Characterization of a multi-function processive endoglucanase CHU_2103 from Cytophaga hutchinsonii.

Cytophaga hutchinsonii is a Gram-negative gliding bacterium which can efficiently degrade crystalline cellulose by an unknown strategy. Genomic analysis suggests the C. hutchinsonii genome lacks homologs to an obvious exoglucanase that previously seemed essential for cellulose degradation. One of the putative endoglucanases, CHU_2103, was successfully expressed in Escherichia coli JM109 and identified as a processive endoglucanase with transglycosylation activity. It could hydrolyze carboxymethyl cellulose (CMC) into cellodextrins and rapidly decrease the viscosity of CMC. When regenerated amorphous cellulose (RAC) was degraded by CHU_2103, the ratio of the soluble to insoluble reducing sugars was 3.72 after 3 h with cellobiose and cellotriose as the main products, indicating that CHU_2103 was a processive endoglucanase. CHU_2103 could degrade cellodextrins of degree of polymerization ≥3. It hydrolyzed p-nitrophenyl ß-D-cellodextrins by cutting glucose or cellobiose from the non-reducing end. Meanwhile, some larger-molecular-weight cellodextrins could be detected, indicating it also had transglycosylation activity. Without carbohydrate-binding module (CBM), CHU_2103 could bind to crystalline cellulose and acted processively on it. Site-directed mutation of CHU_2103 demonstrated that the conserved aromatic amino acid W197 in the catalytic domain was essential not only for its processive activity, but also its cellulose binding ability.

[Factors affecting adhesion of Cytophaga hutchinsonii to cellulose].

OBJECTIVE: The aim of the study was to understand the mechanism of Cytophaga hutchinsonii adhension to cellulose. METHODS: The effects of different factors on the bacterial adhesion to cellulose were studied, including bacterial age, pH, temperature, cell surface charge, cell viability, cell surface protein, extracellular polysaccharides, and cellulose derivates. RESULTS: Treatments with heat and protease reduced the adhesion remarkably. But treatments with NaN3, formalin, glutaraldehyde, Congo red and NaIO4 had only slight effect on the adhesion. The adhension of Cytophaga hutchinsonii cells to microcrystalline cellulose was specific and not inhibited by cellobiose or carboxymethyl cellulose. CONCLUSION: The adhesion of Cytophaga hutchinsonii to cellulose was closely related to cell surface proteins, while cellular metabolic activity and extracellular polysaccharides had only slight effect on it. It is speculated that there might be some specific cellulose binding proteins on the cell surface.

[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.

Automated image-based method for laboratory screening of coating libraries for adhesion of algae and bacterial biofilms.

Assessment and down-selection of non-biocidal coatings that prevent the adhesion of fouling organisms in the marine environment requires a hierarchy of laboratory methods to reduce the number of experimental coatings for field testing. Automated image-based methods are described that facilitate rapid, quantitative biological screening of coatings generated through combinatorial polymer chemistry. Algorithms are described that measure the coverage of bacterial and algal biofilms on coatings prepared in 24-well plates and on array panels, respectively. The data are used to calculate adhesion strength of organisms on experimental coatings. The results complement a number of physical and mechanical methods developed to screen large numbers of samples.

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.

Identification, synthesis, and conformation of tri- and tetrathiacycloalkanes from marine bacteria.

Seven new cyclic natural polysulfides 1-7 were identified in extracts of two bacterial Cytophaga strains (CFB-phylum) isolated from biofilms from the North Sea. Their structures are based on mono- and dimeric-cyclization products of 2-methylpropane-1,2-dithiol 8, which was also present in the extract in trace amounts. The structures were deduced by analysis of their mass spectra and confirmed by synthesis. The 1H NMR spectra of some these compounds suggested a high flexibility of the trithiepane and tetrathiocane systems. Therefore, their conformation was further analyzed by DFT calculations and dynamic NMR spectroscopy. While thiepane 4 possesses a twist-chair lowest energy conformation, its isomers 2 and 3 adopt a chairlike conformation, as does the tetrathiocane 5. In contrast, tetrathiocane 6 favors again a twisted chair conformation.

Pharacine, a natural p-cyclophane and other indole derivatives from Cytophaga sp. strain AM13.1.

