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At least 235 records · Page 13Linked to original sources
PubMed2006

Comparative genomic evidence for a close relationship between the dimorphic prosthecate bacteria Hyphomonas neptunium and Caulobacter crescentus.

The dimorphic prosthecate bacteria (DPB) are alpha-proteobacteria that reproduce in an asymmetric manner rather than by binary fission and are of interest as simple models of development. Prior to this work, the only member of this group for which genome sequence was available was the model freshwater organism Caulobacter crescentus. Here we describe the genome sequence of Hyphomonas neptunium, a marine member of the DPB that differs from C. crescentus in that H. neptunium uses its stalk as a reproductive structure. Genome analysis indicates that this organism shares more genes with C. crescentus than it does with Silicibacter pomeroyi (a closer relative according to 16S rRNA phylogeny), that it relies upon a heterotrophic strategy utilizing a wide range of substrates, that its cell cycle is likely to be regulated in a similar manner to that of C. crescentus, and that the outer membrane complements of H. neptunium and C. crescentus are remarkably similar. H. neptunium swarmer cells are highly motile via a single polar flagellum. With the exception of cheY and cheR, genes required for chemotaxis were absent in the H. neptunium genome. Consistent with this observation, H. neptunium swarmer cells did not respond to any chemotactic stimuli that were tested, which suggests that H. neptunium motility is a random dispersal mechanism for swarmer cells rather than a stimulus-controlled navigation system for locating specific environments. In addition to providing insights into bacterial development, the H. neptunium genome will provide an important resource for the study of other interesting biological processes including chromosome segregation, polar growth, and cell aging.

Alphaproteobacteria↗
PubMed2006

Genetic analysis of a novel pathway for D-xylose metabolism in Caulobacter crescentus.

Genetic data suggest that the oligotrophic freshwater bacterium Caulobacter crescentus metabolizes D-xylose through a pathway yielding alpha-ketoglutarate, comparable to the recently described L-arabinose degradation pathway of Azospirillum brasilense. Enzymes of the C. crescentus pathway, including an NAD(+)-dependent xylose dehydrogenase, are encoded in the xylose-inducible xylXABCD operon (CC0823-CC0819).

Arabinose↗
PubMed2007

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.

Bacterial Proteins↗
PubMed2006

Holdfast formation in motile swarmer cells optimizes surface attachment during Caulobacter crescentus development.

The adhesive holdfast is required for irreversible surface anchoring of Caulobacter crescentus cells. The holdfast is synthesized early during swarmer cell development and, together with pili and a functional flagellum, contributes to optimal attachment during cell differentiation. We present evidence that the timing of holdfast formation in swarmer cells is regulated posttranslationally and is dependent on the diguanylate cyclase PleD.

Bacterial Adhesion↗
PubMed1992

Early Caulobacter crescentus genes fliL and fliM are required for flagellar gene expression and normal cell division.

The biogenesis of the Caulobacter crescentus polar flagellum requires the expression of more than 48 genes, which are organized in a regulatory hierarchy. The flbO locus is near the top of the hierarchy, and consequently strains with mutations in this locus are nonmotile and lack the flagellar basal body complex. In addition to the motility phenotype, mutations in this locus also cause abnormal cell division. Complementing clones restore both motility and normal cell division. Sequence analysis of a complementing subclone revealed that this locus encodes at least two proteins that are homologs of the Salmonella typhimurium and Escherichia coli flagellar proteins FliL and FliM. FliM is thought to be a switch protein and to interface with the flagellum motor. The C. crescentus fliL and fliM genes form an operon that is expressed early in the cell cycle. Tn5 insertions in the fliM gene prevent the transcription of class II and class III flagellar genes, which are lower in the regulatory hierarchy. The start site of the fliLM operon lies 166 bp from the divergently transcribed flaCBD operon that encodes several basal body genes. Sequence comparison of the fliL transcription start site with those of other class I genes, flaS and flaO, revealed a highly conserved 29-bp sequence in a potential promoter region that differs from sigma 70, sigma 54, sigma 32, and sigma 28 promoter sequences, suggesting that at least three class I genes share a unique 5' regulatory region.

Amino Acid Sequence↗
PubMed1992

Molecular genetics of the flgI region and its role in flagellum biosynthesis in Caulobacter crescentus.

The differentiating bacterium Caulobacter crescentus has been studied extensively to understand how a relatively simple life form can govern the timing of expression of genes needed for the production of stage-specific structures. In this study, a clone containing the 5.3-kb flaP region was shown to contain the flgI, cheL, and flbY genes arranged in an operon with transcription proceeding from flgI to flbY. The predicted flgI polypeptide shows remarkable identity (44%) to the flagellar basal body P-ring protein encoded by the flgI gene of Salmonella typhimurium. flgI mutations case a reduction in the levels of flagellin production and the overproduction of the hook proteins. Therefore, the flgI-encoded P-ring protein is required for normal flagellin and hook protein synthesis, suggesting that basal body assembly may play a role in the regulation of flagellar gene expression. The flbY gene probably is a basal body component as well, since flbY mutants have flagellin and hook protein synthesis patterns similar to those exhibited by other basal body mutants. The smaller cheL gene complements a mutant that is unable to respond to chemotactic signals despite possessing a functional flagellum. This is the first example of an operon containing both flagellar and chemotaxis genes in C. crescentus.

