement of the midcell division site in Bacillus subtilis', EMBO Reports, vol. 3, no. 12, pp. 1163-1167.View/Download from: Publisher's site
In bacteria, the Min system plays a role in positioning the midcell division site by inhibiting the formation of the earliest precursor of cell division, the Z ring, at the cell poles. However, whether the Min system also contributes to establishing the precise placement of the midcell Z ring is unresolved. We show that the Z ring is positioned at midcell with a high degree of precision in Bacillus subtilis, and this is completely maintained in the absence of the Min system. Min is therefore not required for correct midcell Z ring placement in B. subtilis. Our results strongly support the idea that the primary role of the Min system is to block Z ring formation at the cell poles and that a separate mechanism must exist to ensure cell division occurs precisely at midcell.
Robson, SA, Michie, KA, Mackay, J, Harry, L & King, GF 2002, 'The Bacillus subtilis cell division proteins FtsL and DivIC are intrinsically unstable and do not interact with one another in the absence of other septasomal components', Molecular Microbiology, vol. 44, no. 3, pp. 663-674.View/Download from: Publisher's site
The bacterial septum appears to comprise a macromolecular assembly of essential cell division proteins (the 'septasome') that are responsible for physically dividing the cell during cytokinesis. FtsL and DivIC are essential components of this division machinery in Bacillus subtilis. We used yeast two-hybrid analysis as well as a variety of biochemical and biophysical methods to examine the proposed interaction between Bacillus subtilis FtsL and DivIC. We show that FtsL and DivIC are thermodynamically unstable proteins that are likely to be unfolded and therefore targeted for degradation unless stabilized by interactions with other components of the septasome. However, we show that this stabilization does not result from a direct interaction between FtsL and DivIC. We propose that the observed interdependence of DivIC and FtsL stability is a result of indirect interactions that are mediated by other septasomal proteins.
The earliest stage of cell division in bacteria is the formation of a Z ring, composed of a polymer of the FtsZ protein, at the division site. Z rings appear to be synthesized in a bi-directional manner from a nucleation site (NS) located on the inside of the cytoplasmic membrane. It is the utilization of a NS specifically at the site of septum formation that determines where and when division will occur. However, a Z ring can be made to form at positions other than at the division site. How does a cell regulate utilization of a NS at the correct location and at the right time? In rod-shaped bacteria such as Escherichia coli and Bacillus subtilis, two factors involved in this regulation are the Min system and nucleoid occlusion. It is suggested that in B. subtilis, the main role of the Min proteins is to inhibit division at the nucleoid-free cell poles. In E. coli it is currently not clear whether the Min system can direct a Z ring to the division site at mid-cell or whether its main role is to ensure that division inhibition occurs away from mid-cell, a role analogous to that in B. subtilis. While the nucleoid negatively influences Z-ring formation in its vicinity in these rod-shaped organisms, the exact relationship between nucleoid occlusion and the ability to form a mid-cell Z ring is unresolved. Recent evidence suggests that in B. subtilis and Caulobacter crescentus, utilization of the NS at the division site is intimately linked to the progress of a round of chromosome replication and this may form the basis of achieving co-ordination between chromosome replication and cell division.
Harry, L 2001, 'Coordinating DNA replication with cell division: Lessons from outgrowing spores', Biochimie: an international journal of biochemistry ..., vol. 83, pp. 75-81.
Progress in solving the long-standing puzzle of how a cell coordinates chromosome replication with cell division is significantly aided by the use of synchronous cell populations. Currently three systems are employed for obtaining such populations: the Escherichia coli `baby machine, the developmentally-controlled cell cycle of Caulobacter crescentus, and Bacillus subtilis germinated and outgrowing spores. This review examines our current understanding of the relationship between replication and division and how the use of B. subtilis outgrowing spores and, more recently its combination with immunofluorescence microscopy, has contributed significantly to this important area of biology. About 20 years ago, and also more recently, this system was used to show convincingly that termination of DNA replication is not essential for a central septum to form, raising the possibility that the early stages of division occur well before termination. It has also been demonstrated that there is no major synthesis of the division initiation proteins, FtsZ and DivIB, linked to initiation, progression or completion of the first round of chromosome replication accompanying spore outgrowth. This has led to the suggestion that the primary link between chromosome replication and cell division at midcell is not likely to occur through a control over the levels of these proteins. Very recent work has employed a combination of the use of B. subtilis outgrowing spores with immunofluorescence microscopy to investigate the relationship between midcell Z ring assembly and the round of chromosome replication linked to it. The results of this work suggest a role for initiation and progression into the round of replication in blocking midcell Z ring formation until the round is complete or almost complete, thereby ensuring that cell division occurs between two equally-partitioned chromosomes.
