Cytophaga hutchinsonii gldN, Encoding a Core Component of the Type IX Secretion System, Is Essential for Ion Assimilation, Cellulose Degradation, and Cell Motility.

The type IX secretion system (T9SS), which is involved in pathogenicity, motility, and utilization of complex biopolymers, is a novel protein secretion system confined to the phylum BacteroidetesCytophaga hutchinsonii, a common cellulolytic soil bacterium belonging to the phylum Bacteroidetes, can rapidly digest crystalline cellulose using a novel strategy. In this study, the deletion mutant of chu_0174 (gldN) was obtained using PY6 medium supplemented with Stanier salts. GldN was verified to be a core component of C. hutchinsonii T9SS, and is indispensable for cellulose degradation, motility, and secretion of C-terminal domain (CTD) proteins. Notably, the ΔgldN mutant showed significant growth defects in Ca2+- and Mg2+-deficient media. These growth defects could be relieved by the addition of Ca2+ or Mg2+ The intracellular concentrations of Ca2+ and Mg2+ were markedly reduced in ΔgldN These results demonstrated that GldN is essential for the acquisition of trace amounts of Ca2+ and Mg2+, especially for Ca2+ Moreover, an outer membrane efflux protein, CHU_2807, which was decreased in abundance on the outer membrane of ΔgldN, is essential for normal growth in PY6 medium. The reduced intracellular accumulation of Ca2+ and Mg2+ in the Δ2807 mutant indicated that CHU_2807 is involved in the uptake of trace amounts of Ca2+ and Mg2+ This study provides insights into the role of T9SS in metal ion assimilation in C. hutchinsoniiIMPORTANCE The widespread Gram-negative bacterium Cytophaga hutchinsonii uses a novel but poorly understood strategy to utilize crystalline cellulose. Recent studies showed that a T9SS exists in C. hutchinsonii and is involved in cellulose degradation and motility. However, the main components of the C. hutchinsonii T9SS and their functions are still unclear. Our study characterized the function of GldN, which is a core component of the T9SS. GldN was proved to play vital roles in cellulose degradation and cell motility. Notably, GldN is essential for the acquisition of Ca2+ and Mg2+ ions under Ca2+- and Mg2+-deficient conditions, revealing a link between the T9SS and the metal ion transport system. The outer membrane abundance of CHU_2807, which is essential for Ca2+ and Mg2+ uptake in PY6 medium, was affected by the deletion of GldN. This study demonstrated that the C. hutchinsonii T9SS has extensive functions, including cellulose degradation, motility, and metal ion assimilation, and contributes to further understanding of the function of the T9SS in the phylum Bacteroidetes.

A new locus in Cytophaga hutchinsonii involved in colony spreading on agar surfaces and individual cell gliding.

Cytophaga hutchinsonii glides rapidly over surfaces by an unknown mechanism without flagella and type IV pili and it can degrade crystalline cellulose efficiently by a novel mechanism. Tn4351 transposon mutagenesis was used to identify a new gene, CHU_1798, essential for colony spreading on agar surfaces. Further study showed that disruption of CHU_1798 caused non-spreading colonies on both soft and hard agar surfaces and individual cells were partially deficient in gliding on glass surfaces. The CHU_1798 mutant could digest cellulose as long as the cells were in direct contact with the cellulose, but it could not degrade cellulose powder buried in the agar plate. Scanning electron microscopy showed that individual mutant cells arranged irregularly on the cellulose fiber surface at an early stage of incubation, but later showed a regular parallel arrangement when there were plenty of cells and could spread along the cellulose fibers. These results suggest that CHU_1798 plays an important role in the motility of C. hutchinsonii and provide insight into the relation between cell motility and cellulose degradation.

Unraveling adaptation of Pontibacter korlensis to radiation and infertility in desert through complete genome and comparative transcriptomic analysis.

The desert is a harsh habitat for flora and microbial life due to its aridness and strong radiation. In this study, we constructed the first complete and deeply annotated genome of the genus Pontibacter (Pontibacter korlensis X14-1(T) = CCTCC AB 206081(T), X14-1). Reconstruction of the sugar metabolism process indicated that strain X14-1 can utilize diverse sugars, including cellulose, starch and sucrose; this result is consistent with previous experiments. Strain X14-1 is also able to resist desiccation and radiation in the desert through well-armed systems related to DNA repair, radical oxygen species (ROS) detoxification and the OstAB and TreYZ pathways for trehalose synthesis. A comparative transcriptomic analysis under gamma radiation revealed that strain X14-1 presents high-efficacy operating responses to radiation, including the robust expression of catalase and the manganese transport protein. Evaluation of 73 novel genes that are differentially expressed showed that some of these genes may contribute to the strain's adaptation to radiation and desiccation through ferric transport and preservation.

Evidence for Autoinduction and Quorum Sensing in White Band Disease-Causing Microbes on Acropora cervicornis.