Amino Acid Sequence↗
PubMed1992

The Caulobacter crescentus flaFG region regulates synthesis and assembly of flagellin proteins encoded by two genetically unlinked gene clusters.

At a specific time in the Caulobacter crescentus cell cycle, a single flagellar filament and multiple receptor sites for the swarmer-specific phage phi Cbk are assembled at one pole of the predivisional cell. One cluster of genes required for this morphogenesis, the flaYG region, includes the flgJKL genes, which encode structural proteins of the flagellar filament. These flagellin genes are flanked by genes required for filament assembly, the flaYE genes at one end and the flaF-flbT-flbA-flaG genes at the other. In this study, we characterized mutants carrying large chromosomal deletions within this region. Several of these strains are phi CbK resistant and produce a novel 22-kDa flagellin that is not assembled into flagella. Merodiploid strains containing either the entire flaFG region or individual fla transcription units from this region were constructed. These strains were used to correlate the presence or absence of specific gene products to changes in flagellin synthesis, filament assembly, or phage sensitivity. As a result of these studies, we were able to conclude that (i) the production of the 22-kDa flagellin results from the absence of the flbA and flaG gene products, which appear to be components of a flagellin-processing pathway common to the 25-, 27-, and 29-kDa flagellins; (ii) flbT negatively modulates the synthesis of the 27- and 25-kDa flagellins from two genetically unlinked gene clusters; (iii) flgL is the only flagellin gene able to encode the 27-kDa flagellin, and this flagellin appears to be required for the efficient assembly of the 25-kDa flagellins; (iv) flaF is required for filament assembly; and (v) phi CbK resistance results from the deletion of at least two genes in the flaFG region.

Caulobacter crescentus↗
PubMed1992

A three-start helical sheath on the flagellar filament of Caulobacter crescentus.

An unusual feature in preparations of the Caulobacter crescentus flagellar filaments is that some filaments are surrounded by a set of three windings that form a sheath. We provide evidence that the sheath is composed of subunits having a molecular mass of 24,000 Da. We suggest that the sheath could be composed of protofilaments of flagellin wound around the filament.

Caulobacter crescentus↗
PubMed1992

The S-layer of Caulobacter crescentus: three-dimensional image reconstruction and structure analysis by electron microscopy.

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 rsaA 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.

Bacterial Outer Membrane Proteins↗
PubMed1992

Identification, isolation, and structural studies of the outer membrane lipopolysaccharide of Caulobacter crescentus.

The lipopolysaccharide (LPS) of the outer membrane of Caulobacter crescentus was purified and analyzed. Two distinct strains of the species, NA 1000 and CB2A, were examined; despite differences in other membrane-related polysaccharides, the two gave similar LPS composition profiles. The LPS was the equivalent of the rough LPS described for other bacteria in that it lacked the ladder of polysaccharide-containing species that results from addition of variable amounts of a repeated sequence of sugars, as detected by gel electrophoresis in smooth LPS strains. The purified LPS contained two definable regions: (i) an oligosaccharide region, consisting of an inner core of three residues of 2-keto-3-deoxyoctonate, two residues of alpha-L-glycero-D-mannoheptose, and one alpha-D-glycero-D-mannoheptose unit and an outer core region containing one residue each of alpha-D-mannose, alpha-D-galactose, and alpha-D-glucose, with the glucose likely phosphorylated and (ii) a region equivalent to the lipid A region of the archetype, consisting primarily of an esterified fatty acid, 3-OH-dodecanoate. The lipid A-like region was resistant to conclusive analysis; in particular, although a variety of analytical methods were used, no amino sugars were detected, as is found in the lipid A of the LPS of most bacteria.

Antigens, Bacterial↗
PubMed1992

Characterization of the Caulobacter crescentus flbF promoter and identification of the inferred FlbF product as a homolog of the LcrD protein from a Yersinia enterocolitica virulence plasmid.