Daniel, RA, Harry, L & Errington, J 2000, 'Role of penicillin-binding protein PBP 2B in assembly and functioning of the division machinery of Bacillus subtilis.', Molecular Microbiology, vol. 35, no. 2, pp. 299-311.View/Download from: Publisher's site
We have characterized the role of the penicillin-binding protein PBP 2B in cell division of Bacillus subtilis. We have shown that depletion of the protein results in an arrest in division, but that this arrest is slow, probably because the protein is relatively stable. PBP 2B-depleted filaments contained, at about their mid-points, structures resembling partially formed septa, into which most, if not all, of the division proteins had assembled. Although clearly deficient in wall material, membrane invagination seemed to continue, indicating that membrane and wall ingrowth can be uncoupled. At other potential division sites along the filaments, no visible ingrowths were observed, although FtsZ rings assembled at regular intervals. Thus, PBP 2B is apparently required for both the initiation of division and continued septal ingrowth. Immunofluorescence microscopy showed that the protein is recruited to the division site. The pattern of localization suggested that this recruitment occurs continually during septal ingrowth. During sporulation, PBP 2B was present transiently in the asymmetrical septum of sporulating cells, and its availability may play a role in the regulation of sporulation septation.
Katis, VL, Wake, G & Harry, L 2000, 'Septal localization of the membrane-bound division proteins of Bacillus subtilis DivIB and DivIC is codependent only at high temperatures and requires FtsZ', Journal Of Bacteriology, vol. 182, no. 12, pp. 3607-3611.View/Download from: Publisher's site
Using immunofluorescence microscopy, we have examined the dependency of localization among three Bacillus subtilis division proteins, FtsZ, DivIB, and DivIC, to the division site. DivIC is required for DivIB localization. However, DivIC localization is dependent on DivIB only at high growth temperatures, at which DivIB is essential for division. FtsZ localization is required for septal recruitment of DivIB and DivIC, but FtsZ can be recruited independently of DivIB. These localization studies suggest a more specific role for DivIB in division, involving interaction with DivIC.
Regamy, A, Harry, L & Wake, G 2000, 'Midcell Z ring assembly in the absence of entry into the elongation phase of the round of replication in bacteria: coordinating chromosome replication with cell division', Molecular Microbiology, vol. 38, no. 3, pp. 423-434.View/Download from: Publisher's site
We have shown previously that, when spores of a thymine-requiring strain of Bacillus subtilis were grown out in the absence of thymine, mid-cell Z rings formed over the nucleoid and much earlier than might be expected with respect to progression into the round of replication. It is now shown that such conditions allow no replication of oriC. Rather than replication, partial degradation of the oriC region occurs, suggesting that the status of this region is connected with the `premature' mid-cell Z ring assembly. A correlation was observed between entry into the replication elongation phase and a block to mid-cell Z rings. The conformation of the nucleoid under various conditions of DNA replication inhibition or limitation suggests that relief of nucleoid occlusion is not primarily responsible for mid-cell Z ring formation in the absence of thymine. We propose the existence of a specific structure at mid-cell that defines the Z ring nucleation site (NS). It is suggested that this NS is normally masked by the replisome upon initiation of replication or soon after entry into the elongation phase, and subsequently unmasked relatively late in the round. During spore outgrowth in the absence of thymine, this checkpoint control over mid-cell Z ring assembly breaks down prematurely.