From the ethyl acetate extract of the bacterial strain Cytophaga sp. AM13.1, among many known compounds, the new natural products 2,5-bis(3-indolylmethyl)pyrazine (2) and a highly symmetrical p-cyclophane named pharacine (5) were identified. In addition, tryptamine isovalerate (1) and p-hydroxyphenylacetamide (4), known as plant metabolites, were isolated and characterized from a microorganism for the first time. The new natural products showed no activity against three microalgae, the fungus Mucor miehei, the yeast Candida albicans, and the bacteria Staphylococcus aureus, Bacillussubtilis, Escherichia coli, and Streptomyces viridochromogenes.

Phylogenetic analysis and taxonomic study of marine Cytophaga-like bacteria: proposal for Tenacibaculum gen. nov. with Tenacibaculum maritimum comb. nov. and Tenacibaculum ovolyticum comb. nov., and description of Tenacibaculum mesophilum sp. nov. and Tenacibaculum amylolyticum sp. nov.

Bacterial strains were isolated from sponge and green algae which were collected on the coast of Japan and Palau. The phylogenetic relationships of these isolates among marine species of the Cytophaga-Flavobacterium-Bacteroides complex were analysed by using their gyrB nucleotide sequences and translated peptide sequences (GyrB) in addition to 16S rDNA sequences. These isolates were closely related to the previously characterized marine Flexibacter species, [Flexibacter] maritimus and [Flexibacter] ovolyticus. These Flexibacter species are distantly related to Flexibacter flexilis, the type species of the genus Flexibacter, and phylogenetically belong to the family Flavobacteriaceae (according to analysis using both 16S rDNA and GyrB sequences). Their phylogenetic, chemotaxonomic and phenotypic characteristics prompted the proposal that these two species should be transferred to the new genus Tenacibaculum, as Tenacibaculum maritimum and Tenacibaculum ovolyticum, respectively. Two additional new species of the genus Tenacibaculum, Tenacibaculum mesophilum gen. nov., sp. nov. (= MBIC 1140T = IFO 16307T) and Tenacibaculum amylolyticum gen. nov., sp. nov. (= MBIC 4355T = IFO 16310T), which were isolated from sponges and macroalgae, are also reported. For taxonomic considerations at the species level, the resolution of gyrB sequences was superior to that of 16S rDNA sequences, and the grouping based on the gyrB phylogram was consistent with DNA-DNA hybridization results.

Polyamine distribution profiles in newly validated genera and species within the Flavobacterium-Flexibacter-Cytophaga-Sphingobacterium complex.

Cellular polyamines of 58 strains belonging to the Flavobacterium-Flexibacter-Cytophaga-Sphingobacterium complex were analysed by HPLC. Homospermidine was found in all species of Flavobacterium, Chryseobacterium, Empedobacter, Myroides, Cellulophaga, Salegentibacter, Psychroserpens and Gelidibacter of the family Flavobacteriaceae. Flavobacterium ferrugineum located outside of this family also contained homospermidine. Cytophaga fermentans and C. xylanolytica belonging to the family Bacteroidaceae contained spermidine. Cytophaga marinoflava and C. latercula belonging to Flavobacteriaceae contained homospermidine. The Cytophaga hutchinsonii/C. aurantiaca group contained homospermidine which was the major polyamine in Flexibacter maritimus/ F. ovolyticus of the family Flavobacteriaceae. The Flexibacter sancti/F filiformis/ Cytophaga arvensicola group, F. elegans, F. ruber, F. canadensis, F. flexilis and F. tractuosus, were located separately in different six clusters, and contained homospermidine. The Flexibacter litoralis/F. polymorphus/F. aggregans group contained spermidine, which was detected in Flexibacter roseolus belonging to a divergent cluster. Sphingobacterium and Pedobacter species of the family Sphingobacteriaceae contained homospermidine. Polyamine profiles serve, as a phenotypic chemotaxonomic marker, for the classification of this complex.

Polyamine distribution profiles in the eighteen genera phylogenetically located within the Flavobacterium-Flexibacter-Cytophaga complex.

