Circular organization of the DNA synthetic pathway in Caulobacter crescentus.

Genetic analysis of the cell cycle of Caulobacter crescentus has identified a DNA synthetic pathway and a cell division pathway (M. A. Osley and A. Newton, J. Mol. Biol. 138:109-128, 1980). The results presented here show that in double-shift experiments the function of the PC2076 gene product, which is required for the initiation of DNA synthesis, depends on completion of a late stage of chromosome replication in the previous cell cycle. These findings suggest a circular organization of steps in the DNA synthetic pathway in C. crescentus.     

Fatty acid degradation in Caulobacter crescentus.

Fatty acid degradation was investigated in Caulobacter crescentus, a bacterium that exhibits membrane-mediated differentiation events. Two strains of C. crescentus were shown to utilize oleic acid as sole carbon source. Five enzymes of the fatty acid beta-oxidation pathway, acyl-coenzyme A (CoA) synthase, crotonase, thiolase, beta-hydroxyacyl-CoA dehydrogenase, and acyl-CoA dehydrogenase, were identified. The activities of these enzymes were significantly higher in C. crescentus than the fully induced levels observed in Escherichia coli. Growth in glucose or glucose plus oleic acid decreased fatty acid uptake and lowered the specific activity of the enzymes involved in beta-oxidation by 2- to 3-fold, in contrast to the 50-fold glucose repression found in E. coli. The mild glucose repression of the acyl-CoA synthase was reversed by exogenous dibutyryl cyclic AMP. Acyl-CoA synthase activity was shown to be the same in oleic acid-grown cells and in cells grown in the presence of succinate, a carbon source not affected by catabolite repression. Thus, fatty acid degradation by the beta-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. Tn5 insertion mutants unable to form colonies when oleic acid was the sole carbon source were isolated. However, these mutants efficiently transported fatty acids and had beta-oxidation enzyme levels comparable with that of the wild type. Our inability to obtain fatty acid degradation mutants after a wide search, coupled with the high constitutive levels of the beta-oxidation enzymes, suggest that fatty acid turnover, as has proven to be the case fatty acid biosynthesis, might play an essential role in membrane biogenesis and cell cycle events in C. crescentus.     

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.     

Isolation and characterization of ilvA, ilvBN, and ilvD mutants of Caulobacter crescentus.

Caulobacter crescentus strains requiring isoleucine and valine (ilv) for growth were shown by transduction and pulsed-field gel electrophoresis to contain mutations at one of two unlinked loci, ilvB and ilvD. Other C. crescentus strains containing mutations at a third locus, ilvA, required either isoleucine or methionine for growth. Biochemical assays for threonine deaminase, acetohydroxyacid synthase, and dihydroxyacid dehydratase demonstrated that the ilvA locus encodes threonine deaminase, the ilvB locus encodes acetohydroxyacid synthase, and the ilvD locus encodes dihydroxyacid dehydratase. C. crescentus strains resistant to the herbicide sulfometuron methyl, which is known to inhibit the action of certain acetohydroxyacid synthases in a variety of bacteria and plants, were shown to contain mutations at the ilvB locus, further suggesting that an acetohydroxyacid synthase gene resides at this locus. Two recombinant plasmids isolated in our laboratory, pPLG389 and pJCT200, were capable of complementing strains containing the ilvB and ilvD mutations, respectively. The DNA in these plasmids hybridized to the corresponding genes of Escherichia coli and Serratia marcescens, confirming the presence of ilvB-like and ilvD-like DNA sequences at the ilvB and ilvD loci, respectively. However, no hybridization was observed between any of the other enteric ilv genes and C. crescentus DNA. These results suggest that C. crescentus contains an isoleucine-valine biosynthetic pathway which is similar to the corresponding pathway in enteric bacteria but that only the ilvB and ilvD genes contain sequences which are highly conserved at the DNA level.     

Identification of a novel response regulator required for the swarmer-to-stalked-cell transition in Caulobacter crescentus.

The onset of motility late in the Caulobacter crescentus cell cycle depends on a signal transduction pathway mediated by the histidine kinase PleC and response regulator DivK. We now show that pleD, whose function is required for the subsequent loss of motility and stalk formation by the motile swarmer cell, encodes a 454-residue protein with tandem N-terminal response regulator domains D1 and D2 and a novel C-terminal GGDEF domain. The identification of pleD301, a semidominant suppressor of the pleC Mot phenotype, as a mutation predicted to result in a D-53-->G change in the D1 domain supports a role for phosphorylation in the PleD regulator. Disruptions constructed in the pleD open reading frame demonstrated that the gene is not essential and that the pleC phenotype can also be suppressed by a recessive, loss-of-function mutation. These results suggest that PleD is part of a signal transduction pathway controlling stalked-cell differentiation early in the C. crescentus cell cycle.     

Transcriptional profiling of Caulobacter crescentus during growth on complex and minimal media

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation.     

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.     

Development of surface adhesion in Caulobacter crescentus

Caulobacter crescentus has a dimorphic life cycle composed of a motile stage and a sessile stage. In the sessile stage, C. crescentus is often found tightly attached to a surface through its adhesive holdfast. In this study, we examined the contribution of growth and external structures to the attachment of C. crescentus to abiotic surfaces. We show that the holdfast is essential but not sufficient for optimal attachment. Rather, adhesion in C. crescentus is a complex developmental process. We found that the attachment of C. crescentus to surfaces is cell cycle regulated and that growth or energy or both are essential for this process. The initial stage of attachment occurs in swarmer cells and is facilitated by flagellar motility and pili. Our results suggest that strong attachment is mediated by the synthesis of a holdfast as the swarmer cell differentiates into a stalked cell.     