Harry, L, Rodwell, JJ & Wake, G 1999, 'Coordinating DNA replication with cell division in bacteria: a link between the early stages of a round of replication and mid-cell Z ring assembly', Molecular Microbiology, vol. 33, no. 1, pp. 33-40.View/Download from: Publisher's site
Spores of a thymine-requiring strain of Bacillus subtilis 168, which is also temperature sensitive for the initiation of chromosome replication, were germinated and allowed to grow out at the permissive temperature in a minimal medium containing no added thymine. Under these conditions, there was no or very limited progression into the elongation phase of the first round of replication. In a significant proportion of the outgrown cells, a Z ring formed precisely at mid-cell and over the centrally positioned nucleoid, leading eventually to the formation of a mature division septum. When initiation of the first round of replication was blocked through a temperature shift and with thymine present, the Z ring was positioned acentrally. The central Z ring that formed in the absence of thymine was blocked by the presence of a DNA polymerase III inhibitor. It is concluded that the very early stages of a round of replication (initiation plus possibly limited progression into the elongation phase) play a key role in the precise positioning of the Z ring at mid-cell and between replicating daughter chromosomes
Daniel, RA, Harry, L, Katis, VL, Wake, G & Errington, J 1998, 'Characterization Of The Essential Cell Division Gene FtsL (ylld) Of Bacillus Subtilis And Its Role In The Assembly Of The Division Apparatus', Molecular Microbiology, vol. 29, no. 2, pp. 593-604.View/Download from: Publisher's site
We have identified the Bacillus subtilis homologue of the essential cell division gene, ftsL, of Escherichia coli, Repression of ftsL in a strain engineered to carry a conditional promoter results in cell filamentation, with a near immediate arrest of ce
Bacterial chromosome segregation involves the separation and movement of two sister chromosomes away from one another to locations where they can be incorporated into separate daughter cells during cell division. Chromosome (nucleoid) segregation involves two distinct processes: (1) the decatenation and resolution of two newly replicated nucleoids to produce two separable sister nucleoids and (2) the movement of these sister chromosomes (partitioning) to reposition them on either side of the division site. Recent work has clarified the first process in molecular terms; however, the process of chromosome movement remains a mystery.
Harry, L & Wake, G 1997, 'The membrane-bound cell division protein DivIB is localized to the division site in Bacillus subtilis', Molecular Microbiology, vol. 25, no. 2, pp. 275-283.View/Download from: Publisher's site
The cell division gene divIB of Bacillus subtilis is essential for the normal rate of growth and division. The gene product, DivIB, is a membrane-bound protein in which the bulk of the protein (at the C-terminal end) is on the exterior surface of the cell membrane. DivIB is involved in the early stages of septum formation, but its exact role in cell division is unknown. To gain more information about the mode of action of DivIB in septum formation, we determined the location of DivIB within the cell membrane using immunofluorescence. This immunolocalization approach established that DivIB becomes localized to the division site before visible septation and remains localized to this site throughout the division process. Various DivIB immunostaining patterns were observed in immunofluorescence experiments and, together with cell length and nucleoid distance measurements, have allowed us to propose two models to describe DivIB localization during the cell cycle.
Katis, VL, Harry, L & Wake, G 1997, 'The Bacillus Subtilis Division Protein Divlc Is A Highly Abundant Membrane-bound Protein That Localizes To The Division Site', Molecular Microbiology, vol. 26, no. 5, pp. 1047-1055.View/Download from: Publisher's site
The Bacillus subtilis divIC gene is involved in the initiation of cell division. It encodes a 14.7 kDa protein, with a potential transmembrane region near the N-terminus. In this paper, we show that DivIC is associated with the cell membrane and, in conj
Harry, L, Pogliano, K & Losick, R 1995, 'Use Of Immunofluorescence To Visualize Cell-specific Gene-expression During Sporulation In Bacillus-subtilis', Journal Of Bacteriology, vol. 177, no. 12, pp. 3386-3393.
