Coordinating Bacterial Cell Division with Nutrient Availability: a Role for Glycolysis

ABSTRACTCell division in bacteria is driven by a cytoskeletal ring structure, the Z ring, composed of polymers of the tubulin-like protein FtsZ. Z-ring formation must be tightly regulated to ensure faithful cell division, and several mechanisms that influence the positioning and timing of Z-ring assembly have been described. Another important but as yet poorly understood aspect of cell division regulation is the need to coordinate division with cell growth and nutrient availability. In this study, we demonstrated for the first time that cell division is intimately linked to central carbon metabolism in the model Gram-positive bacteriumBacillus subtilis. We showed that a deletion of the gene encoding pyruvate kinase (pyk), which produces pyruvate in the final reaction of glycolysis, rescues the assembly defect of a temperature-sensitiveftsZmutant and has significant effects on Z-ring formation in wild-typeB. subtiliscells. Addition of exogenous pyruvate restores normal division in the absence of the pyruvate kinase enzyme, implicating pyruvate as a key metabolite in the coordination of bacterial growth and division. Our results support a model in which pyruvate levels are coupled to Z-ring assembly via an enzyme that actually metabolizes pyruvate, the E1α subunit of pyruvate dehydrogenase. We have shown that this protein localizes over the nucleoid in a pyruvate-dependent manner and may stimulate more efficient Z-ring formation at the cell center under nutrient-rich conditions, when cells must divide more frequently.IMPORTANCEHow bacteria coordinate cell cycle processes with nutrient availability and growth is a fundamental yet unresolved question in microbiology. Recent breakthroughs have revealed that nutritional information can be transmitted directly from metabolic pathways to the cell cycle machinery and that this can serve as a mechanism for fine-tuning cell cycle processes in response to changes in environmental conditions. Here we identified a novel link between glycolysis and cell division inBacillus subtilis. We showed that pyruvate, the final product of glycolysis, plays an important role in maintaining normal division. Nutrient-dependent changes in pyruvate levels affect the function of the cell division protein FtsZ, most likely by modifying the activity of an enzyme that metabolizes pyruvate, namely, pyruvate dehydrogenase E1α. Ultimately this system may help to coordinate bacterial division with nutritional conditions to ensure the survival of newborn cells.

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Noc Corrals Migration of FtsZ Protofilaments during Cytokinesis in Bacillus subtilis

mBio ◽

10.1128/mbio.02964-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yuanchen Yu ◽

Jinsheng Zhou ◽

Frederico J. Gueiros-Filho ◽

Daniel B. Kearns ◽

Stephen C. Jacobson

Keyword(s):

Bacillus Subtilis ◽

Cell Division ◽

Fluorescence Microscopy ◽

Control Cell ◽

Time Lapse ◽

Ring Formation ◽

Microfluidic Channels ◽

Content Type ◽

The Future ◽

Z Ring

ABSTRACT Bacteria that divide by binary fission form FtsZ rings at the geometric midpoint of the cell between the bulk of the replicated nucleoids. In Bacillus subtilis, the DNA- and membrane-binding Noc protein is thought to participate in nucleoid occlusion by preventing FtsZ rings from forming over the chromosome. To explore the role of Noc, we used time-lapse fluorescence microscopy to monitor FtsZ and the nucleoid of cells growing in microfluidic channels. Our data show that Noc does not prevent de novo FtsZ ring formation over the chromosome nor does Noc control cell division site selection. Instead, Noc corrals FtsZ at the cytokinetic ring and reduces migration of protofilaments over the chromosome to the future site of cell division. Moreover, we show that FtsZ protofilaments travel due to a local reduction in ZapA association, and the diffuse FtsZ rings observed in the Noc mutant can be suppressed by ZapA overexpression. Thus, Noc sterically hinders FtsZ migration away from the Z-ring during cytokinesis and retains FtsZ at the postdivisional polar site for full disassembly by the Min system. IMPORTANCE In bacteria, a condensed structure of FtsZ (Z-ring) recruits cell division machinery at the midcell, and Z-ring formation is discouraged over the chromosome by a poorly understood phenomenon called nucleoid occlusion. In B. subtilis, nucleoid occlusion has been reported to be mediated, at least in part, by the DNA-membrane bridging protein, Noc. Using time-lapse fluorescence microscopy of cells growing in microchannels, we show that Noc neither protects the chromosome from proximal Z-ring formation nor determines the future site of cell division. Rather, Noc plays a corralling role by preventing protofilaments from leaving a Z-ring undergoing cytokinesis and traveling over the nucleoid.

