Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase of Caulobacter crescentus

Deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) was purified from the dimorphic bacterium Caulobacter crescentus at three stages in development. Enzyme from pure populations of stalked cells, as well as populations enriched in swarmer and predivisional cells, appeared identical in subunit structure and template requirements. The molecular weights of the enzyme subunits were 165,000, 155,000, 101,000, and 44,000, respectively. By analogy with RNA polymerase from other bacterial sources, they are considered to be components of the C. crescentus holoenzyme, beta', beta, sigma, and alpha, respectively. The C. crescentus enzyme appeared similar to the Pseudomonas aeruginosa enzyme and unlike the Escherichia coli enzyme with respect to subunit molecular weights and failure to separate into core and sigma components upon phosphocellulose chromatography. In addition, the effects of ionic strength on the time course of polymerization varied both with the sources of bacterial polymerase and bacteriophage DNA.     

Characterization of heat-shock response of the marine bacterium Vibrio harveyi

We have investigated heat-shock response in a marine bacterium Vibrio harveyi. We have found that 39 C was the highest tempature at which V. harveyi was able to grow steadily. A shift from 30° C to 39° C caused increased synthesis of at least 10 proteins, as judged by SDS-PAGE, with molecular masses of 90, 70, 58, 41, 31, 27, 22, 15, 14.5 and 14kDa. The 70, 58, 41 and 14.5 kDa proteins were immunologically homologous to DnaK, GroEL, DnaJ and GroES heat-shock proteins of Escherichia coli, respectively. V. harveyi GroES protein had a lower molecular mass (14.5 kDa) than E. coli GroES, migrating in SDS-PAGE as 15 kDa protein. We showed that a protein of ～43 kDa, immunologically reactive with antiserum against E. coli sigma 32 subunit (σ³²) of RNA polymerase, was induced by heat-shock and co-purified with V. harveyi RNA polymerase. These results suggest that the 43 kDa protein is a heat-shock sigma protein of V. harveyi. Preparation containing the V. harveyi sigma 32 homologue, supplemented with core RNA polymerase of E. coli, was able to transcribe heat-shock promoters of E. coli in vitro.     

Cell cycle arrest of a Caulobacter crescentus secA mutant.

Cell differentiation is an inherent component of the Caulobacter crescentus cell cycle. The transition of a swarmer cell, with a single polar flagellum, into a sessile stalked cell includes several morphogenetic events. These include the release of the flagellum and pili, the proteolysis of chemotaxis proteins, the biogenesis of the polar stalk, and the initiation of DNA replication. We have isolated a group of temperature-sensitive mutants that are unable to complete this process at the restrictive temperature. We show here that one of these strains has a mutation in a homolog of the Escherichia coli secA gene, whose product is involved in protein translocation at the cell membrane. This C. crescentus secA mutant has allowed the identification of morphogenetic events in the swarmer-to-stalked cell transition that require SecA-dependent protein translocation. Upon shift to the nonpermissive temperature, the mutant secA swarmer cell is able to release the polar flagellum, degrade chemoreceptors, and initiate DNA replication, but it is unable to form a stalk, complete DNA replication, or carry out cell division. At the nonpermissive temperature, the cell cycle blocks prior to the de novo synthesis of flagella and chemotaxis proteins that normally occurs in the predivisional cell. Although interactions between the chromosome and the cytoplasmic membrane are believed to be a functional component of the temporal regulation of DNA replication, the ability of this secA mutant to initiate replication at the nonpermissive temperature suggests that SecA-dependent events are not involved in this process. However, both cell division and stalk formation, which is analogous to a polar division event, require SecA function.     

Caulobacter crescentus pili: structure and stage-specific expression.

Pili are functionally expressed during the predivisional and swarmer stages of the Caulobacter crescentus differentiation cycle. They appear on the developing swarmer pole and at the same cellular location as flagella and the phiCbK receptor sites. Pili disappear when the swarmer cell differentiates into a stalked cell; this occurs with the loss of flagella and the disappearance of phage receptor sites. C. crescentus CB13B1a pili have been purified and characterized. Monomeric pilin is a protein with an apparent molecular weight of 8,500 that stains weakly with periodic acid-Schiff reagent. The amino acid composition of purified pilin reveals very low quantities of basic amino acids and a complete absence of methionine. Pilin is synthesized throughout the C. crescentus differentiation cycle. Neither free pili nor pilin monomers are detectable in the growth media, suggesting that loss of piliation in the swarmer- to stalked-cell transition occurs via pilus retraction.     