We have adapted immunofluorescence microscopy for use in Bacillus subtilis and have employed this procedure for visualizing cell-specific gene expression at early to intermediate stages of sporulation. Sporangia were doubly stained with propidium iodide
Hofmeister, AE, Londono-Vallejo, A, Harry, L, Stragier, P & Losick, R 1995, 'Extracellular signal protein triggering the proteolytic activation of a developmental transcription factor in B. subtilis', Cell, vol. 83, no. 2, pp. 219-226.View/Download from: Publisher's site
We present biochemical evidence for an intercellular signal transduction pathway in B. subtilis. This pathway governs the conversion of the proprotein pro-Î¸E to the mature transcription factor Î¸E. Proteolytic processing is mediated by the membrane protein SpoIIGA and is triggered by the inferred extracellular signal protein SpoIIR. A factor in conditioned medium from B. subtilis cells engineered to produce SpoIIR during growth triggered processing in protoplasts of B. subtilis cells that had been engineered to produce SpoIIGA and pro-ÏE The factor was also detected in, and partially purified from, extracts of SpoIIR-producing cells of E. coli. We speculate that SpoIIGA is both a receptor and a protease and that SpoIIR interacts with SpolIGA on the outside of the cytoplasmic membrane, activating the intracellular protease domain of SpolIGA.
Pogliano, K, Harry, L & Losick, R 1995, 'Visualisation of the subcellular location of sporulation proteins in Bacillus subtilis using immunofluorescence microscopy', Molecular Microbiology, vol. 18, no. 3, pp. 459-470.View/Download from: Publisher's site
Harry, L, Partridge, SR, Weiss, AS & Wake, G 1994, 'Conservation Of The 168-divIB Gene In Bacillus subtilis W23 And B. licheniformis, And Evidence For Homology To FtsQ Of Escherichia coli', Gene, vol. 147, no. 1, pp. 85-89.View/Download from: Publisher's site
The chromosomal regions of Bacillus subtilis (Bs) W23 and Bacillus licheniformis (Bl), which span the sequence encoding the homolog of the division initiation gene, divIB, of Bs168 were cloned and sequenced. The high level of conservation of the amino ac
Harry, L, Rowland, SL, Malo, MS & Wake, G 1994, 'Expression Of DivIB Of Bacillus-subtilis During Vegetative Growth', Journal Of Bacteriology, vol. 176, no. 4, pp. 1172-1179.
Expression of the division initiation gene, divIB, of Bacillus subtilis during vegetative growth was examined. lacZ fusion studies and transcription start point mapping have established that a sigma(A) promoter proximal to divIB is utilized in vivo. The
Harry, L, Stewart, B & Wake, G 1993, 'Characterization Of Mutations In Divlb Of Bacillus-subtilis And Cellular-localization Of The Divlb Protein', Molecular Microbiology, vol. 7, no. 4, pp. 611-621.View/Download from: Publisher's site
Four temperature-sensitive mutations in the divIB gene of Bacillus subtilis have been localized to the region corresponding to the C-terminal half of the 263-residue DivIB protein. Antiserum was raised to the 80% C-terminal portion lying on one side of a
Harry, L & Wake, G 1989, 'Cloning And Expression Of A Bacillus-subtilis Division Initiation Gene For Which A Homolog Has Not Been Identified In Another Organism', Journal Of Bacteriology, vol. 171, no. 12, pp. 6835-6839.
The Bacillus subtilis 168 division initiation genes defined by the temperature-sensitive mutations ts-1 and ts-12 were cloned into a 10.5-kilobase EcoRI fragment of DNA in the lambda EMBL4 vector. The two genes were separated by approximately 3 kilobases. The gene in which the ts-1 mutation resides was shown to be the same as the B. subtilis homolog of the Escherichia coli ftsZ gene. The other gene was named divIB. It showed no homology to any previously identified gene and coded for a protein of 30.1 kilodaltons which was probably membrane bound.
Bottomley, AL, Turnbull, L, Whitchurch, CB & Harry, EJ 2017, 'Immobilization Techniques of Bacteria for Live Super-resolution Imaging Using Structured Illumination Microscopy.' in Pontus Nordenfelt and Mattias Collin (ed), Bacterial Pathogenesis, pp. 197-209.View/Download from: Publisher's site
Advancements in optical microscopy technology have allowed huge progression in the ability to understand protein structure and dynamics in live bacterial cells using fluorescence microscopy. Paramount to high-quality microscopy is good sample preparation to avoid bacterial cell movement that can result in motion blur during image acquisition. Here, we describe two techniques of sample preparation that reduce unwanted cell movement and are suitable for application to a number of bacterial species and imaging methods.
