RESUMO
Bacterial growth and division rely on intricate regulation of morphogenetic complexes to remodel the cell envelope without compromising envelope integrity. Significant progress has been made in recent years towards understanding the regulation of cell wall metabolic enzymes. However, other cell envelope components play a role in morphogenesis as well. A primary factor required to protect envelope integrity in low osmolarity environments is OpgH, the synthase of osmoregulated periplasmic glucans (OPGs). Here, we demonstrate that OpgH is essential in the α-proteobacterium Caulobacter crescentus. Unexpectedly, depletion of OpgH or attempted complementation with a catalytically dead OpgH variant results in striking asymmetric bulging and cell lysis. These shape defects are accompanied by reduced cell wall synthesis and mislocalization of morphogenetic complexes. Interestingly, overactivation of the CenKR two-component system that has been implicated in cell envelope stress homeostasis in α-proteobacteria phenocopies the morphogenetic defects associated with OpgH depletion. Each of these perturbations leads to an increase in the levels of the elongasome protein, MreB, and decreases in the levels of divisome proteins FtsZ and MipZ as well as OpgH, itself. Constitutive production of OpgH during CenKR overactivation prevents cell bulging, but cells still exhibit morphogenetic defects. We propose that OPG depletion activates CenKR, leading to changes in the expression of cell envelope-related genes, but that OPGs also exert CenKR-independent effects on morphogenesis. Our data establish a surprising function for an OpgH homolog in morphogenesis and reveal an essential role of OpgH in maintaining cell morphology in Caulobacter.IMPORTANCEBacteria must synthesize and fortify the cell envelope in a tightly regulated manner to orchestrate growth and adaptation. Osmoregulated periplasmic glucans (OPGs) are important, but poorly understood, constituents of Gram-negative cell envelopes that contribute to envelope integrity and protect against osmotic stress. Here, we determined that the OPG synthase OpgH plays a surprising, essential role in morphogenesis in Caulobacter crescentus. Loss of OpgH causes asymmetric cell bulging and lysis via misregulation of the localization and activity of morphogenetic complexes. Overactivation of the CenKR two-component system involved in envelope homeostasis phenocopies OpgH depletion, suggesting that depletion of OpgH activates CenKR. Because cell envelope integrity is critical for bacterial survival, understanding how OpgH activity contributes to morphogenesis and maintenance of envelope integrity could aid in the development of antibiotic therapies.
RESUMO
Bacterial growth and division rely on intricate regulation of morphogenetic complexes to remodel the cell envelope without compromising envelope integrity. Significant progress has been made in recent years towards understanding the regulation of cell wall metabolic enzymes. However, other cell envelope components play a role in morphogenesis as well. Components required to maintain osmotic homeostasis are among these understudied envelope-associated enzymes that may contribute to cell morphology. A primary factor required to protect envelope integrity in low osmolarity environments is OpgH, the synthase of osmoregulated periplasmic glucans (OPGs). Here, we demonstrate that OpgH is essential in the α-proteobacterium Caulobacter crescentus. Unexpectedly, depletion of OpgH results in striking asymmetric bulging and cell lysis, accompanied by misregulation of cell wall insertion and mislocalization of morphogenetic complexes. The enzymatic activity of OpgH is required for normal cell morphology as production of an OpgH mutant that disrupts a conserved glycosyltransferase motif phenocopies the depletion. Our data establish a surprising function for an OpgH homolog in morphogenesis and reveal an essential role of OpgH in maintaining proper cell morphology during normal growth and division in Caulobacter.
RESUMO
Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the âº-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell-wall-targeting antibiotics, EstG does not demonstrate biochemical activity toward cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which are important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.
Assuntos
Caulobacter crescentus , Caulobacter , Caulobacter/metabolismo , Esterases , Membrana Celular/metabolismo , Parede Celular/metabolismo , Caulobacter crescentus/metabolismo , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
A peptidoglycan (PG) cell wall is an essential component of nearly all bacteria, providing protection against turgor pressure. Metabolism of this PG meshwork must be spatially and temporally regulated in order to support cell growth and division. Despite being an active area of research for decades, we have only recently identified the primary PG synthesis complexes that function during cell elongation (RodA-PBP2) and cell division (FtsW-FtsI), and we are still uncovering the importance of the other seemingly redundant cell wall enzymes. In this minireview, we highlight the discovery of the monofunctional glycosyltransferases RodA and FtsW and describe how these findings have prompted a re-evaluation of the auxiliary role of the bifunctional class A penicillin-binding proteins (aPBPs) as well as the L,D-transpeptidases (LDTs). Specifically, recent work indicates that the aPBPs and LDTs function independently of the primary morphogenetic complexes to support growth, provide protection from stresses, mediate morphogenesis, and/or allow adaptation to different growth conditions. These paradigm-shifting studies have reframed our understanding of bacterial cell wall metabolism, which will only become more refined as emerging technology allows us to tackle the remaining questions surrounding PG biosynthesis.
