John Smit

Complete genome sequence of Caulobacter crescentus

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 of Caulobacter species. Proposal of Maricaulis gen. nov. with Maricaulis maris (Poindexter) comb. nov. as the type species, and emended description of the genera Brevundimonas and Caulobacter.

The 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 16S rDNA sequencing, profiling restriction fragments of 16S-23S 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 comb. 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 comb. 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.

V. Functions of S‐layers

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.

Monitoring microbiol adhesion and biofilm formation by attenuated total reflection/Fourier transform infrared spectroscopy

Abstract A major problem in accurately defining bacterial adhesion mechanisms and processes occurring in biofilms on surfaces is the lack of techniques that nondestructively provide on-line information about the microorganisms, their extracellular polymers, and metabolites. The attenuated total reflectance (ATR) technique of Fourier transform infrared spectroscopy (FT-IR) is ideally suited to monitor molecular interactions at the solution/internal reflection element (IRE) interface, and we report its application to biofilm research. Two methodologies were utilized to obtain the ATF/FT-IR spectra of living Caulobacter crescentus cells attached to germanium crystals. Initially, spectra of attached bacteria in high purity water produced molecular details of the attachment process without spectral interferences from components of the medium. A growth medium, utilized in the second method, allowed direct examination of the infrared absorption bands associated with the actively growing microorganisms on the surface of the IRE in the spectral region of 2000 to 1200 cm −1 . Using the amide II band as a marker for biofilm biomass, the detection limit was determined to be approximately 5 × 10 5 cells·cm −2 . These results proved that the ATR-FT/IR methodologies can be utilized to provide chemical information from bacteria and bacterial products located within approximately 1 μm of the surface without spectral interferences due to components of the medium.

Cell-surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S-layer of Caulobacter crescentus

The paracrystalline surface (S)‐layer of Caulobacter crescentus is composed of a single secreted protein (RsaA) that interlocks in a hexagonal pattern to completely envelop the bacterium. Using a genetic approach, we inserted a 12 amino acid peptide from Pseudomonas aeruginosa strain K pilin at numerous semirandom positions in RsaA. We then used an immunological screen to identify those sites that presented the inserted pilin peptide on the C. crescentus cell surface as a part of the S‐layer. Eleven such sites (widely separated in the primary sequence) were identified, demonstrating for the first time that S‐layers can be readily exploited as carrier proteins to display ‘epitope‐size’ heterologous peptides on bacterial cell surfaces. Whereas intact RsaA molecules carrying a pilin peptide could always be found on the surface of C. crescentus regardless of the particular insertion site, introduction of the pilin peptide at 9 of the 11 sites resulted in some proteolytic cleavage of RsaA. Two types of proteolytic phenomena were observed. The first was characterized by a single cleavage within the pilin peptide insert with both fragments of the S‐layer protein remaining anchored to the outer membrane. The other proteolytic phenomenon was characterized by cleavage of the S‐layer protein at a point distant from the site of the pilin peptide insertion. This cleavage always occurred at the same location in RsaA regardless of the particular insertion site, yielding a surface‐anchored 26 kDa proteolytic fragment bearing the RsaA N‐terminus; the C‐terminal cleavage product carrying the pilin peptide was released into the growth medium. When the results of this work were combined with the results of a previous study, the RsaA primary sequence could be divided into three regions with respect to the location of a peptide insertion and its effect on S‐layer biogenesis: (i) insertions in the extreme N‐terminus of RsaA either produce no apparent effect on S‐layer biogenesis or disrupt surface‐anchoring of the protein; (ii) insertions in the extreme C‐terminus either produce no apparent effect on S‐layer biogenesis or disrupt protein secretion; and (iii) insertions more centrally located in the protein either have no apparent effect on S‐layer biogenesis or result in proteolytic cleavage of RsaA. These data are discussed in relation to our previous assignment of the RsaA N‐ and C‐terminus as regions that are important for surface anchoring and secretion respectively.

S-Layer-Mediated Display of the Immunoglobulin G-Binding Domain of Streptococcal Protein G on the Surface of Caulobacter crescentus: Development of an Immunoactive Reagent

ABSTRACT The immunoglobulin G (IgG)-binding streptococcal protein G is often used for immunoprecipitation or immunoadsorption-based assays, as it exhibits binding to a broader spectrum of host species IgG and IgG subclasses than the alternative, Staphylococcus aureus protein A. Caulobacter crescentus produces a hexagonally arranged paracrystalline protein surface layer (S-layer) composed of a single secreted protein, RsaA, that is notably tolerant of heterologous peptide insertions while maintaining the surface-attached crystalline character. Here, a protein G IgG-binding domain, GB1, was expressed as an insertion into full-length RsaA on the cell surface to produce densely packed immunoreactive particles. GB1 insertions at five separate sites were expressed, and all bound rabbit and goat IgG, but expression levels were reduced compared to those of wild-type RsaA and poor binding to mouse IgG was noted. To remedy this, we used the 20-amino-acid Muc1 peptide derived from human mucins as a spacer, since insertions of multiple tandem repeats were well tolerated for RsaA secretion and assembly. This strategy worked remarkably well, and recombinant RsaA proteins, containing up to three GB1 domains, surrounded by Muc1 peptides, not only were secreted and assembled but did so at wild-type levels. The ability to bind IgG (including mouse IgG) increased as GB1 units were added, and those with three GB1 domains bound twice as much rabbit IgG per cell as S. aureus cells (Pansorbin). The ability of recombinant protein G-Caulobacter cells to function as immunoactive reagents was assessed in an immunoprecipitation assay using a FLAG-tagged protein and anti-FLAG mouse monoclonal antibody; their performance was comparable to that of protein G-Sepharose beads. This work demonstrates the potential for using cells expressing recombinant RsaA/GB1 in immunoassays, especially considering that protein G-Caulobacter cells are more cost-effective than protein G beads and exhibit a broader species and IgG isotype binding range than protein A.

