Purification and characterization of flagella from Roseburia cecicola, an obligately anaerobic bacterium

Flagella from Roseburia cecicola, an obligately anaerobic bacterium originally isolated from murine caecal mucosa, were purified by mechanical shearing followed by differential centrifugation. Purity of the flagellar preparation was determined by polyacrylamide gel electrophoresis, electron microscopy and chemical analysis. The flagella were composed of a single protein subunit (flagellin) with an estimated molecular weight of 42 000. The amino acid composition of the flagellin was similar to that of some facultatively anaerobic and aerobic bacteria.     

Analysis of the flagellin (hag) gene of alkalophilic Bacillus sp. C-125.

Motility of the alkalophilic Bacillus sp. C-125, a flagellate bacterium, was demonstrated to be Na(+)- and pH-dependent. Flagellin protein from this strain was purified to homogeneity and the N-terminal sequence determined. Using the hag gene of Bacillus subtilis as a probe, the hag gene of Bacillus sp. C-125 was identified and cloned into Escherichia coli. Sequencing of this hag gene revealed that it encodes a protein of 272 amino acids (M(r) 29,995). The predicted N terminal sequence of this protein was identical to that determined by N-terminal sequencing of the flagellin protein from strain C-125. The alkalophilic Bacillus sp. C-125 flagellin shares homology with other known flagellins in both the N- and C-terminal regions. The middle portion, however, shows considerable differences, even from that of flagellin from the related species, B. subtilis.     

A new type of flagellin gene in Pseudomonas putida

Previously established PCR amplification and Southern hybridization procedures were developed for the isolation of the 0.8-kb flagellin gene in Pseudomonas putida. The deduced protein sequence has significant homology to the N- and C-terminal sequences of other bacterial flagellins. We propose that P. putida flagellin genes can be divided at least into three size groups: type I (2.0 kb), type II (1.4 kb), and type III (0.8 kb). Type I and type II flagellin genes have been reported. The new 0.8-kb type III gene was expressed in E. coli, and the resulting protein was purified and used to raise polyclonal antibody to study whether this small gene encodes flagellin. The antiserum reacted with purified flagellin monomers from representatives of each flagellin type, as well as proteins of the same sizes in lysates of these organisms, on Western immunoblots. This antiserum was determined to be functional in a motility inhibition assay. Similar results were obtained from antiserum directed against purified type III flagellin, indicating that a new type of flagellin gene in P. putida has been found. Preliminary electron microscopic study revealed that P. putida isolate with the smaller flagellin gene type appeared to have a thinner flagellar filament.     

Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vitro-derived deletion mutation.

The flagellin promoter and structural gene from Bacillus subtilis I168 was cloned and sequenced. The amino-terminal protein sequence deduced from the coding sequence of the cloned gene was identical to that of the amino terminus of purified flagellin, indicating that the export of this protein is not directed by a posttranslationally processed N-terminal signal peptide. A sequence that was homologous to that of a consensus sigma 28 RNA polymerase recognition site lay upstream of the proposed translational start site. Amplification of this promoter region on a multicopy plasmid resulted in the formation of long, filamentous cells that accumulated flagellin intracellularly. The chromosomal locus containing the wild-type flagellin allele was replaced with a defective allele of the gene (delta hag-633) that contained a 633-base-pair deletion. Transport analysis of various flagellin gene mutations expressed in the hag deletion strain suggest that the extreme C-terminal portion of flagellin is functionally involved in export of the protein.     

TetZ, a new tetracycline resistance determinant discovered in gram-positive bacteria, shows high homology to gram-negative regulated efflux systems

The complete nucleotide sequence of the tetracycline resistance plasmid pAG1 from the gram-positive soil bacterium Corynebacterium glutamicum 22243 (formerly Corynebacterium melassecola 22243) was determined. The R-plasmid has a size of 19,751 bp and contains at least 18 complete open reading frames. The resistance determinant of pAG1 revealed homology to gram-negative tetracycline efflux and repressor systems of Tet classes A through J. The highest levels of amino acid sequence similarity were observed to the transmembrane tetracycline efflux protein TetA(A) and to the tetracycline repressor TetR(A) of transposon Tn1721 with 64 and 56% similarity, respectively. This is the first time a repressor-regulated tet gene has been found in gram-positive bacteria. A new class of tetracycline resistance and repressor proteins, termed TetA(Z) and TetR(Z), is proposed.     

Oar, a 115-kilodalton membrane protein required for development of Myxococcus xanthus.

Myxococcus xanthus is a developmental gram-negative bacterium which forms multicellular fruiting bodies upon nutrient starvation. This bacterium was found to contain a 115-kDa membrane protein which separated with the inner membrane fraction by sucrose density gradient centrifugation. The gene for this protein was cloned, and its DNA sequence was determined. The deduced amino acid sequence consists of 1,061 residues. This protein contains a putative signal sequence and many short segments, found scattered throughout the entire protein, that have sequence similarities with OmpA, a major outer membrane protein of Escherichia coli. Thus, the gene was designated oar (OmpA-related protein). A second open reading frame was found 36 bases downstream of the oar termination codon. This open reading frame encodes a protein of 236 residues and contains a putative lipoprotein signal sequence. An aor disruption mutation (delta oar) showed no effect on vegetative growth but caused abnormal morphogenesis during development and reduced myxospore formation. When examined with a light microscope, delta oar cells were unable to aggregate on developmental agar, indicating that Oar is required for cellular adhesiveness during development.     

Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from Matsuebacter chitosanotabidus 3001

The extracellular chitosanase (34,000 M(r)) produced by a novel gram-negative bacterium Matsuebacter chitosanotabidus 3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40 degrees C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag(+). Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire choA gene encoding chitosanase. Sequencing of the choA gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in Escherichia coli. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.     

Evidence for posttranslational modification and gene duplication of Campylobacter flagellin.

A gene encoding a flagellin protein of Campylobacter coli VC167 has been cloned and sequenced. The gene was identified in a pBR322 library by hybridization to a synthetic oligonucleotide probe corresponding to amino acids 4 to 9 of the N-terminal sequence obtained by direct chemical analysis (S. M. Logan, L. A. Harris, and T. J. Trust, J. Bacteriol. 169:5072-5077, 1987). The DNA was sequenced and shown to contain an open reading frame encoding a protein with a molecular weight of 58,945 and a length of 572 amino acids. The deduced amino acid sequence was identical to the published N-terminal amino acid sequence of VC167 flagellin and to four internal regions whose partial sequences were obtained by direct chemical analysis of two tryptic and two cyanogen bromide peptides of VC167 flagellin. The C. coli flagellin protein contains posttranslationally modified serine residues, most of which occur within a region containing two 9-amino-acid repeating peptides separated by 34 unique amino acids. Comparisons with the sequences of flagellins from other bacterial species revealed conserved residues at the amino- and carboxy-terminal regions. Hybridization data suggest the presence of a second flagellin copy located adjacent to the first on the VC167 chromosome.     

Flagellin gene (fliC) of Thermus thermophilus HB8: characterization of its product and involvement to flagella assembly and microbial motility

Thermus thermophilus HB8 flagellin protein (FliC) is encoded by the TTHC004 (fliC) gene, which is located in the pTT8 plasmid of the bacterium. Flagellin monomer and flagella fibres were isolated from a culture of T. thermophilus grown in rich medium, or in mineral salt medium with sodium gluconate as the carbon source. Western blot immunodetection with anti-FliC revealed a stable complex (FliC)(1)(FliS)(2) of flagellin (FliC, 27.7 kDa) with a homodimer of FliS (FliS, 18.2 kDa) that are encoded by TTHC004 and TTHC003 genes, respectively. The complex is dissociable at low pHs and/or by heat treatment. Glycan staining of purified flagella and treatment with N-glycosidase F suggested that flagellin of T. thermophilus is a glycosylated protein. Size exclusion chromatography revealed that flagellar filaments (FliC) have a molecular mass higher than 200 kDa. The formation of flagella is enhanced after prolonged cultivation time where phosphate and other nutrient were depleted, giving in the bacterium considerable swimming motility in low viscosity media.     

Spiralins of Spiroplasma citri and Spiroplasma melliferum: amino acid sequences and putative organization in the cell membrane

Spiralin is the major membrane protein of the helical mollicute Spiroplasma citri. A similar protein occurs in the membrane of Spiroplasma melliferum, an organism related to S. citri. The gene encoding spiralin has been sequenced. A restriction fragment of the spiralin gene has been used as a probe to detect the gene encoding S. melliferum spiralin. A 4.6-kilobase-pair ClaI DNA fragment from S. melliferum strongly hybridized with the probe. This fragment was inserted in pBR322 and cloned in Escherichia coli. It was further subcloned in the replicative forms of M13mp18 and M13mp19, and its nucleotide sequence was determined (GenBank accession number M33991). An open reading frame showing 88.6% base sequence homology with the S. citri spiralin gene could be identified and was assumed to be the gene encoding S. melliferum spiralin. The deduced amino acid sequence of the protein had 75% homology with the spiralin sequence. In particular, the two proteins possess a stretch of 20 amino acids which can form an alpha-helix, in which all polar amino acids occupy approximately one-third of the axial projection down the helix. On the basis of these data and published data, we propose a topological model for the structural organization of the spiralin in the cell membrane of spiroplasmas.     