Utilization of histidine by Caulobacter crescentus

Caulobacter crescentus has an inducible pathway which is responsible for the degradation of histidine. Induction of this pathway occurs in the presence of both glucose and ammonia. Growth yield experiments indicate that only two of the three available nitrogens are used for growth suggesting that formamide may be produced as a waste product. However, formamide was not detected in the culture fluid and formate was formed instead. These results suggest that histidine may be degraded in a novel pathway which results in the production of 1 mol each of ammonia, glutamate and formate per mol of histidine. The third nitrogen from histidine appears to be sequestered in some kind of secondary metabolite.     

Use of Pulsed Field Gel Electrophoresis and Transposon Mutagenesis to Estimate the Minimal Number of Genes Required for Motility in Caulobacter Crescentus

To facilitate the mapping of transposon insertion mutations in Caulobacter crescentus, we have used pulsed field gel electrophoresis to construct a detailed physical and genetic map of the C. crescentus genome. Restriction fragments were generated by DraI, AseI, or SpeI which cleave the C. crescentus 40, 13, and 26 times, respectively, and Tn5 insertions were used to align the restriction fragments generated by each of the enzymes. The utility of the resulting map was demonstrated by determining the chromosomal locations of a collection of flagellar mutations. As a result of this study, we were able to identify ten new flagellar genes at various locations on the chromosome. Thus, at least 48 genes are required for the assembly of a functional flagellum in C. crescentus.     

Copper-zinc superoxide dismutase of Caulobacter crescentus: cloning, sequencing, and mapping of the gene and periplasmic location of the enzyme.

Although widely found in the cytoplasm of eucaryotes, the copper-zinc form of superoxide dismutase (CuZnSOD) has been identified in only a small number of bacterial species. One species is the freshwater bacterium Caulobacter crescentus, which also contains an SOD with iron as the metal cofactor (FeSOD). To investigate the function of this CuZnSOD and its structural relationship to the eucaryotic CuZnSODs, the gene encoding CuZnSOD (sodC) of C. crescentus CB15 was cloned and sequenced. By hybridization to pulsed-field electrophoresis gels, sodC was mapped near cysE in the C. crescentus chromosome. Through analysis of spheroplasts, the two SODs of C. crescentus were shown to be differently localized, CuZnSOD in the periplasm and FeSOD in the cytoplasm. In its natural habitat, C. crescentus is frequently associated with blue-green algae (cyanobacteria). The oxygen evolved by these photosynthetic algae may create an extracellular oxidative stress against which the periplasmic CuZnSOD may defend more effectively than the cytoplasmic FeSOD. Amino acid sequence alignments of C. crescentus CuZnSOD with eucaryotic CuZnSODs and with CuZnSOD of Photobacterium leiognathi (the only other bacterium from which CuZnSOD has been isolated and sequenced) suggest similar supersecondary structures for bacterial and eucaryotic CuZnSODs but reveal four novel substitutions in C. crescentus CuZnSOD: a phenylalanine critical to intrasubunit hydrophobic bonding replaced by alanine, a histidine ligand of zinc replaced by aspartate, and substitutions of two other previously invariant residues that stabilize zinc or both copper and zinc. These amino acid substitutions in C. crescentus CuZnSOD may have implications for its catalysis and stability.     

Expression in Caulobacter crescentus of the phosphate-starvation-inducible porin OprP of Pseudomonas aeruginosa

The gene for the phosphate-starvation-inducible outer membrane protein OprP, of Pseudomonas aeruginosa was introduced into Caulobacter crescentus CB2A on a plasmid vector. As is the case in P. aeruginosa and Escherichia coli the oprP gene was inducible under conditions of limiting phosphate in C. crescentus. However, the maximal medium concentration of phosphate which still permitted induction of OprP was lower in C. crescentus (50 microM) than in P. aeruginosa (200 microM). Induction of OprP was coincident with the process of stalk elongation, known to occur in C. crescentus under phosphate starvation conditions. When induced, OprP was localized to the cell envelope and became a major membrane protein, indicating that the Pseudomonas promoter was efficiently recognized in C. crescentus and that the gene product was targeted to the appropriate region of the cell. Our data provide support for the hypothesis that the mechanism for regulation of phosphate-starvation-inducible genes is highly conserved amongst the eubacteria.     

Three-dimensional reconstruction of the flagellar filament of Caulobacter crescentus. A flagellin lacking the outer domain and its amino acid sequence lacking an internal segment

We obtained a three-dimensional reconstruction of the flagellar filament of Caulobacter crescentus CB15 from electron micrographs of negatively stained preparations. The C. crescentus filament appears, both in negative stain and in the frozen-hydrated state, significantly smoother and narrower than other filaments. Its helical symmetry, and unit cell size, however, are similar to that of other filaments. Although the molecular weight of the C. crescentus flagellin is about half that of other plain flagellins, there is only one monomer per unit cell as indicated by diffraction studies and by linear mass density measurements with the scanning transmission electron microscope. Alignment of the primary amino acid sequences of Salmonella typhimurium (serotype i) and C. crescentus (29,000 Mr) flagellins shows that whereas there is homology at the amino and carboxyterminal ends of the two sequences, the central segment of the S. typhimurium sequence has no homology to that of C. crescentus. A correlated comparison between the three-dimensional reconstructions of the two filaments and primary amino acid sequences of the two flagellins suggests that: (1) the C. crescentus subunit is missing the outer molecular domain but is, otherwise, similar to that of S. typhimurium; (2) the outer molecular domain in S. typhimurium corresponds, therefore, to a central stretch of the primary amino acid sequence; and (3) the outer molecular domain, missing in C. crescentus, is not obligatory for flagellar motility.     

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.