Coral reefs have entered a state of global decline party due to an increasing incidence of coral disease. However, the diversity and complexity of coral-associated bacterial communities has made identifying the mechanisms underlying disease transmission and progression extremely difficult. This study explores the effects of coral cell-free culture fluid (CFCF) and autoinducer (a quorum sensing signaling molecule) on coral-associated bacterial growth and on coral tissue loss respectively. All experiments were conducted using the endangered Caribbean coral Acropora cervicornis. Coral-associated microbes were grown on selective media infused with CFCF derived from healthy and white band disease-infected A. cervicornis. Exposure to diseased CFCF increased proliferation of Cytophaga-Flavobacterium spp. while exposure to healthy CFCF inhibited growth of this group. Exposure to either CFCF did not significantly affect Vibrio spp. growth. In order to test whether disease symptoms can be induced in healthy corals, A. cervicornis was exposed to bacterial assemblages supplemented with exogenous, purified autoinducer. Incubation with autoinducer resulted in complete tissue loss in all corals tested in less than one week. These findings indicate that white band disease in A. cervicornis may be caused by opportunistic pathogenesis of resident microbes.

FLP-FRT-based method to obtain unmarked deletions of CHU_3237 (porU) and large genomic fragments of Cytophaga hutchinsonii.

Cytophaga hutchinsonii is a widely distributed cellulolytic bacterium in the phylum Bacteroidetes. It can digest crystalline cellulose rapidly without free cellulases or cellulosomes. The mechanism of its cellulose utilization remains a mystery. We developed an efficient method based on a linear DNA double-crossover and FLP-FRT recombination system to obtain unmarked deletions of both single genes and large genomic fragments in C. hutchinsonii. Unmarked deletion of CHU_3237 (porU), an ortholog of the C-terminal signal peptidase of a type IX secretion system (T9SS), resulted in defects in colony spreading, cellulose degradation, and protein secretion, indicating that it is a component of the T9SS and that T9SS plays an important role in cellulose degradation by C. hutchinsonii. Furthermore, deletions of four large genomic fragments were obtained using our method, and the sizes of the excised fragments varied from 9 to 19 kb, spanning from 6 to 22 genes. The customized FLP-FRT method provides an efficient tool for more rapid progress in the cellulose degradation mechanism and other physiological aspects of C. hutchinsonii.

Deletion of the Cytophaga hutchinsonii type IX secretion system gene sprP results in defects in gliding motility and cellulose utilization.

Cytophaga hutchinsonii glides rapidly over surfaces and employs a novel collection of cell-associated proteins to digest crystalline cellulose. HimarEm1 transposon mutagenesis was used to isolate a mutant with an insertion in CHU_0170 (sprP) that was partially deficient in gliding motility and was unable to digest filter paper cellulose. SprP is similar in sequence to the Porphyromonas gingivalis type IX secretion system (T9SS) protein PorP that is involved in the secretion of gingipain protease virulence factors and to the Flavobacterium johnsoniae T9SS protein SprF that is needed to deliver components of the gliding motility machinery to the cell surface. We developed an efficient method to construct targeted nonpolar mutations in C. hutchinsonii and deleted sprP. The deletion mutant was defective in gliding and failed to digest cellulose, and complementation with sprP on a plasmid restored both abilities. Sequence analysis predicted that CHU_3105 is secreted by the T9SS, and deletion of sprP resulted in decreased levels of extracellular CHU_3105. The results suggest that SprP may function in protein secretion. The T9SS may be required for motility and cellulose utilization because cell surface proteins predicted to be involved in both processes have C-terminal domains that are thought to target them to this secretion system. The efficient genetic tools now available for C. hutchinsonii should allow a detailed analysis of the cellulolytic, gliding motility, and protein secretion machineries of this common but poorly understood bacterium.

A novel locus essential for spreading of Cytophaga hutchinsonii colonies on agar.

Cytophaga hutchinsonii is an aerobic cellulolytic gliding bacterium. The mechanism of its cell motility over surfaces without flagella and type IV pili is not known. In this study, mariner-based transposon mutagenesis was used to identify a new locus CHU_1797 essential for colony spreading on both hard and soft agar surfaces through gliding. CHU_1797 encodes a putative outer membrane protein of 348 amino acids with unknown function, and proteins which have high sequence similarity to CHU_1797 were widespread in the members of the phylum Bacteroidetes. The disruption of CHU_1797 suppressed spreading toward glucose on an agar surface, but had no significant effect on cellulose degradation for cells already in contact with cellulose. SEM observation showed that the mutant cells also regularly arranged on the surface of cellulose fiber similar with that of the wild type strain. These results indicated that the colony spreading ability on agar surfaces was not required for cellulose degradation by C. hutchinsonii. This was the first study focused on the relationship between cell motility and cellulose degradation of C. hutchinsonii.

[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.

A new locus affects cell motility, cellulose binding, and degradation by Cytophaga hutchinsonii.