Secretion of the Caulobacter crescentus S-Layer Protein: Further Localization of the C-Terminal Secretion Signal and Its Use for Secretion of Recombinant Proteins

The secretion signal of the Caulobacter crescentus S-layer protein (RsaA) was localized to the C-terminal 82 amino acids of the molecule. Protein yield studies showed that 336 or 242 C-terminal residues of RsaA mediated secretion of >50 mg of a cellulase passenger protein per liter to the culture fluids.

Phospho- and sulfolipids as biomarkers of Caulobacter sensu lato, Brevundimonas and Hyphomonas.

Summary Phospholipids of type strains and isolates of Caulobacter, Brevundimonas, Hyphomonas and Mycoplana bullata were studied by chromatographic and spectroscopic methods. Using tandem mass spectrometry individual phospholipids were analyzed and their structures elucidated. From the 69 strains included in this study 26 new phosphoglucolipids and two new sulfoquinovosyl-diacylglycerols could be identified. While more than 30 different fatty acids were found in the phospholipid fraction of Caulobacter and Brevundimonas only seven different fatty acids occur within the 26 different phosphoglucolipids identified. The distribution of the different types of phospholipids, especially of the sulfoquinovosyl-, glucoronotaurine amide- and phosphatidyl-glucopyranosyl-diacylglycerols, leads to the identification of four different phenotypic groups of these bacteria in agreement with the phylogeny published by Stahl et al. in 1992. One comprises Caulobacter sensu stricto with the type strain C. vibrioides , the second the type strains of Brevundimonas together with a considerable number of Caulobacter species and the third the halophilic Caulobacter spp . around C. maris . The latter group lacks the phosphatidyl-glucopyranosyl-diacylglycerols but contains sulfoquinovosyl- and glucuronotaurine amide diacylgly- cerols. They can be discerned from Hyphomonas by the occurrence of sulfoquinovosyl-diacylglycerols which are absent in strains of the genus Hyphomonas .

S-Layer Anchoring and Localization of an S-Layer-Associated Protease in Caulobacter crescentus

The S-layer of the gram-negative bacterium Caulobacter crescentus is composed of a single protein, RsaA, that is secreted and assembled into a hexagonal crystalline array that covers the organism. Despite the widespread occurrence of comparable bacterial S-layers, little is known about S-layer attachment to cell surfaces, especially for gram-negative organisms. Having preliminary indications that the N terminus of RsaA anchors the monomer to the cell surface, we developed an assay to distinguish direct surface attachment from subunit-subunit interactions where small RsaA fragments are incubated with S-layer-negative cells to assess the ability of the fragments to reattach. In doing so, we found that the RsaA anchoring region lies in the first approximately 225 amino acids and that this RsaA anchoring region requires a smooth lipopolysaccharide species found in the outer membrane. By making mutations at six semirandom sites, we learned that relatively minor perturbations within the first approximately 225 amino acids of RsaA caused loss of anchoring. In other studies, we confirmed that only this N-terminal region has a direct role in S-layer anchoring. As a by-product of the anchoring studies, we discovered that Sap, the C. crescentus S-layer-associated protease, recognized a cleavage site in the truncated RsaA fragments that is not detected by Sap in full-length RsaA. This, in turn, led to the discovery that Sap was an extracellular membrane-bound protease, rather than intracellular, as previously proposed. Moreover, Sap was secreted to the cell surface primarily by the S-layer type I secretion apparatus.

The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex.

The β‐barrel assembly machine (BAM) complex is an essential feature of all bacteria with an outer membrane. The core subunit of the BAM complex is BamA and, in Escherichia coli, four lipoprotein subunits: BamB, BamC, BamD and BamE, also function in the BAM complex. Hidden Markov model analysis was used to comprehensively assess the distribution of subunits of the BAM lipoproteins across all subclasses of proteobacteria. A patchwork distribution was detected which is readily reconciled with the evolution of the α‐, β‐, γ‐, δ‐ and ε‐proteobacteria. Our findings lead to a proposal that the ancestral BAM complex was composed of two subunits: BamA and BamD, and that BamB, BamC and BamE evolved later in a distinct sequence of events. Furthermore, in some lineages novel lipoproteins have evolved instead of the lipoproteins found in E. coli. As an example of this concept, we show that no known species of α‐proteobacteria has a homologue of BamC. However, purification of the BAM complex from the model α‐proteobacterium Caulobacter crescentus identified a novel subunit we refer to as BamF, which has a conserved sequence motif related to sequences found in BamC. BamF and BamD can be eluted from the BAM complex under similar conditions, mirroring the BamC:D module seen in the BAM complex of γ‐proteobacteria such as E. coli.