scholarly journals SmdA is a novel cell morphology determinant in Staphylococcus aureus

2021 ◽  
Author(s):  
Ine Storaker Myrbråten ◽  
Gro A. Stamsås ◽  
Helena Chan ◽  
Danae Morales Angeles ◽  
Tiril Mathiesen Knutsen ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Division ◽  
Cell Morphology ◽  
Spherical Shape ◽  
Major Influence ◽  
Cell Wall Synthesis ◽  
Division Plane ◽  
Microscopy Techniques ◽  
Two Hybrid

Cell division and cell wall synthesis in staphylococci need to be precisely coordinated and controlled to allow the cell to multiply while maintaining their nearly spherical shape. The mechanisms ensuring correct placement of the division plane and synthesis of new cell wall have been studied intensively, however, hitherto unknown factors and proteins are likely to play key roles in this complex interplay. Starting from a subcellular localization- and gene knockdown screen of essential genes with unknown functions in Staphylococcus aureus, we identified a protein with major influence on cell morphology in S. aureus. The protein, here named SmdA (for staphylococcal morphology determinant A), is a membrane-protein with septum-enriched localization. By smdA silencing and overexpression, we have used different microscopy techniques to show that SmdA is critical for cell division, including septum formation and cell splitting. We also identified conserved residues in SmdA that are critical for functionality. Pulldown- and bacterial two-hybrid interaction experiments showed that SmdA interacts with several known cell division- and cell wall synthesis proteins, including penicillin binding proteins (PBPs) and EzrA. Notably, SmdA also affects susceptibility to cell wall targeting antibiotics, particularly in methicillin-resistant S. aureus (MRSA). Together, our results show that S. aureus is dependent on balanced amounts of membrane-attached SmdA in order to carry out proper cell division.

scholarly journals CozEa and CozEb play overlapping and essential roles in controlling cell division in Staphylococcus aureus

2018 ◽  
Author(s):  
Gro Anita Stamsås ◽  
Ine Storaker Myrbråten ◽  
Daniel Straume ◽  
Zhian Salehian ◽  
Jan-Willem Veening ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Normal Cell ◽  
Double Mutant ◽  
Spherical Shape ◽  
Cell Wall Synthesis ◽  
Morphological Defects ◽  
Cell Division Protein

SummaryStaphylococcus aureus needs to control the position and timing of cell division and cell wall synthesis to maintain its spherical shape. We identified two membrane proteins, named CozEa and CozEb, which together are important for proper cell division in S. aureus. CozEa and CozEb are homologs of the cell elongation regulator CozESpn of Streptococcus pneumoniae. While cozEa and cozEb were not essential individually, the ΔcozEaΔcozEb double mutant was lethal. To study the functions of cozEa and cozEb, we constructed a CRISPR interference (CRISPRi) system for S. aureus, allowing transcriptional knockdown of essential genes. CRISPRi knockdown of cozEa in the ΔcozEb strain (and vice versa) causes cell morphological defects and aberrant nucleoid staining, showing that cozEa and cozEb have overlapping functions and are important for normal cell division. We found that CozEa and CozEb interact with the cell division protein EzrA, and that EzrA-GFP mislocalizes in the absence of CozEa and CozEb. Furthermore, the CozE-EzrA interaction is conserved in S. pneumoniae, and cell division is mislocalized in cozESpn-depleted S. pneumoniae cells. Together, our results show that CozE proteins mediate control of cell division in S. aureus and S. pneumoniae, likely via interactions with key cell division proteins such as EzrA.

scholarly journals Bacteriophage SP01 Gene Product 56 (gp56) Inhibits Bacillus subtilis Cell Division by Interacting with DivIC/FtsL to Prevent Pbp2B/FtsW Recruitment

2020 ◽  
Author(s):  
Amit Bhambhani ◽  
Isabella Iadicicco ◽  
Jules Lee ◽  
Syed Ahmed ◽  
Max Belfatto ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Gene Product ◽  
Lytic Bacteriophage ◽  
Cell Wall Synthesis ◽  
Ftsz Ring ◽  
Ring Assembly ◽  
Ring Shaped Structure ◽  
Two Hybrid

