The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living α-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii , the plant pathogen Agrobacterium tumefaciens , and the bovine and human pathogen Brucella abortus .

Phylogeny and polyphasic taxonomy ofThe genus Caulobacter is composed of prosthecate bacteria often specialized for oligotrophic environments. The taxonomy of Caulobacter has relied primarily upon morphological criteria: a strain that visually appeared to be a member of the Caulobacter has generally been called one without challenge. A polyphasic approach, comprising 165 rDNA sequencing, profiling restriction fragments of 165-235 rDNA interspacer regions, lipid analysis, immunological profiling and salt tolerance characterizations, was used to clarify the taxonomy of 76 strains of the genera Caulobacter, Brevundimonas, Hyphomonas and Mycoplana. The described species of the genus Caulobacter formed a paraphyletic group with Caulobacter henricii, Caulobacter fusiformis, Caulobacter vibrioides and Mycoplana segnis (Caulobacter segnis com b. nov.) belonging to Caulobacter sensu stricto. Caulobacter bacteroides (Brevundimonas bacteroides comb. nov.), C. henricii subsp. aurantiacus (Brevundimonas aurantiaca comb. nov.), Caulobacter intermedius (Brevundimonas intermedia comb. nov.), Caulobacter subvibrioides (Brevundimonas subvibrioides com b. nov.), C. subvibrioides subsp. albus (Brevundimonas alba comb. nov.), Caulobacter variabilis (Brevundimonas variabilis comb. nov.) and Mycoplana bullata belong to the genus Brevundimonas. The halophilic species Caulobacter maris and Caulobacter halobacteroides are different from these two genera and form the genus Maricaulis gen. nov. with Maricaulis maris as the type species. Caulobacter leidyia was observed to cluster with species of the genus Sphingomonas. Caulobacter crescentus is synonymous with C. vibrioides and C. halobacteroides is synonymous with Maricaulis maris as determined by these analyses and DNA-DNA hybridization. Biomarkers discerning these different genera were determined. The necessary recombinations have been proposed and a description of Maricaulis is presented.

Caulobacters are prosthecate (stalked) bacteria that elaborate an attachment organelle called a holdfast at the tip of the cellular stalk. We examined the binding of lectins to the holdfasts of 16 marine Caulobacter strains and 10 freshwater species or strains by using a panel of fluorescein-conjugated lectins and fluorescence microscopy. The holdfasts of all the marine isolates bound to only wheat germ agglutinin (WGA) and other lectins that bind N-acetylglucosamine (GlcNac) residues. The freshwater caulobacters showed more variability in holdfast composition. Some bound only to WGA and comparable lectins as the marine strains did. Others bound additional or other lectins, and some did not bind to the lectins tested. The binding of WGA appeared to involve the regions of the holdfast involved with adhesion; a holdfast bound to WGA was significantly less adhesive to glass. Competition experiments with WGA-binding holdfasts and oligomers of GlcNac demonstrated that trimers of GlcNac (the preferred substrate for WGA binding) were more effective than dimers or monomers in preventing WGA binding to holdfasts, suggesting that stretches of contiguous GlcNac residues occur in the WGA-binding holdfasts. In addition, differences between freshwater and marine holdfasts in the strength of WGA binding were noted. The eect of a number of proteolytic and glycolytic enzymes on holdfast integrity was examined; the proteases had no,effect for all caulobacters. None of the glycolytic enzymes had an effect on marine caulobacter holdfasts, but chitinase and lysozyme (both attack oligomers of GlcNac) disrupted the holdfasts of those freshwater caulobacters that bound WGA. Despite some similarity to chitin, holdfasts did not bind Calcofluor and no measurable effects on holdfast production were detectable after cell growth in the presence of diflubenzuron or polyoxin D, inhibitors of chitin synthesis in other systems. Finally, the holdfasts of all caulobacters bound to colloidal gold particles, without regard to the coating used to stabilize the gold particles. This binding was stronger or more specific than WGA binding; treatment with colloidal gold particles prevented WGA binding, but the reverse was not the case.

Caulobacters attach to surfaces in the environment via their holdfasts, attachment organelles located at the base of the flagellum in swarmer cells and later at the end of the cellular stalk in the stalked cells which develop from the swarmer cells. There seems to be little specificity with respect to the types of surfaces to which holdfasts adhere. A notable exception is that the holdfast of one cell does not adhere to the cell surface of another caulobacter, except by joining holdfasts, typically forming "rosettes" of stalked cells. Thus, the localized adhesion of the holdfasts to the cells is in some way a specialized attachment. We investigated this holdfast-cell attachment by developing an adhesion screening assay and analyzing several mutants of Caulobacter crescentus CB2A selected to be defective in adhesion. One class of mutants made a normal holdfast by all available criteria, yet the attachment to the cell was very weak, such that the holdfast was readily shed. Another class of mutants made no holdfast at all, but when mixed with a wild-type strain, a mutant of this class participated in rosette formation. The mutant could also attach to the discarded holdfast produced by a shedding mutant. In addition, when rosettes composed of holdfast-defective and wild-type cells were examined, an increase in the number of holdfast-defective cells was correlated with a decrease in the ability of the holdfast material at the center of the rosette to bind colloidal gold particles. Gold particles are one type of surface to which holdfasts adhere well, suggesting that the stalk end and the colloidal gold particles occupy the same sites on the holdfast substance. Taken together, the data support the interpretation that there is a specialized attachment site for the holdfast at the base of the flagellum which later becomes the end of the stalk, but not a specialized region of the holdfast for attachment to this site. Also, attachment to the cell is accomplished by bond formations that occur not only at the time of holdfast production. Thus, we propose that the attachment of the holdfast to the cell is a true adhesion process and that the stalk tip and base of the flagellum must have compositions distinctly different from that of the remainder of the caulobacter cell surface.