Monahan, LG, D'Elia, M & Harry, L 2011, 'Mining bacterial cell division for new antibacterial drugs' in Miller, AA & Miller, PF (eds), Emerging Trends in Antibacterial Discovery: Answering the Call to Arms, Caister Academic Press, United Kingdom, pp. 35-75.
As bacterial antibiotic resistance continues to exhaust our supply of effective antibiotics, a global public health disaster appears likely. Poor financial investment in antibiotic research has exacerbated the situation. A call to arms raised by several prestigious scientific organisations a few years ago rallied the scientific community, and now the scope of antibacterial research has broadened considerably. Multi-disciplinary approaches have yielded a wealth of new data on areas ranging from the identification of novel antibacterial targets to the use of biological agents for antibacterial therapy.
Herbert, BR & Harry, L 2009, 'Difficult proteins' in Sheehan, D & Tyther, R (eds), Two-dimensional electrophoresis protocols, Humana Press, New York, pp. 47-63.
Harry, L, Monahan, LG & Thompson, L 2006, 'Bacterial cell division: the mechanism and its precision' in Jeon, KW (ed), International Review of Cytology: A Survey of Cell biology Volume 253, Elsevier, The netherlands, pp. 27-94.
The recent devlopment of cell biolofy technques for bacteria to allow visualisation of fundamental processes in time and space, and their use in synchronous populations of cells, has resulted in a dramatic increase in our understanding of cell division and it regulation in these tiny cells. The first stage of cell division is the formation of a Z ring, composed of apolymerised tubulin-like protein, FtZ,at the division site precisely at midcell. Several membrane-associated division proteins are then recruited to this ring to forma complex, the divisome, which causes invagination of the cell envelope layers to form a division septum. The z Ring marks the future division site, and the timing of assembly and positioning of this structure are important in determining where and when division will take place in the cell. Z ring assembly is controlled bnu many factors including negative regulatory mechanisms such as Min and nucleoid occlusion that influence Z ring positioning and FtZ accessory proteins that bind to FtZ directly and modulate its polymerisation behaviour. The replication status of the cell also influences the positionin of the Z ring,w hich may allow the tight coordination between DNA replication and cell division required toproduce two identical newborn cells.
Campbell, LT, Simonin, AR, Padula, MP, Harry, E, Herbert, BR & Carter, DA 2012, 'The fungal secretome and virulence: Analysis of the proteins secreted by Cryptococcus gattii strains with different virulence profiles', MYCOSES, WILEY-BLACKWELL, pp. 163-163.
Chong, HS, Campbell, LT, Padula, M, Harry, E, Hill, C, Li, S, Herbert, B, Wilkins, MR & Carter, DA 2012, 'The dynamic response of Cryptococcus gattii cells to fluconazole: a time-course analysis of the proteome and interactome', MYCOSES, WILEY-BLACKWELL, pp. 115-116.
Mueller, P, Turnbull, L, Schlothauer, R, Whitchurch, C & Harry, E 2012, 'NEW ZEALAND MANUKA HONEY HELPS HUMAN KERATINOCYTES TO SURVIVE IN PRESENCE OF S-AUREUS', WOUND REPAIR AND REGENERATION, WILEY-BLACKWELL, pp. A75-A75.
Theis, T, Quach, P, Lock, RL, Lindner, R & Harry, L 2007, 'Divide and forfeit: Cell division as an antibacterial target', BacPath 9 The Molecular Biology of Bacterial Pathogens, Lorne, Victoria.
Using the crystal structure of S. aureus FtsZ with a co-crystallised known inhibitor, a pharmacophore was developed that could be utilized in the design of novel inhibitors. A library of 19 molecules were synthesized and structurally elucidated that contain a pyrazole linker (Scheme 1). These molecules were screened against S. aureus ATCC 25923 and Escherichia coli (E. coli) MG1556 cells to determine their antibacterial activity.