ABSTRACTPrevious work identified gp56, encoded by the lytic bacteriophage SP01, as responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here we show that expression of the predicted 9.3-kDa gene product 56 (gp56) of SP01 inhibits latter stages of B. subtilis cell division without altering FtsZ ring assembly. GFP-tagged gp56 localizes to the membrane at the site of division. While its localization permits recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analysis suggest that gp56 localization and activity depends on its interaction with mid-recruited proteins DivIC and/or FtsL. Together these data support a model where gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCEResearch over the past decades has uncovered bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. Phage factors that cause cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanism of several identified phage factors that inhibit cytokinesis remain unexplored, including gp56 of bacteriophage SP01 of Bacillus subtilis. Here, we show that unlike related published examples of phage inhibition of cyotkinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and block recruitment of proteins needed for the septal cell wall synthesis.

scholarly journals Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division

2016 ◽  
Author(s):  
Alexandre W. Bisson Filho ◽  
Yen-Pang Hsu ◽  
Georgia R. Squyres ◽  
Erkin Kuru ◽  
Fabai Wu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Division Ring ◽  
Bacterial Cell ◽  
Cytoplasmic Membrane ◽  
Bacterial Cell Division ◽  
Cell Wall Synthesis ◽  
Division Plane ◽  
Peptidoglycan Synthesis

AbstractHow bacteria produce a septum to divide in two is not well understood. This process is mediated by periplasmic cell-wall producing enzymes that are positioned by filaments of the cytoplasmic membrane-associated actin FtsA and the tubulin FtsZ (FtsAZ). To understand how these components act in concert to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We find that the division septum is built at discrete sites that move around the division plane. Furthermore, FtsAZ filaments treadmill in circumferential paths around the division ring, pulling along the associated cell-wall-synthesizing enzymes. We show that the rate of FtsZ treadmilling controls both the rate of cell wall synthesis and cell division. The coupling of both the position and activity of the cell wall synthases to FtsAZ treadmilling guides the progressive insertion of new cell wall, synthesizing increasingly small concentric rings to divide the cell.One-sentence summaryBacterial cytokinesis is controlled by circumferential treadmilling of FtsAZ filaments that drives the insertion of new cell wall.

Antibiotic resistance of glycine-resistant variants of Staphylococcus aureus

1968 ◽  
Vol 14 (7) ◽  
pp. 811-812
Author(s):  
Joseph T. Parisi ◽  
William J. Suling
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Antibiotic Resistance ◽  
Cross Resistance ◽  
Cell Wall Synthesis ◽  
Minimal Inhibitory Concentrations

Glycine-resistant variants of Staphylococcus aureus were obtained by successive cultivation of parent strains in increasing concentrations of glycine, and the minimal inhibitory concentrations of glycine of the parents and variants were determined. Although it has been reported that growth in glycine or certain antibiotics causes the accumulation of nucleotides involved in cell wall synthesis, a lack of cross resistance of the variants to some of these antibiotics was observed.

scholarly journals A Staphylococcus aureus clpX Mutant Used as a Unique Screening Tool to Identify Cell Wall Synthesis Inhibitors that Reverse β-Lactam Resistance in MRSA

2021 ◽  
Vol 8 ◽  
Author(s):  
Kristoffer T. Bæk ◽  
Camilla Jensen ◽  
Maya A. Farha ◽  
Tobias K. Nielsen ◽  
Ervin Paknejadi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
High Throughput Screening ◽  
Bacterial Infections ◽  
Screening Tool ◽  
Acquired Resistance ◽  
Teichoic Acid ◽  
Acid Synthesis ◽  
Biologically Active ◽  
Cell Wall Synthesis