Assuntos
Bactérias/enzimologia , Parede Celular/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Glicosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação às Penicilinas/análise , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/biossíntese , Peptidoglicano/química , Peptidoglicano/metabolismoRESUMO
Bacterial growth and division require regulated synthesis of the macromolecules used to expand and replicate components of the cell. Transcription of housekeeping genes required for metabolic homeostasis and cell proliferation is guided by the sigma factor σ70. The conserved CarD-like transcriptional regulator, CdnL, associates with promoter regions where σ70 localizes and stabilizes the open promoter complex. However, the contributions of CdnL to metabolic homeostasis and bacterial physiology are not well understood. Here, we show that Caulobacter crescentus cells lacking CdnL have severe morphological and growth defects. Specifically, ΔcdnL cells grow slowly in both rich and defined media, and are wider, more curved, and have shorter stalks than WT cells. These defects arise from transcriptional downregulation of most major classes of biosynthetic genes, leading to significant decreases in the levels of critical metabolites, including pyruvate, α-ketoglutarate, ATP, NAD+, UDP-N-acetyl-glucosamine, lipid II, and purine and pyrimidine precursors. Notably, we find that ΔcdnL cells are glutamate auxotrophs, and ΔcdnL is synthetic lethal with other genetic perturbations that limit glutamate synthesis and lipid II production. Our findings implicate CdnL as a direct and indirect regulator of genes required for metabolic homeostasis that impacts morphogenesis through availability of lipid II and other metabolites.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Homeostase , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Caulobacter crescentus/metabolismo , Caulobacter crescentus/fisiologia , Divisão Celular , Sequência Conservada , Metaboloma , Fatores de Transcrição/genéticaRESUMO
Bacterial growth and division require insertion of new peptidoglycan (PG) into the existing cell wall by PG synthase enzymes. Emerging evidence suggests that many PG synthases require activation to function; however, it is unclear how activation of division-specific PG synthases occurs. The FtsZ cytoskeleton has been implicated as a regulator of PG synthesis during division, but the mechanisms through which it acts are unknown. Here, we show that FzlA, an FtsZ-binding protein and essential regulator of constriction in Caulobacter crescentus, helps link FtsZ to PG synthesis to promote division. We find that hyperactive mutants of the PG synthases FtsW and FtsI specifically render fzlA, but not other division genes, non-essential. However, FzlA is still required to maintain proper constriction rate and efficiency in a hyperactive PG synthase background. Intriguingly, loss of fzlA in the presence of hyperactivated FtsWI causes cells to rotate about the division plane during constriction and sensitizes cells to cell-wall-specific antibiotics. We demonstrate that FzlA-dependent signaling to division-specific PG synthesis is conserved in another α-proteobacterium, Agrobacterium tumefaciens. These data establish that FzlA helps link FtsZ to cell wall remodeling and is required for signaling to both activate and spatially orient PG synthesis during division. Overall, our findings support the paradigm that activation of SEDS-PBP PG synthases is a broadly conserved requirement for bacterial morphogenesis.
Assuntos
Proteínas de Bactérias/genética , Caulobacter crescentus/fisiologia , Divisão Celular/fisiologia , Proteínas do Citoesqueleto/genética , Ligases/metabolismo , Peptidoglicano/metabolismo , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Divisão Celular/genética , Parede Celular/metabolismo , Proteínas do Citoesqueleto/metabolismoRESUMO
During its life cycle, Caulobacter crescentus undergoes a series of coordinated shape changes, including generation of a polar stalk and reshaping of the cell envelope to produce new daughter cells through the process of cytokinesis. The mechanisms by which these morphogenetic processes are coordinated in time and space remain largely unknown. Here we demonstrate that the conserved division complex FtsEX controls both the early and late stages of cytokinesis in C. crescentus, namely initiation of constriction and final cell separation. ΔftsE cells display a striking phenotype: cells are chained, with skinny connections between cell bodies resulting from defects in inner membrane fusion and cell separation. Surprisingly, the thin connections in ΔftsE cells share morphological and molecular features with C. crescentus stalks. Our data uncover unanticipated morphogenetic plasticity in C. crescentus, with loss of FtsE causing a stalk-like program to take over at failed division sites.