[Morphologic changes in the life cycle of Cytophaga hutchinsonii].

OBJECTIVE: To study the morphological changes of Cytophaga hutchinsonii cell during its life circle. METHODS: Cytophaga hutchinsonii cell was observed under light microscope, fluorescence microscope and scanning electron microscope. The nucleoids in the cell were stained with fluorescent dye Hoechst33342 and examined by fluorescence microscopy. RESULTS: We discovered that under starvation conditions, the long, flexible rod cell of Cytophaga hutchinsonii would bend and turn into circular cell. The circular cell failed to produce carboxymethyl cellulase. Some of the circular cells might further wind around and turn into tiny spherical cells. The tiny spherical cell similar to the microcyst of sporocytophaga could germ into long flexible rods again under certain circumstances. When growing cultures to logarithmic phase of cell growth, Cytophaga hutchinsonii cell with three nucleoids in it was occasionally observed, which indicated that the two strands of DNA might act differently in the initiation of DNA replication. CONCLUSION: This is the first detailed description of the formation process of circular cell and tiny spherical cell in the life circle of Cytophaga hutchinsonii. The result will help to further reveal the relation between morphologic change and cellulose degradation ability of the strain.

Characterization, identification, and cloning of the S-layer protein from Cytophaga sp.

We characterized, identified, and cloned a major protein which comprised 16% of the total proteins from Cytophaga sp. cell lysate. After French pressing, the fraction of cell envelope was treated with 0.2% Triton X-100 to remove cell membranes. Subsequent SDS-PAGE analysis of the Triton X-100-insoluble cell wall revealed a protein of 120 kDa with a pI of 5.4, which was identified by gold immunostaining as the surface (S)-layer protein of this soil bacterium. The nucleotide sequence of the cloned S-layer protein gene (slp) encoding this protein consisted of 3144 nucleotides with an ORF for 1047 amino acids, which included a typical 32-amino acid leader peptide sequence. Amino acid sequence alignment revealed 29-48% similarity between this protein and the S-layer proteins from other prokaryotic organisms. The 120-kDa protein from the Cytophaga sp. cell lysate has been characterized as a member of the S-layer proteins, and the slp gene was cloned and expressed in Escherichia coli. E. coli harboring the plasmid containing the 600- or 800-bp DNA fragment upstream of the initiation codon of the slp gene, in the presence of the reporter gene rsda (raw starch digesting amylase), showed amylase activity in starch containing plate. The putative promoter region of slp located 600 bp upstream of the initiation codon might be used for foreign gene expression.

Genome sequence of the cellulolytic gliding bacterium Cytophaga hutchinsonii.

The complete DNA sequence of the aerobic cellulolytic soil bacterium Cytophaga hutchinsonii, which belongs to the phylum Bacteroidetes, is presented. The genome consists of a single, circular, 4.43-Mb chromosome containing 3,790 open reading frames, 1,986 of which have been assigned a tentative function. Two of the most striking characteristics of C. hutchinsonii are its rapid gliding motility over surfaces and its contact-dependent digestion of crystalline cellulose. The mechanism of C. hutchinsonii motility is not known, but its genome contains homologs for each of the gld genes that are required for gliding of the distantly related bacteroidete Flavobacterium johnsoniae. Cytophaga-Flavobacterium gliding appears to be novel and does not involve well-studied motility organelles such as flagella or type IV pili. Many genes thought to encode proteins involved in cellulose utilization were identified. These include candidate endo-beta-1,4-glucanases and beta-glucosidases. Surprisingly, obvious homologs of known cellobiohydrolases were not detected. Since such enzymes are needed for efficient cellulose digestion by well-studied cellulolytic bacteria, C. hutchinsonii either has novel cellobiohydrolases or has an unusual method of cellulose utilization. Genes encoding proteins with cohesin domains, which are characteristic of cellulosomes, were absent, but many proteins predicted to be involved in polysaccharide utilization had putative D5 domains, which are thought to be involved in anchoring proteins to the cell surface.

Cytophaga-flavobacterium gliding motility.