Staphylococcus aureus is a leading cause of bacterial infections world-wide. Staphylococcal infections are preferentially treated with β-lactam antibiotics, however, methicillin-resistant S. aureus (MRSA) strains have acquired resistance to this superior class of antibiotics. We have developed a growth-based, high-throughput screening approach that directly identifies cell wall synthesis inhibitors capable of reversing β-lactam resistance in MRSA. The screen is based on the finding that S. aureus mutants lacking the ClpX chaperone grow very poorly at 30°C unless specific steps in teichoic acid synthesis or penicillin binding protein (PBP) activity are inhibited. This property allowed us to exploit the S. aureus clpX mutant as a unique screening tool to rapidly identify biologically active compounds that target cell wall synthesis. We tested a library of ∼50,000 small chemical compounds and searched for compounds that inhibited growth of the wild type while stimulating growth of the clpX mutant. Fifty-eight compounds met these screening criteria, and preliminary tests of 10 compounds identified seven compounds that reverse β-lactam resistance of MRSA as expected for inhibitors of teichoic acid synthesis. The hit compounds are therefore promising candidates for further development as novel combination agents to restore β-lactam efficacy against MRSA.

scholarly journals Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment

2020 ◽  
Vol 203 (2) ◽  
pp. e00463-20
Author(s):  
Amit Bhambhani ◽  
Isabella Iadicicco ◽  
Jules Lee ◽  
Syed Ahmed ◽  
Max Belfatto ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Gene Product ◽  
Fluorescent Protein ◽  
Lytic Bacteriophage ◽  
Cell Wall Synthesis ◽  
Content Type ◽  
Ftsz Ring ◽  
Green Fluorescent

ABSTRACTPrevious work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

scholarly journals Daptomycin Exerts Bactericidal Activity without Lysis of Staphylococcus aureus

2008 ◽  
Vol 52 (6) ◽  
pp. 2223-2225 ◽  
Author(s):  
Nicole Cotroneo ◽  
Robert Harris ◽  
Nancy Perlmutter ◽  
Terry Beveridge ◽  
Jared A. Silverman
Keyword(s):  
Electron Microscopy ◽  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Division ◽  
Bactericidal Activity ◽  
Membrane Integrity ◽  
Cell Lysis

ABSTRACT The ability of daptomycin to produce bactericidal activity against Staphylococcus aureus while causing negligible cell lysis has been demonstrated using electron microscopy and the membrane integrity probes calcein and ToPro3. The formation of aberrant septa on the cell wall, suggestive of impairment of the cell division machinery, was also observed.

scholarly journals Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis

2001 ◽  
Vol 155 (2) ◽  
pp. 239-250 ◽  
Author(s):  
Maren Heese ◽  
Xavier Gansel ◽  
Liliane Sticher ◽  
Peter Wick ◽  
Markus Grebe ◽  
...  
Keyword(s):  
Cell Division ◽  
Membrane Fusion ◽  
Functional Characterization ◽  
Cell Plate ◽  
Yeast Two Hybrid ◽  
Division Plane ◽  
Plant Cytokinesis ◽  
Cell Plate Formation ◽  
Plate Formation ◽  
Two Hybrid

Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.

scholarly journals High-Level β-Lactam Resistance and Cell Wall Synthesis Catalyzed by the mecA Homologue of Staphylococcus sciuri Introduced into Staphylococcus aureus

2005 ◽  
Vol 187 (19) ◽  
pp. 6651-6658 ◽  
Author(s):  
Anatoly Severin ◽  
Shang Wei Wu ◽  
Keiko Tabei ◽  
Alexander Tomasz
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
High Performance ◽  
Protein Product ◽  
Spectrometric Analysis ◽  
Cell Wall Synthesis ◽  
Absolute Dependence ◽  
A Cell ◽  
High Concentrations ◽  
High Level