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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 ◽

10.1128/jb.187.18.6536-6544.2005 ◽

2005 ◽

Vol 187 (18) ◽

pp. 6536-6544 ◽

Cited By ~ 63

Author(s):

S. O. Jensen ◽

L. S. Thompson ◽

E. J. Harry

Keyword(s):

Bacillus Subtilis ◽

Cell Division ◽

Ring Formation ◽

Division Site ◽

Ring Assembly ◽

Synchronous Cell ◽

Z Ring ◽

Membrane Bound ◽

Chemical Cross Linking

ABSTRACT 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 directly with FtsZ. Its function remains unknown. It is generally believed that FtsA localization to the division site occurs immediately after Z-ring formation or concomitantly with it and that FtsA is responsible for recruiting the later-assembling membrane-bound division proteins to the division site. Here, we report the development of an in vivo chemical cross-linking assay to examine the association between FtsZ and FtsA in Bacillus subtilis cells. We subsequently use this assay in a synchronous cell cycle to show that these two proteins can interact prior to Z-ring formation. We further show that in a B. subtilis strain containing an ftsA deletion, FtsZ localized at regular intervals along the filament but the majority of Z rings were abnormal. FtsA in this organism is therefore critical for the efficient formation of functional Z rings. This is the first report of abnormal Z-ring formation resulting from the loss of a single septation protein. These results suggest that in this organism, and perhaps others, FtsA ensures recruitment of the membrane-bound division proteins by ensuring correct formation of the Z ring.

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Loss of PodJ in Agrobacterium tumefaciens Leads to Ectopic Polar Growth, Branching, and Reduced Cell Division

Journal of Bacteriology ◽

10.1128/jb.00198-16 ◽

2016 ◽

Vol 198 (13) ◽

pp. 1883-1891 ◽

Cited By ~ 13

Author(s):

James C. Anderson-Furgeson ◽

John R. Zupan ◽

Romain Grangeon ◽

Patricia C. Zambryski

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Agrobacterium Tumefaciens ◽

Cell Envelope ◽

Critical Factor ◽

Polar Growth ◽

Content Type ◽

Growth Pole ◽

Envelope Material ◽

Z Ring

ABSTRACTAgrobacterium tumefaciensis a rod-shaped Gram-negative bacterium that elongates by unipolar addition of new cell envelope material. Approaching cell division, the growth pole transitions to a nongrowing old pole, and the division site creates new growth poles in sibling cells. TheA. tumefacienshomolog of theCaulobacter crescentuspolar organizing protein PopZ localizes specifically to growth poles. In contrast, theA. tumefacienshomolog of theC. crescentuspolar organelle development protein PodJ localizes to the old pole early in the cell cycle and accumulates at the growth pole as the cell cycle proceeds. FtsA and FtsZ also localize to the growth pole for most of the cell cycle prior to Z-ring formation. To further characterize the function of polar localizing proteins, we created a deletion ofA. tumefacienspodJ(podJAt). ΔpodJAtcells display ectopic growth poles (branching), growth poles that fail to transition to an old pole, and elongated cells that fail to divide. In ΔpodJAtcells,A. tumefaciensPopZ-green fluorescent protein (PopZAt-GFP) persists at nontransitioning growth poles postdivision and also localizes to ectopic growth poles, as expected for a growth-pole-specific factor. Even though GFP-PodJAtdoes not localize to the midcell in the wild type, deletion ofpodJAtimpacts localization, stability, and function of Z-rings as assayed by localization of FtsA-GFP and FtsZ-GFP. Z-ring defects are further evidenced by minicell production. Together, these data indicate that PodJAtis a critical factor for polar growth and that ΔpodJAtcells display a cell division phenotype, likely because the growth pole cannot transition to an old pole.IMPORTANCEHow rod-shaped prokaryotes develop and maintain shape is complicated by the fact that at least two distinct species-specific growth modes exist: uniform sidewall insertion of cell envelope material, characterized in model organisms such asEscherichia coli, and unipolar growth, which occurs in several alphaproteobacteria, includingAgrobacterium tumefaciens. Essential components for unipolar growth are largely uncharacterized, and the mechanism constraining growth to one pole of a wild-type cell is unknown. Here, we report that the deletion of a polar development gene,podJAt, results in cells exhibiting ectopic polar growth, including multiple growth poles and aberrant localization of cell division and polar growth-associated proteins. These data suggest that PodJAtis a critical factor in normal polar growth and impacts cell division inA. tumefaciens.