Gamma-ray sensitivity during synchronous cell differentiation in Caulobacter crescentus.

Gamma-ray sensitivity of Caulobacter crescentus during its cell cycle was examined. Survival curves of the swarmer and stalked cells were similar and exponential in shape, whereas that of the predivisional cell was sigmoidal, with an extrapolation number of 1.8.     

Differential expression and positioning of chemotaxis methylation proteins in Caulobacter

Proteins involved in chemotaxis methylation reactions have been identified in Caulobacter crescentus and their activities, times of synthesis and cellular positions have been determined. The methyl-accepting chemotaxis proteins, the methyl-transferase and the methylesterase were all shown to be active in the flagella-bearing swarmer cell, but all three activities were lost after the swarmer cells shed their flagellum and differentiated into a stalked cell. The membrane methyl-accepting chemotaxis proteins were shown to be synthesized before cell division, coincident with the synthesis of the components of the flagellum, and to be specifically localized in the membrane of the incipient swarmer cell portion of the predivisional cell. The cytoplasmic methylesterase was also found to be differentially synthesized coincident with the period of flagellar biogenesis. Furthermore, methyltransferase activity, present in the predivisional cell, was detected only in the swarmer cell upon cell division. These results demonstrate that the chemotaxis methylation machinery is positionally biased toward one portion of the predivisional cell, and that the time of expression of a set of fla and che genes is correlated with the positioning of their gene products within the cell.     

Plasmid and chromosomal DNA replication and partitioning during the Caulobacter crescentus cell cycle

Cell division in Caulobacter crescentus yields a swarmer and a stalked cell. Only the stalked cell progeny is able to replicate its chromosome, and the swarmer cell progeny must differentiate into a stalked cell before it too can replicate its chromosome. In an effort to understand the mechanisms that limit chromosomal replication to the stalked cell, plasmid DNA synthesis was analyzed during the developmental cell cycle of C. crescentus, and the partitioning of both the plasmids and the chromosomes to the progeny cells was examined. Unlike the chromosome, plasmids from the incompatibility groups Q and P replicated in all C. crescentus cell types. However, all plasmids tested showed a ten- to 20-fold higher replication rate in the stalked cells than the swarmer cells. We observed that all plasmids replicated during the C. crescentus cell cycle with comparable kinetics of DNA synthesis, even though we tested plasmids that encode very different known (and putative) replication proteins. We determined the plasmid copy number in both progeny cell types, and determined that plasmids partitioned equally to the stalked and swarmer cells. We also reexamined chromosome partitioning in a recombination-deficient strain of C. crescentus, and confirmed an earlier report that chromosomes partition to the progeny stalked and swarmer cells in a random manner that does not discriminate between old and new DNA strands.     

Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway

Proteolysis is involved in cell differentiation and the progression through the cell cycle in Caulobacter crescentus. We have constitutively expressed the transmembrane chemoreceptor McpA from a multicopy plasmid to demonstrate that McpA degradation is modulated during the cell cycle. The level of McpA protein starts to decrease only when the swarmer cells differentiate into stalked cells. The reduction in McpA protein levels is maintained until the stalked cells develop into predivisional cells, at which point the level returns to that observed in swarmer cells. The cell-cycle-regulated degradation of McpA does not require the last 12 C-terminal amino acids, but it does require three amino acids (AAL) located 15 residues away from the C terminus. The ClpXP protease is essential in C. crescentus for viability, and thus, we tested McpA degradation in xylose conditional mutants. The effect on McpA degradation occurred within two generations from the start of ClpX depletion. The conditional mutants' growth rate was only slightly affected, suggesting that ClpX is directly involved in McpA proteolysis.     

Differential membrane phospholipid synthesis during the cell cycle of Caulobacter crescentus.