ABSTRACT A close homologue of mecA, the determinant of broad-spectrum β-lactam resistance in Staphylococcus aureus was recently identified as a native gene in the animal commensal species Staphylococcus sciuri. Introduction of the mecA homologue from a methicillin-resistant strain of S. sciuri into a susceptible strain of S. aureus caused an increase in drug resistance and allowed continued growth and cell wall synthesis of the bacteria in the presence of high concentrations of antibiotic. We determined the muropeptide composition of the S. sciuri cell wall by using a combination of high-performance liquid chromatography, mass spectrometric analysis, and Edman degradation. Several major differences between the cell walls of S. aureus and S. sciuri were noted. The pentapeptide branches in S. sciuri were composed of one alanine and four glycine residues in contrast to the pentaglycine units in S. aureus. The S. sciuri wall but not the wall of S. aureus contained tri- and tetrapeptide units, suggesting the presence of dd- and ld-carboxypeptidase activity. Most interestingly, S. aureus carrying the S. sciuri mecA and growing in methicillin-containing medium produced a cell wall typical of S. aureus and not S. sciuri, in spite of the fact that wall synthesis under these conditions had an absolute dependence on the heterologous S. sciuri gene product. The protein product of the S. sciuri mecA can efficiently participate in cell wall biosynthesis and build a cell wall using the cell wall precursors characteristic of the S. aureus host.

scholarly journals The Antituberculosis Drug Ethambutol Selectively Blocks Apical Growth in CMN Group Bacteria

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Karin Schubert ◽  
Boris Sieger ◽  
Fabian Meyer ◽  
Giacomo Giacomelli ◽  
Kati Böhm ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Cell Growth ◽  
Antibiotic Treatment ◽  
Bacterial Infections ◽  
Apical Cell ◽  
Combined Treatment ◽  
Mycolic Acid ◽  
Cell Wall Synthesis ◽  
Beta Lactam

ABSTRACT Members of the genus Mycobacterium are the most prevalent cause of infectious diseases. Mycobacteria have a complex cell envelope containing a peptidoglycan layer and an additional arabinogalactan polymer to which a mycolic acid bilayer is linked; this complex, multilayered cell wall composition (mAGP) is conserved among all CMN group bacteria. The arabinogalactan and mycolic acid synthesis pathways constitute effective drug targets for tuberculosis treatment. Ethambutol (EMB), a classical antituberculosis drug, inhibits the synthesis of the arabinose polymer. Although EMB acts bacteriostatically, its underlying molecular mechanism remains unclear. Here, we used Corynebacterium glutamicum and Mycobacterium phlei as model organisms to study the effects of EMB at the single-cell level. Our results demonstrate that EMB specifically blocks apical cell wall synthesis, but not cell division, explaining the bacteriostatic effect of EMB. Furthermore, the data suggest that members of the family Corynebacterineae have two dedicated machineries for cell elongation (elongasome) and cytokinesis (divisome). IMPORTANCE Antibiotic treatment of bacterial pathogens has contributed enormously to the increase in human health. Despite the apparent importance of antibiotic treatment of bacterial infections, surprisingly little is known about the molecular functions of antibiotic actions in the bacterial cell. Here, we analyzed the molecular effects of ethambutol, a first-line antibiotic against infections caused by members of the genus Mycobacterium. We find that this drug selectively blocks apical cell growth but still allows for effective cytokinesis. As a consequence, cells survive ethambutol treatment and adopt a pneumococcal cell growth mode with cell wall synthesis only at the site of cell division. However, combined treatment of ethambutol and beta-lactam antibiotics acts synergistically and effectively stops cell proliferation. IMPORTANCE Antibiotic treatment of bacterial pathogens has contributed enormously to the increase in human health. Despite the apparent importance of antibiotic treatment of bacterial infections, surprisingly little is known about the molecular functions of antibiotic actions in the bacterial cell. Here, we analyzed the molecular effects of ethambutol, a first-line antibiotic against infections caused by members of the genus Mycobacterium. We find that this drug selectively blocks apical cell growth but still allows for effective cytokinesis. As a consequence, cells survive ethambutol treatment and adopt a pneumococcal cell growth mode with cell wall synthesis only at the site of cell division. However, combined treatment of ethambutol and beta-lactam antibiotics acts synergistically and effectively stops cell proliferation.

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