DipM,a new factor required for peptidoglycan remodelling during cell division in Caulobacter crescentus

In bacteria, cytokinesis is dependent on lytic enzymes that facilitate remodelling of the cell wall during constriction. In this work, we identify a thus far uncharacterized periplasmic protein, DipM, that is required for cell division and polarity in Caulobacter crescentus. DipM is composed of four peptidoglycan binding (LysM) domains and a C‐terminal lysostaphin‐like (LytM) peptidase domain. It binds to isolated murein sacculi in vitro, and is recruited to the site of constriction through interaction with the cell division protein FtsN. Mutational analyses showed that the LysM domains are necessary and sufficient for localization of DipM, while its peptidase domain is essential for function. Consistent with a role in cell wall hydrolysis, DipM was found to interact with purified murein sacculi in vitro and to induce cell lysis upon overproduction. Its inactivation causes severe defects in outer membrane invagination, resulting in a significant delay between cytoplasmic compartmentalization and final separation of the daughter cells. Overall, these findings indicate that DipM is a periplasmic component of the C. crescentus divisome that facilitates remodelling of the peptidoglycan layer and, thus, coordinated constriction of the cell envelope during the division process.     

Amino acid composition of peptidoglycan in Caulobacter crescentus.

Peptidoglycan of a gram-negative stalked bacterium, Caulobacter crescentus CB13, contained alanine, diaminopimelic acid, and glutamic acid, in molar ratios of 2 : 1 : 1. The amino acid compositions of peptidoglycans isolated from cultures enriched in swarmer and stalked cells, and from a stalk-less mutant were similar. This finding conflicts with a previous observation that swarmer peptidoglycan does not contain diaminopimelic acid (Goodwin and Shedlarski (1975) Arch. Biochem. Biophys. 170, 23-36). It appears that, despite the morphological differences, the Caulobacter cells all contain a similar peptidoglycan in the cell wall.     

A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus

In most bacteria, the tubulin‐like GTPase FtsZ forms an annulus at midcell (the Z‐ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z‐ring assembly and early FtsZ‐directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C‐terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane‐anchored FtsZ in the regulation of cell wall hydrolysis.     

Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus

The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod‐shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme‐resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell body peptidoglycan contained primarily DD‐crosslinks between meso‐diaminopimelic acid and D‐alanine residues, whereas stalk peptidoglycan had more LD‐transpeptidation (meso‐diaminopimelic acid‐meso‐diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD‐transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD‐crosslinking. Despite no obvious selection pressure for LD‐crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP‐DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme.     

Growth‐driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus

All living cells must cope with protein aggregation, which occurs as a result of experiencing stress. In previously studied bacteria, aggregated protein is collected at the cell poles and is retained throughout consecutive cell divisions only in old pole‐inheriting daughter cells, resulting in aggregation‐free progeny within a few generations. In this study, we describe the in vivo kinetics of aggregate formation and elimination following heat and antibiotic stress in the asymmetrically dividing bacterium Caulobacter crescentus. Unexpectedly, in this bacterium, protein aggregates form as multiple distributed foci located throughout the cell volume. Time‐lapse microscopy revealed that under moderate stress, the majority of these protein aggregates are short‐lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality control machinery induces the formation of long‐lived aggregates. Importantly, the majority of persistent aggregates neither collect at the cell poles nor are they partitioned to only one daughter cell type. Instead, we show that aggregates are distributed to both daughter cells in the same ratio at each division, which is driven by the continuous elongation of the growing mother cell. Therefore, our study has revealed a new pattern of protein aggregate inheritance in bacteria.     