The pattern of phospholipid synthesis during the cell cycle of Caulobacter crescentus has been determined. Although the phospholipid composition of swarmer and stalked cells was indistinguishable in continuously labeled cultures if the two cell types were pulse-labeled for a short time period, marked differences in the pattern of phospholipid synthesis were detected. Pulse-labeled swarmer cells exhibited a higher proportion of phosphatidic acid and a lower proportion of phosphatidylglycerol. In addition, minor phospholipids were detected in the swarmer cells that were not detected in stalked cells. Stalked cells that developed directly from swarmer cells showed that same phospholipid profile as the swarmer cells. The switch to the second phospholipid profile was observed to occur at the predivisional cell stage. Because cell division then yielded a swarmer cell with a different phospholipid profile than its sibling stalked cell, the cell division process may trigger a mechanism which alters the pattern of phospholipid synthesis.     

Differential localization of two histidine kinases controlling bacterial cell differentiation

The bacterium C. crescentus coordinates cellular differentiation and cell cycle progression via a network of signal transduction proteins. Here, we demonstrate that the antagonistic DivJ and PleC histidine kinases that regulate polar differentiation are differentially localized as a function of the cell cycle. The DivJ kinase localizes to the stalked pole in response to a signal at the G1-to-S transition, while the PleC kinase is localized to the flagellar pole in swarmer and predivisional cells but is dispersed throughout the cell in the stalked cell. PleC, which is required for DivJ localization, may provide the cue at the G1-to-S transition that directs the polar positioning of DivJ. The dynamic positioning of signal transduction proteins may contribute to the regulation of polar differentiation at specific times during the bacterial cell cycle.     

The chaperonin GroEL and other heat-shock proteins, besides DnaK, participate in ribosome biogenesis in Escherichia coli

It has been shown that in Escherichia coli the chaperone DnaK is necessary for the late stages of 50S and 30S ribosomal subunit assembly in vivo. Here we focus on the roles of other HSPs (heat-shock proteins), including the chaperonin GroEL, in addition to DnaK, in ribosome biogenesis at high temperature. GroEL is shown to be required for the very late 45S-->50S step in the biogenesis of the large ribosome subunit, but not for 30S assembly. Interestingly, overproduction of GroES/GroEL can partially compensate for a lack of DnaK/DnaJ at 44 degrees C.     

The Caulobacter crescentus polar organelle development protein PodJ is differentially localized and is required for polar targeting of the PleC development regulator

Regulation of polar development and cell division in Caulobacter crescentus relies on the dynamic localization of several proteins to cell poles at specific stages of the cell cycle. The polar organelle development protein, PodJ, is required for the synthesis of the adhesive holdfast and pili. Here we show the cell cycle localization of PodJ and describe a novel role for this protein in controlling the dynamic localization of the developmental regulator PleC. In swarmer cells, a short form of PodJ is localized at the flagellated pole. Upon differentiation of the swarmer cell into a stalked cell, full length PodJ is synthesized and localizes to the pole opposite the stalk. In late predivisional cells, full length PodJ is processed into a short form which remains localized at the flagellar pole after cell division and is degraded during swarmer to stalked cell differentiation. Polar localization of the developmental regulator PleC requires the presence of PodJ. In contrast, the polar localization of PodJ is not dependent on the presence of PleC. These results indicate that PodJ is an important determinant for the localization of a major regulator of cell differentiation. Thus, PodJ acts directly or indirectly to target PleC to the incipient swarmer pole, to establish the cellular asymmetry that leads to the synthesis of holdfasts and pili at their proper subcellular location.     

Cell cycle and positional constraints on FtsZ localization and the initiation of cell division in Caulobacter crescentus

Swarmer cells of Caulobacter crescentus are devoid of the cell division initiation protein FtsZ and do not replicate DNA. FtsZ is synthesized during the differentiation of swarmer cells into replicating stalked cells. We show that FtsZ first localizes at the incipient stalked pole in differentiating swarmer cells. FtsZ subsequently localizes at the mid-cell early in the cell cycle. In an effort to understand whether Z-ring formation and cell constriction are driven solely by the cell cycle-regulated increase in FtsZ concentration, FtsZ was artificially expressed in swarmer cells at a level equivalent to that found in predivisional cells. Immunofluorescence microscopy showed that, in these swarmer cells, simply increasing FtsZ concentration was not sufficient for Z-ring formation; Z-ring formation took place only in stalked cells. Expression of FtsZ in swarmer cells did not alter the timing of cell constriction initiation during the cell cycle but, instead, caused additional constrictions and a delay in cell separation. These additional constrictions were confined to sites close to the original mid-cell constriction. These results suggest that the timing and placement of Z-rings is tightly coupled to an early cell cycle event and that cell constriction is not solely dependent on a threshold level of FtsZ.     