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Transcription Regulation of ezrA and Its Effect on Cell Division of Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.186.17.5926-5932.2004 ◽

2004 ◽

Vol 186 (17) ◽

pp. 5926-5932 ◽

Cited By ~ 11

Author(s):

Kuei-Min Chung ◽

Hsin-Hsien Hsu ◽

Suresh Govindan ◽

Ban-Yang Chang

Keyword(s):

Bacillus Subtilis ◽

Cell Division ◽

Critical Concentration ◽

In Vitro Transcription ◽

Ring Formation ◽

Negative Regulator ◽

Cell Length ◽

Intracellular Level ◽

Z Ring

ABSTRACT The EzrA protein of Bacillus subtilis is a negative regulator for FtsZ (Z)-ring formation. It is able to modulate the frequency and position of Z-ring formation during cell division. The loss of this protein results in cells with multiple Z rings located at polar as well as medial sites; it also lowers the critical concentration of FtsZ required for ring formation (P. A. Levin, I. G. Kurster, and A. D. Grossman, Proc. Natl. Acad. Sci. USA 96:9642-9647, 1999). We have studied the regulation of ezrA expression during the growth of B. subtilis and its effects on the intracellular level of EzrA as well as the cell length of B. subtilis. With the aid of promoter probing, primer extension, in vitro transcription, and Western blotting analyses, two overlapping σA-type promoters, P1 and P2, located about 100 bp upstream of the initiation codon of ezrA, have been identified. P1, supposed to be an extended −10 promoter, was responsible for most of the ezrA expression during the growth of B. subtilis. Disruption of this promoter reduced the intracellular level of EzrA very significantly compared with disruption of P2. Moreover, deletion of both promoters completely abolished EzrA in B. subtilis. More importantly, the cell length and percentage of filamentous cells of B. subtilis were significantly increased by disruption of the promoter(s). Thus, EzrA is required for efficient cell division during the growth of B. subtilis, despite serving as a negative regulator for Z-ring formation.

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Escherichia coli Division Inhibitor MinCD Blocks Septation by Preventing Z-Ring Formation

Journal of Bacteriology ◽

10.1128/jb.183.22.6630-6635.2001 ◽

2001 ◽

Vol 183 (22) ◽

pp. 6630-6635 ◽

Cited By ~ 89

Author(s):

Sebastien Pichoff ◽

Joe Lutkenhaus

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Direct Interaction ◽

Ring Formation ◽

Ring Assembly ◽

Z Ring ◽

Alternative Fixation ◽

Green Fluorescent

ABSTRACT The min system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD was previously shown to inhibit division by preventing assembly of the Z ring (E. Bi and J. Lutkenhaus, J. Bacteriol. 175:1118–1125, 1993); however, this was questioned in a recent report (S. S. Justice, J. Garcia-Lara, and L. I. Rothfield, Mol. Microbiol. 37:410–423, 2000) which indicated that MinCD acted after Z-ring formation and prevented the recruitment of FtsA to the Z ring. This discrepancy was due in part to alternative fixation conditions. We have therefore reinvestigated the action of MinCD and avoided fixation by using green fluorescent protein (GFP) fusions to division proteins. MinCD prevented the localization of both FtsZ-GFP and ZipA-GFP, consistent with it preventing Z-ring assembly. Consistent with a direct interaction between FtsZ and the MinCD inhibitor, we find that increased FtsZ, but not FtsA, suppresses MinCD-induced lethality. Furthermore, strains carrying various alleles offtsZ, selected on the basis of resistance to the inhibitor SulA, displayed variable resistance to MinCD. These results are consistent with FtsZ as the target of MinCD and confirm that this inhibitor prevents Z-ring assembly.

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The Min System Disassembles FtsZ Foci and Inhibits Polar Peptidoglycan Remodeling in Bacillus subtilis

mBio ◽

10.1128/mbio.03197-19 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 1

Author(s):

Yuanchen Yu ◽

Jinsheng Zhou ◽

Felix Dempwolff ◽

Joshua D. Baker ◽

Daniel B. Kearns ◽

...