Flavobacterium johnsoniae, like many other members of the Cytophaga-Flavobacterium-Bacteroides group, displays rapid gliding motility. Cells of F. johnsoniae glide over surfaces at rates of up to 10 microm/s. Latex spheres added to F. johnsoniae bind to and are rapidly propelled along cells, suggesting that adhesive molecules move laterally along the cell surface during gliding. Genetic analyses have identified a number of gld genes that are required for gliding. Three Gld proteins are thought to be components of an ATP-binding-cassette transporter. Five other Gld proteins are lipoproteins that localize to the cytoplasmic membrane or outer membrane. Disruption of gld genes results not only in loss of motility, but also in resistance to bacteriophages that infect wild-type cells, and loss of the ability to digest the insoluble polysaccharide chitin. Two models that attempt to incorporate the available data to explain the mechanism of F. johnsoniae gliding are presented.

Viable cytophaga-like bacterium in the 0.2 microm-filtrate seawater.

A strain of the Cytophaga-like bacterium (CLB), Nano-1, was isolated from the 0.2 microm-filtrate of natural seawater. Both cellular fatty acid and 16S rDNA sequence analyses indicated that Nano-1 is closely affiliated to the marine gliding CLB genus, Microscilla. Nano-1 was observed to undergo cyclic morphological change typical of the genus Microscilla, and sub-0.2-microm cells were formed in the late stationary phase. The sub-0.2-microm cells were repeatedly revived and subcultured. Formation of the sub-0.2-microm cells seems to be adaptive for oligotrophic growth and starvation survival.

Bacterial filament formation, a defense mechanism against flagellate grazing, is growth rate controlled in bacteria of different phyla.

A facultatively filamentous bacterium was isolated from eutrophic lake water and was identified as Flectobacillus sp. strain MWH38 (a member of the Cytophaga-Flavobacterium-Bacteroides phylum) by comparative 16S rRNA gene sequence analysis. Filament formation by Flectobacillus sp. strain MWH38 and filament formation by Flectobacillus major, the closest known relative of strain MWH38, were studied in chemostat cultures under grazing pressure by the bacterivorous flagellate Ochromonas sp. strain DS and without predation at several growth rates. The results clearly demonstrated that filament formation by the two flectobacilli is growth rate controlled and thus independent of the presence of a predator. However, flagellate grazing positively influenced bacterial growth rates by decreasing bacterial biomass and thus indirectly stimulated filament formation. The results of investigations of cell elongation and filament formation by Comamonas acidovorans PX54 (a member of the beta subclass of the class Proteobacteria) supported the recent proposal that in this species the mechanism of filament formation is growth rate controlled. The finding that the grazing defense mechanism consisting of filament formation is growth rate controlled in the flectobacilli investigated and C. acidovorans PX54 (i.e., in bacteria belonging to divergent evolutionary phyla) may indicate that this mechanism is a phylogenetically widely distributed defense strategy against grazing.

Polaribacter gen. nov., with three new species, P. irgensii sp. nov., P. franzmannii sp. nov. and P. filamentus sp. nov., gas vacuolate polar marine bacteria of the Cytophaga-Flavobacterium-Bacteroides group and reclassification of 'Flectobacillus glomeratus' as Polaribacter glomeratus comb. nov.

Several psychrophilic, gas vacuolate strains of the Cytophage-Flavobacterium-Bacteroides (CFB) phylogenetic group were isolated from sea ice and water from the Arctic and the Antarctic. The closest taxonomically defined species by 16S rRNA sequence analysis is 'Flectobacillus glomeratus'. However, 'Flc. glomeratus' is phylogenetically distant from the Flectobacillus type species, Flc. major. On the basis of phenotypic, genotypic and 16S rRNA sequence analyses we propose a new genus, Polaribacter, with three new species, Polaribacter irgensii strain 23-P (ATCC 700398), Polaribacter franzmannii strain 301 (ATCC 700399) and Polaribacter filamentus strain 215 (ATCC 700397). P. filamentus is the type species of the genus. None of these species exhibits a cosmopolitan or bipolar distribution. This is the first taxonomic description of gas vacuolate bacteria in the CFB group. Additionally, we propose that 'Flc. glomeratus' be reclassified to the genus Polaribacter as P. glomeratus, comb. nov.

Biodiversity of gas vacuolate bacteria from Antarctic sea ice and water.