The regular surface protein structure (S-layer) of Caulobacter crescentus was analyzed by electron microscopy and three-dimensional image reconstruction to a resolution of 2 nm. Projections showed that the S-layer is an array of ring structures, each composed of six subunits that are arranged on a lattice with p6 symmetry. Three-dimensional reconstructions showed that the ring subunits were approximately rod-shaped structures and were perpendicular to the plane of the array, with a linker arm emanating from approximately the middle of the rod, accounting for the connections between the rings. The calculated subunit mass was ca. 100 kDa, very close to the size of RsaA (the protein known to be at least the predominant species in the S-layer) predicted from the DNA sequence of the rsaA4 gene. The core region of the rings creates an open pore 2.5 to 3.5 nm in diameter. The size of the gaps between the neighboring unit cells is in the same range, suggesting a uniform porosity predicted to exclude molecules larger than ca. 17 kDa. Attempts to remove membrane material from S-layer preparations with detergents revealed that the structure spontaneously rearranged into a mirror-image double layer. Negative-stain and thin-section electron microscopy examination of colonies of C. crescentus strains with a mutation in a surface molecule involved in the attachment of the S-layer showed that shed RsaA protein organized into large sheets. The sheets in turn organized into stacks that tended to accumulate near the upper surface of the colony. Image reconstruction indicated that these sheets were also precise mirror-image double layers, and thickness measurements obtained from thin sections were consistent with this finding. The sheets were absent when these mutant strains were grown without calcium, supporting other data that calcium is involved in attachment of the S-layer to a surface molecule and perhaps in subunit-subunit interactions. We propose that when the membrane is removed from S-layer fragments by detergents or the attachment-related surface molecule is absent, the attachment sites of the S-layer align precisely to form a double layer via a calcium interaction.

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

The entire nucleotide sequence of the rsaA gene, encoding the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A, was determined. The rsaA gene encoded a protein of 1026 amino acids, with a predicted molecular weight of 98,132. Protease cleavage of mature RsaA protein and amino acid sequencing of retrievable peptides yielded two peptides: one aligned with a region approximately two-thirds the way into the predicted amino acid sequence and the second peptide corresponded to the predicted carboxy terminus. Thus, no cleavage processing of the carboxy portion of the RsaA protein occurred during export, and with the exception of the removal of the initial methionine residue, the protein was not processed by cleavage to produce the mature protein. The predicted RsaA amino acid profile was unusual, with small neutral residues predominating. Excepting aspartate, charged amino acids were in relatively low proportion, resulting in an especially acidic protein, with a predicted pI of 3.46. As with most other sequenced S-layer proteins, RsaA contained no cysteine residues. A homology scan of the Swiss Protein Bank 17 produced no close matches to the predicted RsaA sequence. However, RsaA protein shared measurable homology with some exported proteins of other bacteria, including the hemolysins. Of particular interest was a specific region of the RsaA protein that was homologous to the repeat regions of glycine and aspartate residues found in several proteases and hemolysins. These repeats are implicated in the binding of calcium for proper structure and biological activity of these proteins. Those present in the RsaA protein may perform a similar function, since S-layer assembly and surface attachment requires calcium. RsaA protein also shared some homology with 10 other S-layer proteins, with the Campylobacter fetus S-layer protein scoring highest.

Phylogeny by a polyphasic approach of the order Caulobacterales, proposal of Caulobacter mirabilis sp. nov., Phenylobacterium haematophilum sp. nov. and Phenylobacterium conjunctum sp. nov., and emendation of the genus Phenylobacterium rRNA genes were determined for the genera Asticcacaulis, Brevundimonas, Caulobacter and Phenylobacterium and the description of the genus Phenylobacterium is emended.The genus Caulobacter was originally composed of dimorphic, prosthecate bacteria. In these prokaryotes, reproduction results in the separation of two cells that are morphologically and behaviourally different from each other. One cell is non-motile and prosthecate (Staley, 1968) and sticks to surfaces by adhesive material located at the tip of the prostheca. The other cell bears one polar flagellum by which it is motile. This mode of reproduction helps these oligotrophic bacteria to disperse the population at each generation, thereby minimizing competition for resources (Poindexter, 1981). Caulobacteria have been known for more than a century and the first isolation of a Caulobacter sp. was reported by Löffler in 1890 (Löffler, 1890). Even then, he noticed the unusual nutrient requirements of the strain due to the oligotrophic nature of the isolate. In 1935, Henrici and Johnson described the genus Caulobacter with Caulobacter vibrioides as the type species ) but they did not isolate C. vibrioides and therefore no type strain was given. Poindexter (1964) proposed strain CB 51 as the neotype strain of C. vibrioides and described nine species and two Figures showing a phylogenetic tree constructed from 16S rRNA gene sequences using the minimal evolution algorithm and the negative fast atom bombardment mass spectra of the polar lipids are available as supplementary material with the online version of this paper. Tables detailing the cellular fatty acid contents, phospho-and sulfolipids, polar lipids, enzyme activities and the results of the Biolog tests for the species examined in this study are given in supplementary tables S1-S5, available with the online version of this paper.