Keyword(s):

Bacillus Subtilis ◽

Cell Division ◽

Microfluidic System ◽

Critical Threshold ◽

Content Type ◽

Daughter Cells ◽

Spatiotemporal Regulation ◽

Powerful Approach ◽

In The Wild ◽

Z Ring

ABSTRACT A microfluidic system coupled with fluorescence microscopy is a powerful approach for quantitative analysis of bacterial growth. Here, we measure parameters of growth and dynamic localization of the cell division initiation protein FtsZ in Bacillus subtilis. Consistent with previous reports, we found that after division, FtsZ rings remain at the cell poles, and polar FtsZ ring disassembly coincides with rapid Z-ring accumulation at the midcell. In cells mutated for minD, however, the polar FtsZ rings persist indefinitely, suggesting that the primary function of the Min system is in Z-ring disassembly. The inability to recycle FtsZ monomers in the minD mutant results in the simultaneous maintenance of multiple Z-rings that are restricted by competition for newly synthesized FtsZ. Although the parameters of FtsZ dynamics change in the minD mutant, the overall cell division time remains the same, albeit with elongated cells necessary to accumulate a critical threshold amount of FtsZ for promoting medial division. Finally, the minD mutant characteristically produces minicells composed of polar peptidoglycan shown to be inert for remodeling in the wild type. Polar peptidoglycan, however, loses its inert character in the minD mutant, suggesting that the Min system not only is important for recycling FtsZ but also may have a secondary role in the spatiotemporal regulation of peptidoglycan remodeling. IMPORTANCE Many bacteria grow and divide by binary fission in which a mother cell divides into two identical daughter cells. To produce two equally sized daughters, the division machinery, guided by FtsZ, must dynamically localize to the midcell each cell cycle. Here, we quantitatively analyzed FtsZ dynamics during growth and found that the Min system of Bacillus subtilis is essential to disassemble FtsZ rings after division. Moreover, a failure to efficiently recycle FtsZ results in an increase in cell size. Finally, we show that the Min system has an additional role in inhibiting cell wall turnover and contributes to the “inert” property of cell walls at the poles.

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Novel Divisome-Associated Protein Spatially Coupling the Z-Ring with the Chromosomal Replication Terminus in Caulobacter crescentus

mBio ◽

10.1128/mbio.00487-20 ◽

2020 ◽

Vol 11 (2) ◽

Author(s):

Shogo Ozaki ◽

Urs Jenal ◽

Tsutomu Katayama

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Caulobacter Crescentus ◽

Enteric Bacteria ◽

Chromosome Replication ◽

Chromosomal Replication ◽

Content Type ◽

Physical Linkage ◽

Z Ring ◽

Replication Terminus

ABSTRACT Cell division requires proper spatial coordination with the chromosome, which undergoes dynamic changes during chromosome replication and segregation. FtsZ is a bacterial cytoskeletal protein that assembles into the Z-ring, providing a platform to build the cell division apparatus. In the model bacterium Caulobacter crescentus, the cellular localization of the Z-ring is controlled during the cell cycle in a chromosome replication-coupled manner. Although dynamic localization of the Z-ring at midcell is driven primarily by the replication origin-associated FtsZ inhibitor MipZ, the mechanism ensuring accurate positioning of the Z-ring remains unclear. In this study, we showed that the Z-ring colocalizes with the replication terminus region, located opposite the origin, throughout most of the C. crescentus cell cycle. Spatial organization of the two is mediated by ZapT, a previously uncharacterized protein that interacts with the terminus region and associates with ZapA and ZauP, both of which are part of the incipient division apparatus. While the Z-ring and the terminus region coincided with the presence of ZapT, colocalization of the two was perturbed in cells lacking zapT, which is accompanied by delayed midcellular positioning of the Z-ring. Moreover, cells overexpressing ZapT showed compromised positioning of the Z-ring and MipZ. These findings underscore the important role of ZapT in controlling cell division processes. We propose that ZapT acts as a molecular bridge that physically links the terminus region to the Z-ring, thereby ensuring accurate site selection for the Z-ring. Because ZapT is conserved in proteobacteria, these findings may define a general mechanism coordinating cell division with chromosome organization. IMPORTANCE Growing bacteria require careful tuning of cell division processes with dynamic organization of replicating chromosomes. In enteric bacteria, ZapA associates with the cytoskeletal Z-ring and establishes a physical linkage to the chromosomal replication terminus through its interaction with ZapB-MatP-DNA complexes. However, because ZapB and MatP are found only in enteric bacteria, it remains unclear how the Z-ring and the terminus are coordinated in the vast majority of bacteria. Here, we provide evidence that a novel conserved protein, termed ZapT, mediates colocalization of the Z-ring with the terminus in Caulobacter crescentus, a model organism that is phylogenetically distant from enteric bacteria. Given that ZapT facilitates cell division processes in C. crescentus, this study highlights the universal importance of the physical linkage between the Z-ring and the terminus in maintaining cell integrity.