We have investigated the organization and expression of the Caulobacter crescentus flbF gene because it occupies a high level in the flagellar gene regulatory hierarchy. The nucleotide sequence comprising the 3' end of the flaO operon and the adjacent flbF promoter and structural gene was determined, and the organization of transcription units within this sequence was investigated. We located the 3' ends of the flaO operon transcript by using a nuclease S1 protection assay, and the 5' end of the flbF transcript was precisely mapped by primer extension analysis. The nucleotide sequence upstream from the 5' end of the flbF transcript contains -10 and -35 elements similar to those found in promoters transcribed by sigma 28 RNA polymerase in other organisms. Mutations that changed nucleotides in the -10 or -35 elements or altered their relative spacing resulted in undetectable levels of flbF transcript, demonstrating that these sequences contain nucleotides essential for promoter function. We identified a 700-codon open reading frame, downstream from the flbF promoter region, that was predicted to be the flbF structural gene. The amino-terminal half of the FlbF amino acid sequence contains eight hydrophobic regions predicted to be membrane-spanning segments, suggesting that the FlbF protein may be an integral membrane protein. The FlbF amino acid sequence is very similar to that of a transcriptional regulatory protein called LcrD that is encoded in the highly conserved low-calcium-response region of virulence plasmid pYVO3 in Yersinia enterocolitica (A.-M. Viitanen, P. Toivanen, and M. Skurnik, J. Bacteriol. 172:3152-3162, 1990).

Amino Acid Sequence↗
PubMed1993

Identification of the promoter and a negative regulatory element, ftr4, that is needed for cell cycle timing of fliF operon expression in Caulobacter crescentus.

The fliF operon of Caulobacter crescentus, which was previously designated the flaO locus, is near the top of the flagellar-gene regulatory hierarchy, and it is one of the earliest transcription units to be expressed in the cell cycle. In this report, we have identified two cis-acting sequences that are required for cell cycle regulation of fliF transcription. The first sequence was defined by the effects of three 2-bp deletions and five point mutations, each of which greatly reduced the level of fliF operon transcript in vivo. These eight mutations lie between -37 and -22 within an 18-bp sequence that matches, at 11 nucleotides, sequences in the 5' regions of the flaQR (flaS locus) and fliLM operons, which are also expressed early and occupy a high level in the regulatory hierarchy (A. Dingwall, A. Zhuang, K. Quon, and L. Shapiro, J. Bacteriol. 174:1760-1768, 1992). We propose that this 18-bp sequence contains all or part of the fliF promoter. We have also identified a second sequence, 17 bp long and centered at -8, which we have provisionally designated ftr4 because of its similarity to the enhancer-like ftr sequences required for regulation of sigma 54 promoters flaN and flbG (D. A. Mullin and A. Newton, J. Bacteriol. 171:3218-3227, 1989). Six of the seven mutations in ftr4 examined resulted in a large increase in fliF operon transcript levels, suggesting a role for ftr4 in negative regulation. A 2-bp deletion at -12 and -13 in ftr4 altered the cell cycle pattern of fliF operon transcription; the transcript was still expressed periodically, but the period of its synthesis was extended significantly. We suggest that the ftr4 sequence may form part of a developmental switch which is required to turn off fliF operon transcription at the correct time in the cell cycle.

Base Sequence↗
PubMed1993

Asymmetric expression of the gyrase B gene from the replication-competent chromosome in the Caulobacter crescentus predivisional cell.

The bacterium Caulobacter crescentus undergoes an asymmetric cell division resulting in the formation of two different daughter cells, a motile swarmer cell and a nonmotile stalked cell. These two cell types differ in their program of gene expression, their ability to replicate DNA, and the physical properties of their nucleoids. We show here that two genes, gyrB (encoding the gyrase B subunit) and orf-1, are specifically transcribed from the chromosome in the portion of the predivisional cell destined for the progeny stalked cell. This is in contrast to a subset of flagellar genes which are transcribed from the chromosome in the incipient swarmer portion of the predivisional cell. gyrB and orf-1 are within a newly identified cluster of genes involved in DNA replication and recombination, including dnaN and recF. The transcription of gyrB and orf1 occurs from the replication-competent chromosome in stalked and predivisional cells and is silenced in swarmer cells. We hypothesize that selective silencing of groups of genes in the chromosomes at the swarmer and stalked poles of the predivisional cell results in the different developmental programs and the difference in replicative ability of the two progeny cells.

Amino Acid Sequence↗
PubMed1993

Function of periplasmic copper-zinc superoxide dismutase in Caulobacter crescentus.

Caulobacter crescentus is one of a small number of bacterial species that contain a periplasmic copper-zinc superoxide dismutase (CuZnSOD). A C. crescentus mutant, with the CuZnSOD gene interrupted by a promoterless cat gene, was constructed and characterized to analyze CuZnSOD function. Periplasmic SOD does not protect against oxyradical damage in the cytosol or play a major role in maintaining the integrity of the cell envelope. Studies of the effect of sodium citrate on plating efficiency suggest that CuZnSOD protects a periplasmic or membrane function(s) requiring magnesium or calcium.

Cations, Divalent↗
PubMed1993

A sigma 54 promoter and downstream sequence elements ftr2 and ftr3 are required for regulated expression of divergent transcription units flaN and flbG in Caulobacter crescentus.