RNA-controlled regulation in Caulobacter crescentus

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Transposon mutagenesis in Caulobacter crescentus

Transposons Tn5 (Km) and Tn7 (Tp and Sm) were transferred to Caulobacter crescentus via P-type antibiotic resistance factors. Transposition was demonstrated by the isolation of chromosomal insertions of each transposon. With C. crescentus strains harboring RP4 aphA::Tn7, the introduction of a wild-type RP4 resulted in the loss of the resident plasmid. Simultaneous selection for Kmr and Smr yielded colonies with chromosomal insertions of Tn7. Examination of over 10,000 chromosomal insertions of Tn7 indicated no auxotrophic or motility mutants. Thus, Tn7 appears to have a high specificity of insertion in C. crescentus. The Mu-containing plasmid pJB4JI transferred Tn5 to C. crescentus, but the plasmid was not maintained. Control experiments showed that recovery of Mu-containing plasmids occurred at very low frequencies in C. crescentus and that the plasmids which were recovered had undergone extensive deletion of plasmid DNA. Presumably, some part of the Mu genome was not tolerated by C. crescentus. The instability of the Mu-containing plasmids makes them excellent vectors for the introduction of transposons, and we have used pJB4JI to isolated chromosomal insertions of Tn5. When several thousand of these insertion mutants were examined, we found auxotrophic and motility mutants at frequencies of 1 and 2%, respectively. These results indicate that Tn5 had a low specificity of insertion in C. crescentus and therefore would be a useful mutagen for obtaining a variety of mutant phenotypes.     

A moving DNA replication factory in Caulobacter crescentus

The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an active process, the replisome appears to be an untethered replication factory that is passively displaced towards the center of the cell by the newly replicated DNA. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation.     

Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP

The commonly used, monomeric EYFP enabled imaging of intracellular protein structures beyond the optical resolution limit ('super-resolution' imaging) in living cells. By combining photoinduced activation of single EYFP fusions and time-lapse imaging, we obtained sub-40 nm resolution images of the filamentous superstructure of the bacterial actin protein MreB in live Caulobacter crescentus cells. These studies demonstrated that EYFP is a useful emitter for in vivo super-resolution imaging.     

Galactose catabolism in Caulobacter crescentus.

Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Spontaneous mutants have been isolated which are able to grow on galactose in the absence of exogenous cyclic nucleotides. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. It is shown here that C. crescentus catabolizes galactose by the Entner-Duodoroff pathway. Galactose is initially converted to galactonate by galactose dehydrogenase and then 2-keto-3-deoxy-6-phosphogalactonate aldolase catalyzes the hydrolysis of 2-keto-3-deoxy-6-phosphogalactonic acid to yield triose phosphate and pyruvate. Two enzymes of galactose catabolism, galactose dehydrogenase and 2-keto-3-deoxy-6-phosphogalactonate aldolase, were shown to be inducible and independently regulated. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes.     

A quantitative study of the division cycle of Caulobacter crescentus stalked cells

Progression of a cell through the division cycle is tightly controlled at different steps to ensure the integrity of genome replication and partitioning to daughter cells. From published experimental evidence, we propose a molecular mechanism for control of the cell division cycle in Caulobacter crescentus. The mechanism, which is based on the synthesis and degradation of three “master regulator” proteins (CtrA, GcrA, and DnaA), is converted into a quantitative model, in order to study the temporal dynamics of these and other cell cycle proteins. The model accounts for important details of the physiology, biochemistry, and genetics of cell cycle control in stalked C. crescentus cell. It reproduces protein time courses in wild-type cells, mimics correctly the phenotypes of many mutant strains, and predicts the phenotypes of currently uncharacterized mutants. Since many of the proteins involved in regulating the cell cycle of C. crescentus are conserved among many genera of α-proteobacteria, the proposed mechanism may be applicable to other species of importance in agriculture and medicine.     

Synchronous Cell Differentiation in Caulobacter crescentus

The growth of a stalked bacterium, Caulobacter crescentus, has been synchronized easily and reproducibly by a new method. When this bacterium is grown to a late log phase in nutrient broth at 30 C with aeration, swarmer cells are accumulated in the culture to 80% of the whole cell population. When this culture is inoculated into fresh pre-warmed broth at twentyfold dilution, it immediately initiates synchronous cell growth. Simultaneously, synchronous cell differentiation is monitored by the susceptibility of the cells to RNA phage infection. The swarmer cells accumulated in the late log phase of growth possess nearly the same susceptibility to RNA phage infection as those in the early log phase of growth while RNA phage-adsorbing capacity is lower in such swarmer cells. It is suggested that the swarmer cells accumulated in the late log phase of growth have lost some pili.