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The Chaperonin GroESL Facilitates Caulobacter crescentus Cell Division by Supporting the Functions of the Z-Ring Regulators FtsA and FzlA

mBio ◽

10.1128/mbio.03564-20 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Kristen Schroeder ◽

Kristina Heinrich ◽

Ines Neuwirth ◽

Kristina Jonas

Keyword(s):

Cell Cycle ◽

Cell Wall ◽

Cell Division ◽

Bacterial Cell ◽

Antimicrobial Agents ◽

Caulobacter Crescentus ◽

Content Type ◽

Growing Cell ◽

Z Ring ◽

Bacterial Cell Cycle

ABSTRACT The highly conserved chaperonin GroESL performs a crucial role in protein folding; however, the essential cellular pathways that rely on this chaperone are underexplored. Loss of GroESL leads to severe septation defects in diverse bacteria, suggesting the folding function of GroESL may be integrated with the bacterial cell cycle at the point of cell division. Here, we describe new connections between GroESL and the bacterial cell cycle using the model organism Caulobacter crescentus. Using a proteomics approach, we identify candidate GroESL client proteins that become insoluble or are degraded specifically when GroESL folding is insufficient, revealing several essential proteins that participate in cell division and peptidoglycan biosynthesis. We demonstrate that other cell cycle events, such as DNA replication and chromosome segregation, are able to continue when GroESL folding is insufficient. We further find that deficiency of two FtsZ-interacting proteins, the bacterial actin homologue FtsA and the constriction regulator FzlA, mediate the GroESL-dependent block in cell division. Our data show that sufficient GroESL is required to maintain normal dynamics of the FtsZ scaffold and divisome functionality in C. crescentus. In addition to supporting divisome function, we show that GroESL is required to maintain the flow of peptidoglycan precursors into the growing cell wall. Linking a chaperone to cell division may be a conserved way to coordinate environmental and internal cues that signal when it is safe to divide. IMPORTANCE All organisms depend on mechanisms that protect proteins from misfolding and aggregation. GroESL is a highly conserved molecular chaperone that functions to prevent protein aggregation in organisms ranging from bacteria to humans. Despite detailed biochemical understanding of GroESL function, the in vivo pathways that strictly depend on this chaperone remain poorly defined in most species. This study provides new insights into how GroESL is linked to the bacterial cell division machinery, a crucial target of current and future antimicrobial agents. We identify a functional interaction between GroESL and the cell division proteins FzlA and FtsA, which modulate Z-ring function. FtsA is a conserved bacterial actin homologue, suggesting that as in eukaryotes, some bacteria exhibit a connection between cytoskeletal actin proteins and chaperonins. Our work further defines how GroESL is integrated with cell wall synthesis and illustrates how highly conserved folding machines ensure the functioning of fundamental cellular processes during stress.

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Antibiotic-induced degradation of FtsZ reveals distinct stages of Bacillus subtilis FtsZ ring assembly and constriction

10.1101/2020.06.30.180018 ◽

2020 ◽

Cited By ~ 2

Author(s):

Nadine Silber ◽

Christian Mayer ◽

Cruz L Matos de Opitz ◽

Peter Sass

Keyword(s):

Bacillus Subtilis ◽

Cell Division ◽

Ring Formation ◽

Ftsz Ring ◽

Ring Assembly ◽

Cell Division Protein

AbstractADEP antibiotics induce the degradation of the cell division protein FtsZ, thereby primarily depleting the cytoplasmic FtsZ pool that is needed for treadmilling FtsZ rings. We here studied the effect of ADEP on FtsZ ring formation. Our data reveal the disintegration of early FtsZ rings during ADEP treatment, while progressed FtsZ rings finalize cytokinesis, thus indicating different roles for FtsZ treadmilling during distinct stages of divisome assembly and constriction.

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Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

Journal of Bacteriology ◽

10.1128/jb.00408-19 ◽

2019 ◽

Vol 202 (2) ◽

Cited By ~ 5

Author(s):

Peter E. Burby ◽

Lyle A. Simmons

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Division ◽

Dna Replication ◽

Cell Cycle Progression ◽

Genome Instability ◽

Sos Response ◽

Bacterial Cells ◽

Cycle Progression ◽

Content Type

ABSTRACT All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

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