Identification of genes required for synthesis of the adhesive holdfast in Caulobacter crescentus

Adhesion to both abiotic and biotic surfaces by the gram-negative prothescate bacterium Caulobacter crescentus is mediated by a polar organelle called the "holdfast," which enables the bacterium to form stable monolayer biofilms. The holdfast, a complex polysaccharide composed in part of N-acetylglucosamine, localizes to the tip of the stalk (a thin cylindrical extension of the cell wall and membranes). We report here the isolation of adhesion mutants with transposon insertions in an uncharacterized gene cluster involved in holdfast biogenesis (hfs) as well as in previously identified polar development genes (podJ and pleC), and the holdfast attachment genes (hfa). Clean deletions of three of the four genes in the hfs gene cluster (hfsDAB) resulted in a severe holdfast biogenesis phenotype. These mutants do not bind to surfaces or to a fluorescently labeled lectin, specific for N-acetylglucosamine. Transmission electron microscopy indicated that the hfsDAB mutants fail to synthesize a holdfast at the stalk tip. The predicted hfs gene products have significant sequence similarity to proteins necessary for exopolysaccharide export in gram-negative bacteria. HfsA has sequence similarity to GumC from Xanthomonas campestris, which is involved in exopolysaccharide export in the periplasm. HfsD has sequence similarity to Wza from Escherichia coli, an outer membrane protein involved in secretion of polysaccharide through the outer membrane. HfsB is a novel protein involved in holdfast biogenesis. These data suggest that the hfs genes play an important role in holdfast export.     

Colonization of gnotobiotic mice by Roseburia cecicola, a motile, obligately anaerobic bacterium from murine ceca.

In scrapings of mouse cecal mucosae, motile bacteria outnumbered nonmotile bacteria by a ratio of 2:1. Obligately anaerobic bacteria were obtained from such scrapings through the use of techniques designed for the selective isolation of motile bacteria. One of the isolates, Roseburia cecicola, was rapidly motile in broth by means of 20 to 35 flagella arranged in a fascicle on each cell. R. cecicola cells colonized germfree mice (3 x 10(9) to 1 x 10(10) CFU/g of cecum) within 11 days after the animals were inoculated intragastrically with 2 x 10(8) CFU per mouse. In such monoassociated gnotobiotes, the bacteria were found primarily in the cecum, dispersed in the lumen among particles of digesta, and in the mucus over the epithelial surface. Between 2 and 3 weeks after birth, offspring of monoassociated adult mice were colonized by the bacterium (2 x 10(9) to 1 x 10(10) CFU/g of cecum). These results indicate that R. cecicola is suitable for studies of the ecology of host-associated microorganisms, particularly for investigation of the role of motility and possibly also chemotaxis in bacterial colonization of the mammalian gastrointestinal tract.     

Genetic and molecular characterization of the polar flagellum of Vibrio parahaemolyticus.

Vibrio parahaemolyticus possesses two alternate flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (swimming), while multiple lateral flagella move the bacterium over surfaces (swarming). Energy to rotate the polar flagellum is derived from the sodium membrane potential, whereas lateral flagella are powered by the proton motive force. Lateral flagella are arranged peritrichously, and the unsheathed filaments are polymerized from a single flagellin. The polar flagellum is synthesized constitutively, but lateral flagella are produced only under conditions in which the polar flagellum is not functional, e.g., on surfaces. This work initiates characterization of the sheathed, polar flagellum. Four genes encoding flagellins were cloned and found to map in two loci. These genes, as well as three genes encoding proteins resembling HAPs (hook-associated proteins), were sequenced. A potential consensus polar flagellar promoter was identified by using upstream sequences from seven polar genes. It resembled the enterobacterial sigma 28 consensus promoter. Three of the four flagellin genes were expressed in Escherichia coli, and expression was dependent on the product of the fliA gene encoding sigma 28. The fourth flagellin gene may be different regulated. It was not expressed in E. coli, and inspection of upstream sequence revealed a potential sigma 54 consensus promoter. Mutants with single and multiple defects in flagellin genes were constructed in order to determine assembly rules for filament polymerization. HAP mutants displayed new phenotypes, which were different from those of Salmonella typhimurium and most probably were the result of the filament being sheathed.     

Analysis of the nitrous oxide reduction genes, nosZDFYL, of Achromobacter cycloclastes.

The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.     

The development of a flagellin surface display expression system in a moderate thermophile, <Emphasis Type="Italic">Bacillus halodurans</Emphasis> Alk36

This study relates to the development of an alkaliphilic, thermotolerant, Gram-positive isolate, Bacillus halodurans Alk36, for the over-production and surface display of chimeric gene products. This bacterium continuously over-produces flagellin. To harness this ability, key genetic tools, such as gene targeted inactivation, were developed for this strain. The hag gene, which codes for flagellin, was inactivated on the chromosome giving rise to the B. halodurans BhFC01 mutant. Polylinkers were inserted as in-frame, chimeric, flagellin sandwich fusions to identify the permissive insertion sites corresponding to the variable regions of the flagellin protein. Flagellin expression and motility were evaluated for these constructs. Two sites were identified for possible peptide insertion in the flagellin gene, one of which produced functional flagella and was able to restore the motility phenotype to a non-motile mutant. Peptides encoding a poly-histidine peptide and the HIV-1 subtype C gp120 epitope were, respectively, incorporated into this site as in-frame fusions. The peptides were found to be successfully displayed on the cell surface and functional through metal binding and immunological studies, respectively.