Cellular polyamines of eighteen genera belonging to the Flavobacterium-Flexibacter-Cytophaga complex were analysed by ion exchange liquid chromatography. Homospermidine was the major polyamine in the genera Bergeyella, Riemerella, Ornithobacterium, Weeksella, Capnocytophaga, Polaribacter and Psychroflexus belonging to the family Flavobacteriaceae. In the family Spirosomaceae, Runella, Spirosoma and Flectobacillus species contained spermidine whereas Cyclobacterium species contained homospermidine. Within a divergent cluster, Haliscomenobacter and Lewinella species contained spermidine whereas Saprospira grandis contained agmatine alone. The major polyamine of Chitinophaga and Sporocytophaga species was homospermidine. Flexithrix dorotheae contained spermidine. Microscilla marina, the type species of the genus Microscilla, contained spermidine and cadaverine. However, 'Microscilla sericea' contained homospermidine, 'Microscilla furvescens' contained spermidine, and 'Microscilla arenaria' lacked all polyamines. Polyamine profiles serve as a phenotypic chemotaxonomic marker for the reclassification of the genera belonging to the complex.

[Isolation and characterization of Cytophaga lytica lipopolysaccharide]. / Vydelenie i kharakteristika lipopolisakharida Cytophaga lytica.

Lipopolysaccharide (LPS) and its structural components: lipid A, O-specific polysaccharide (O-PS) and oligosaccharide core (OG-core) have been isolated from Cytophaga lytica. 3-Oxytetradecanoic (40.8%) and dodecanoic (28.7%) are the predominant fatty acids of lipid A; pentadecanoic (6.8%), 3-oxyhexadecanoic (6.5%) as well as hexadecanoic (5.4%) acids have been found as well. The content of the rest of fatty acids is inconsiderable (2.3 to 0.5%). OG-core contained monosaccharides both typical of the most Gram-negative bacteria (glucose, mannose, galactose, rhamnose, glucosamine) and rarely occurring one-arabinose. O-PS is represented by glucose, mannose, rhamnose, glucosamine, galactosamine as well as unidentified hexosamine.

Description of Cellulophaga algicola sp. nov., isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov.

A group of strains with potent extracellular enzymic activity were isolated from the surfaces of the chain-forming sea-ice diatom Melosira and from an unidentified macrophyte collected from the Eastern Antarctic coastal zone. 16S rDNA sequence analysis indicated that the strains belonged to the genus Cellulophaga and showed greatest similarity to the species Cellulophaga baltica (sequence similarity 97%). Phenotypic characteristics, DNA base composition and DNA-DNA hybridization values clearly separate the Antarctic strains from Cellulophaga baltica and other Cellulophaga species. Thus, the strains form a distinct and novel species and have the proposed name Cellulophaga algicola sp. nov. (type strain IC166T = ACAM 630T). In addition, it was recognized that the species Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989, a species phylogenetically remote from the type species of the genus Cytophaga, possessed 16S rDNA sequences and phenotypic and chemotaxonomic traits similar to those of other Cellulophaga species. Thus, it was proposed that the species Cytophaga uliginosa be renamed as Cellulophaga uliginosa comb. nov.

N-type calcium channel blockers from a marine bacterium, Cytophaga sp. SANK 71996.

N-(3-Acyloxyacyl)glycines were isolated as N-type calcium channel blockers from a marine bacterium Cytophaga sp. SANK 71996. The identification and fermentation of the producing strain and structure characterization of N-(3-acyloxyacyl)glycines by spectral analyses and chemical syntheses are described together with their antagonistic activities.

Gliding bacterial adjuvant stimulates feline cytokines in vitro and antigen-specific IgG in vivo.