Principal sigma subunit of the Caulobacter crescentus RNA polymerase.

We have identified the gene encoding the Caulobacter crescentus principal sigma subunit, RpoD. The rpoD gene codes for a polypeptide of 653 amino acids with a predicted molecular mass of 72,623 Da (sigma 73). The C. crescentus sigma subunit has extensive amino acid sequence homology with the principal sigma factors of a number of divergent procaryotes. In particular, the segments designated region 2 that are involved in core polymerase binding and promoter recognition were identical among these bacteria despite the fact that the -10 region recognized by the C. crescentus sigma 73 differs significantly from that of the other bacteria. Thus, it appears that additional sigma factor regions must be involved in -10 region recognition. This conclusion was strengthened by a heterologous complementation assay in which C. crescentus sigma 73 was capable of complementing the Escherichia coli rpoD285 temperature-sensitive mutant. Furthermore, C. crescentus sigma 73 conferred new specificity on the E. coli RNA polymerase, allowing the expression of C. crescentus promoters in E. coli. Thus, the C. crescentus sigma 73 appears to have a broader specificity than does the sigma 70 of the enteric bacteria.     

GroES/GroEL and DnaK/DnaJ have distinct roles in stress responses and during cell cycle progression in Caulobacter crescentus

Misfolding and aggregation of protein molecules are major threats to all living organisms. Therefore, cells have evolved quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins. DnaK/DnaJ and GroES/GroEL are the best-characterized molecular chaperone systems in bacteria. In Caulobacter crescentus these chaperone machines are the products of essential genes, which are both induced by heat shock and cell cycle regulated. In this work, we characterized the viabilities of conditional dnaKJ and groESL mutants under different types of environmental stress, as well as under normal physiological conditions. We observed that C. crescentus cells with GroES/EL depleted are quite resistant to heat shock, ethanol, and freezing but are sensitive to oxidative, saline, and osmotic stresses. In contrast, cells with DnaK/J depleted are not affected by the presence of high concentrations of hydrogen peroxide, NaCl, and sucrose but have a lower survival rate after heat shock, exposure to ethanol, and freezing and are unable to acquire thermotolerance. Cells lacking these chaperones also have morphological defects under normal growth conditions. The absence of GroE proteins results in long, pinched filamentous cells with several Z-rings, whereas cells lacking DnaK/J are only somewhat more elongated than normal predivisional cells, and most of them do not have Z-rings. These findings indicate that there is cell division arrest, which occurs at different stages depending on the chaperone machine affected. Thus, the two chaperone systems have distinct roles in stress responses and during cell cycle progression in C. crescentus.     

Heat shock response of Pseudomonas aeruginosa.

The general properties of the heat shock response in Pseudomonas aeruginosa were characterized. The transfer of cells from 30 to 45 degrees C repressed the synthesis of many cellular proteins and led to the enhanced production of 17 proteins. With antibodies raised against the Escherichia coli proteins, two polypeptides of P. aeruginosa with apparent molecular weights of 76,000 and 61,000 (76K and 61K proteins) were shown to be analogous to the DnaK and GroEL heat shock proteins of E. coli due to their immunologic cross-reactivity. The major sigma factor (sigma 87) of P. aeruginosa was shown to be a heat shock protein that was immunologically related to the sigma 70 of E. coli by using polyclonal antisera. A hybridoma was produced, and the monoclonal antibody MP-S-1 was specific for the sigma 87 and did not cross-react with sigma 70 of E. coli. A smaller 40K protein was immunoprecipitated with RNA polymerase antisera from cells that had been heat shocked. The 40K protein was also associated with RNA polymerase which had been purified from heat-shocked cells and may be the heat shock sigma factor of P. aeruginosa. Exposure to ethanol resulted in the production of seven new proteins, three of which appeared to be heat shock proteins.