In this study, we investigated the cis-acting sequences required for transcription of the divergent, cell cycle-regulated flaN and flbG operons of Caulobacter crescentus. Previous work showed that transcription of flbG in vivo depends on a sigma 54 promoter and a sequence element called ftr1 that is located about 100 bp upstream from the transcription start site (D. A. Mullin and A. Newton, J. Bacteriol. 171:3218-3227, 1989). We now show that regulation of flaN transcription in vivo depends on a sigma 54 promoter and two ftr elements located downstream of the transcription start site at +86 (ftr2) and +120 (ftr3). Mutations in or between the conserved elements at -24 and -12 in this sigma 54 promoter reduced or abolished flaN transcription, and one mutation that eliminated flaN expression led to an increased level of flbG transcript. Mutations in ftr2 resulted in greatly reduced levels of flaN transcript but had no noticeable effect on flbG transcript levels. All three mutations constructed in ftr3 resulted in elevated flaN and flbG transcript levels. We conclude that ftr2 is required for positive regulation of flaN, whereas ftr3 appears to play a negative regulatory role in flaN and flbG expression. To explain the coordinated positive activation and negative autoregulation of these two transcription units and the effect of mutations on gene expression, we propose a model in which the flaN and flbG promoters interact through alternative DNA looping to form structures that are transcriptionally active or inactive.

Bacterial Proteins↗
PubMed1994

Regulation of Caulobacter crescentus ilvBN gene expression.

As part of an effort to determine the mechanisms employed by Caulobacter crescentus to regulate gene expression, the ilvBN genes encoding the two subunits of an acetohydroxy acid synthase (AHAS) have been characterized. Analysis of the DNA sequences indicated that the C. crescentus AHAS was highly homologous to AHAS isozymes from other organisms. S1 nuclease and primer extension studies demonstrated that transcription initiation occurred 172 bp upstream of the AHAS coding region. The region between the AHAS coding region and the transcription initiation site was shown to have the properties of a transcription attenuator. Deletion analysis of the region containing the stem-loop structure of the proposed attenuator resulted in the derepression of ilvBN expression. Thus, it appears that C. crescentus uses attenuation to regulate the expression of the ilvBN operon.

Acetolactate Synthase↗
PubMed1994

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.

Adenosine Triphosphatases↗
PubMed1994

FlbD has a DNA-binding activity near its carboxy terminus that recognizes ftr sequences involved in positive and negative regulation of flagellar gene transcription in Caulobacter crescentus.

FlbD is a transcriptional regulatory protein that negatively autoregulates fliF, and it is required for expression of other Caulobacter crescentus flagellar genes, including flaN and flbG. In this report we have investigated the interaction between carboxy-terminal fragments of FlbD protein and enhancer-like ftr sequences in the promoter regions of fliF, flaN, and flbG. FlbDc87 is a glutathione S-transferase (GST)-FlbD fusion protein that carries the carboxy-terminal 87 amino acids of FlbD, and FlbDc87 binds to restriction fragments containing the promoter regions of fliF, flaN, and flbG, whereas a GST-FlbD fusion protein carrying the last 48 amino acids of FlbD failed to bind to these promoter regions. DNA footprint analysis demonstrated that FlbDc87 is a sequence-specific DNA-binding protein that makes close contact with 11 nucleotides in ftr4, and 6 of these nucleotides were shown previously to function in negative regulation of fliF transcription in vivo (S. M. Van Way, A. Newton, A. H. Mullin, and D. A. Mullin, J. Bacteriol. 175:367-376, 1993). Three DNA fragments, each carrying an ftr4 mutation that resulted in elevated fliF transcript levels in vivo, were defective in binding to FlbDc87 in vitro. We also found that a missense mutation in the recognition helix of the putative helix-turn-helix DNA-binding motif of FlbDc87 resulted in defective binding to ftr4 in vitro. These data suggest that the binding of FlbDc87 to ftr4 is relevant to negative transcriptional regulation of fliF and that FlbD functions directly as a repressor. Footprint analysis showed that FlbDc87 also makes close contacts with specific nucleotides in ftr1, ftr2, and ftr3 in the flaN-flbG promoter region, and some of these nucleotides were shown previously to be required for regulated transcription of flaN and flbG (D. A. Mullin and A. Newton, J. Bacteriol. 175:2067-2076, 1993). Footprint analysis also revealed a new ftr-like sequence, ftr5, at -136 from the transcription start site of flbG. Our results demonstrate that FlbD contains a sequence-specific DNA-binding activity within the 87 amino acids at its carboxy terminus, and the results suggest that FlbD exerts its effect as a positive and negative regulator of C. crescentus flagellar genes by binding to ftr sequences.

Amino Acid Sequence↗