Gliding bacterial adjuvant (GBA) has been previously characterized as a potent immune modulator, stimulating the growth of murine B lymphocytes, inducing murine NK cell activity, and promoting the release of several murine cytokines. Based on these studies and our interest in potentiating the effectiveness of feline vaccines, GBA was tested for its ability to stimulate feline T cells in vitro and act as a vaccine adjuvant in vivo. In vitro, GBA stimulated feline PBLs to proliferate and release interferon (IFN) and IL-2. Unlike IFN, the release of IL-2 appeared to be unaffected by prior depletion of macrophages, indicating GBA directly stimulated feline T cells. In vivo GBA was co-administered with Keyhole Limpet Hemacyanin (KLH) and the anti-KLH antibody response was compared to cats receiving KLH emulsified in complete Freund's adjuvant (CFA). Fourteen days after the third immunization and continuing for a 30-day observation period, KLH-specific IgG titers in cats receiving GBA were significantly higher than those given CFA. However, when cats were subsequently boosted with KLH alone, those cats receiving CFA demonstrated significantly higher antibody titers throughout a second 30-day observation period. The anti-KLH antibody memory response was greatly enhanced when GBA was emulsified with incomplete Freunds adjuvant (IFA) prior to injection. Serum titers of cats given KLH in an oil-based GBA preparation were significantly higher than cats receiving KLH adjuvanted with IFA or CFA, an effect which persisted 38 days after boosting with KLH alone. Finally, GBA significantly enhanced the feline humoral response to a recombinant protein of Dirofilaria immitis, the causative agent of feline heartworm. Serum titers of cats inoculated with recombinant antigen in GBA were significantly greater than cats given recombinant antigen adjuvanted with Titermax, alum, or NAGO. These studies indicate that GBA induces T cell proliferation and the release of IL-2 and IFN in vitro and can be used to enhance the recall antibody response to both a T cell dependent antigen and an immunogen derived from Dirofilaria immitis.

Phylogenetic diversity of the genus Cytophaga revealed by 16S rRNA sequencing and menaquinone analysis.

To clarify the intra- and intergeneric relationships of the genus Cytophaga, 16S rRNA sequences and respiratory isoprenoid quinones were determined for the type strains of the 21 validly published species and one isolate in the genus Cytophaga. The sequence analysis revealed extreme heterogeneity of this genus, which diverged into nine distinct lines of descent. Each lineage of Cytophaga was characterized by possessing either menaquinone-6 (MK-6) or MK-7. The MK-6-possessing species were located in the two lineages that were remote from MK-7 species. One of the MK-6 lineages was composed only of terrestrial species and the other only of marine species. Flavobacterium aquatile, the type species of the genus Flavobacterium, was located in the MK-6 terrestrial lineage. The terrestrial Cytophaga species with MK-6 should be transferred to the genus Flavobacterium. The marine facultative anaerobes with MK-7 were located in the bacteroides branch, and possessed signature sequences with features intermediate between the bacteroides and the flavobacteria subdivisions. Cytophaga hutchinsonii, the type species of the genus Cytophaga, had a close relationship only with Cytophaga aurantiaca. The genus Cytophaga should be restricted to these two cellulose-degrading species. The genus Cytophaga is so heterogeneous that it should be divided into several genera and higher taxa in accordance with the phylogenetic relationships.

TAN-1057 A-D, new antibiotics with potent antibacterial activity against methicillin-resistant Staphylococcus aureus. Taxonomy, fermentation and biological activity.

Two Gram-negative bacteria were found to produce the new antibacterial antibiotics TAN-1057 A, B, C and D. The producing bacteria were characterized and designated as Flexibacter sp. PK-74 and PK-176. These antibiotics were active against Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. TAN-1057 A inhibited protein biosynthesis in Escherichia coli and S. aureus. It showed excellent protective effects against an experimental methicillin-resistant S. aureus infection in mice.

Polyamine distributions in the Falvobacterium-Cytophaga-Sphingobacterium complex.

Homospermidine was found as the major polyamine in one newly described species of Flavobacterium (F. indologenes), in three species of Sphingobacterium (S. mizutae, S. multivorium, and S. spiritivorum), and in 10 species of Cytophaga (C. aquatilis, C. arvensicola, C. heparina, C. hutchinsonii, C. johnsonae, "C. keratolytica," C. lytica, C. marinoflava, C. uliginosa, and "C. xantha"). These bacteria also all contain putrescine and agmatine as minor components. Flavobacterium indologenes and C. johnsonae contain an unusual diamine, 2-hydroxyputrescine, as a major polyamine. The polyamine distributions of four other species originally included in Flavobacterium (F. acidurans, "F. dormitator," "F. tirrenicum," and Halomonas halmophila), whose taxonomic positions are or were uncertain, were different from the group mentioned above. They either contain spermidine as the major polyamine or lack any polyamine. These results suggest that homospermidine can serve as a chemotaxonomic marker to delineate true members of the Flavobacterium-Cytophaga-Sphingobacterium complex.