Psychrophilic, gas vacuolate, heterotrophic bacteria indigenous to sea ice communities in Antarctica have been isolated. Phylogenetic analysis of representative members of these bacteria shows that they belong to the alpha, beta, and gamma Proteobacteria and the Flavobacteria-Cytophaga group. This is the first report of gas vacuolate bacteria from the beta Proteobacteria and the Flavobacteria-Cytophaga groups.

Topostin, a novel inhibitor of mammalian DNA topoisomerase I from Flexibacter topostinus sp. nov. I. Taxonomy, and fermentation of producing strain.

We found a new inhibitor of mammalian DNA topoisomerase I, named topostin, from a bacterial culture. The bacteria was identified as Flexibacter topostinus sp. nov., B-572. Morphological and physiological characteristics, and utilization of sugars were examined. Comparison of the strain with known species of the genus Flexibacter was made and indicates that the strain is a new species of the genus Flexibacter. The bacteria produced the inhibitor in parallel with their growth up to 72 hours.

Gliding motility in Cytophaga.

Gliding motility in Cytophaga depends on a motility machinery that keeps surfaces of cells in motion and on a surface slime that allows motile cells to translocate over a substratum. The unusual nature of the moving cell surfaces appears to be responsible for several cell-surface properties being motility dependent.

Coaggregation of human oral Cytophaga species and Actinomyces israelii.

A total of 19 strains of oral Cytophaga sp. obtained from subgingival plaque deposits were tested for their ability to coaggregate with strains of Actinomyces israelii, A. viscosus, A. naeslundii, Streptococcus sanguis, S. mutans, S. salivarius, and S. mitis. Coaggregation was observed only with A. israelii. Based on their coaggregation patterns with eight A. israelii strains, the Cytophaga strains were distributed among three distinct groups: those that coaggregated with A. israelii PK16 but not with A. israelii W1011 (ATCC 29322), those that coaggregated with A. israelii ATCC 29322 but not with A. israelii PK16, and those that coaggregated with none of the eight A. israelii strains. In each of the coaggregations, prior heat treatment (85 degrees C, 30 min) of the Cytophaga cells prevented coaggregation, whereas identical treatment of the A. israelii cells had no effect. The ability of A. israelii PK16 to form adherent plaque on a tooth surface previously coated with Cytophaga plaque was tested with one of the coaggregating Cytophaga strains. White patches of A. israelii plaque were found covering both the amber-colored Cytophaga plaque on the cementum surface as well as the enamel surface to which Cytophaga strains do not adhere. Electron micrographs of thin-sectioned mixed-plaque material revealed both cell types in close proximity. In addition, electron micrographs of negatively stained coaggregated cells showed interbacterial adherence between surface fimbrae on A. israelii and outer membrane blebs on the gram-negative Cytophaga sp. The kinetics of binding of A. israelii to spheroidal hydroxyapatite and to root powder were indicative of a high-affinity binding system with comparatively large numbers of available binding sites on both substrata. These results indicate the highly specific nature of Cytophaga sp.--A. israelii recognition. The contribution of such recognition toward the mechanisms that are responsible for the indigenous nature of these oral bacteria is discussed.

Fimbriation in gliding bacteria.

Of twenty-two strains of gliding prokaryotes examined, all but three were found to possess polar fimbriae. Fimbriae were not observed on two gliders, while Chloroflexus aurantiacus bore abundant peritrichous fimbriae. In some gliding bacteria, fimbriae were associated with 'holes' surrounded by an electron-transparent collar bearing 12 spike-like projections.

Examination of fimbriation of some gram-negative rods with and without twitching and gliding motility.

Negatively stained preparations of 30 different strains of gram-negative rods representing 20 different taxa were examined in the electron microscope. Thirteen of the strains studied exhibited twitching and six of the strains exhibited motility. Additionally, non-twitching substrains of two of the twitching strains and a non-gliding substrain of one of the gliding strains were examined. A variety of cultural media, preparations for negative straining and negative strains were used. It was found that all strains with twitching motility possessed fimbriae, the diameter of which was approximately 50 A in all but one strain; the fimbriae of this strain had a diameter of approximately 40 A. The fimbriae were judged to be of polar origin in all cases where the origin could be determined with certainty. On none of the strains without twitching motility could fimbriae be demonstrated. Only one of the six strains with gliding motility possessed fimbriae.