Cytophaga hutchinsonii is a Gram-negative gliding bacterium, which can rapidly degrade crystalline cellulose via a novel strategy without any recognizable processive cellulases. Its mechanism of cellulose binding and degradation is still a mystery. In this study, the mutagenesis of C. hutchinsonii with the mariner-based transposon HimarEm3 and gene complementation with the oriC-based plasmid carrying the antibiotic resistance gene cfxA or tetQ were reported for the first time to provide valuable tools for mutagenesis and genetic manipulation of the bacterium. Mutant A-4 with a transposon mutation in gene CHU_0134, which encodes a putative thiol-disulfide isomerase exhibits defects in cell motility and cellulose degradation. The cellulose binding ability of A-4 was only half of that of the wild-type strain, while the endo-cellulase activity of the cell-free supernatants and on the intact cell surface of A-4 decreased by 40%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins binding to cellulose in the outer membrane showed that most of them were significantly decreased or disappeared in A-4 including some Gld proteins and hypothetical proteins, indicating that these proteins might play an important role in cell motility and cellulose binding and degradation by the bacterium.

A meta-analysis of 16S rRNA gene clone libraries from the polymicrobial black band disease of corals.

Black band disease (BBD) is a polymicrobial disease affecting corals on reefs worldwide. Since 2002, researchers have constructed clone libraries from the BBD consortium using 16S rRNA gene primers targeting a variety of phyla. In the present study, a meta-analysis was conducted of published libraries from 2002 through the present that contain bacterial sequence data associated with individual clones and BBD samples. The libraries analyzed were from 87 BBD samples, collected from 16 species of scleractinian corals in 10 different geographic locations, and included 327 unique operational taxonomic units (OTUs). One OTU (cyanobacterial) was present in 62 (71%) samples, and three (one Cytophaga-Flavobacter-Bacteriodetes and two alphaproteobacterial) were present in 11 (13%) of the samples. The remaining 323 unique OTUs were present in <10% of the samples. The Alphaproteobacteria was the most diversely represented group. Analysis of clone libraries using nonmetric multidimensional scaling indicated strong regional specificity of BBD microbial populations, but limited host coral specificity. The results of this analysis support the hypotheses that: (1) a specific cyanobacterium may be the primary pathogen of BBD; (2) additional functional groups, required for BBD pathobiology, are represented by variable opportunistic species; and (3) opportunistic BBD microorganisms are primarily derived from the environment.

Characterization of the bacterial community associated with body wall lesions of Tripneustes gratilla (Echinoidea) using culture-independent methods.

The bacterial community associated with skin lesions of the sea urchin Tripneustes gratilla was investigated using 16S ribosomal RNA gene cloning and fluorescent in situ hybridization (FISH). All clones were classified in the Alphaproteobacteria, Gammaproteobacteria and Cytophaga-Flexibacter-Bacteroides (CFB) bacteria. Most of the Alphaproteobacteria were related to the Roseobacter lineage and to bacteria implicated in marine diseases. The majority of the Gammaproteobacteria were identified as Vibrio while CFB represented only 9% of the total clones. FISH analyses showed that Alphaproteobacteria, CFB bacteria and Gammaproteobacteria accounted respectively for 43%, 38% and 19% of the DAPI counts. The importance of the methods used is emphasized.

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.

Quantitative analysis of biofilm EPS uronic acid content.

The uronic acids assay was evaluated for its ability to measure the amount of uronic acids contained within a biofilm exopolysaccharide matrix. Cytophaga lytica, a marine bacterium isolated from a naturally occurring biofilm, was used to form single-species biofilms for the method assessment. The assay was found to be simple, reproducible, and sensitive to 1 microg levels, suggesting its potential for application as a screening technique for compounds that inhibit the production of microbial biofilm exopolysaccharide containing uronic acids.

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.

In vitro cultivation and antibiotic susceptibility of a Cytophaga-like intracellular symbiote isolated from the tick Ixodes scapularis.

A Cytophaga-like organism (CLO), isolated from the tick Ixodes scapularis (IsCLO), was adapted to growth in insect cell lines and its antibiotic sensitivity was tested. IsCLO were introduced to four insect cell lines, and their growth was measured by quantitative polymerase chain reaction. IsCLO propagated well in a mosquito cell line, AeAl-2, and caused cytopathic effects in host cells. A lepidopteran cell line, HZ-AM1, was also suitable for propagation of IsCLO and kept a steady state with bacterial growth. Using IsCLO-infected AeAl-2, antibiotics effective against the bacteria included ampicillin, chloramphenicol, penicillin-G, rifampicin, and tetracycline. These antibiotics will be useful for eliminating CLO from host arthropods, which is necessary for in vivo studies of the intracellular facultative symbiotes.

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.

The microbial community analysis of a 5-stage BNR process with step feed system.

The microbial communities of 5-stage BNR activated sludge samples were analyzed using fluorescence in-situ hybridization (FISH) and 16S rDNA characterization. The total cell numbers of each reactor were from 2.36 x 10(9) cells/ml to 2.83 x 10(9) cells/ml. From 56.5% to 62.0% of total DAPI cell counts were hybridized to the most bacterial specific probe EUB 338. Among them, beta-proteobacteria were most dominant in each tank. The number of phosphate accumulating organisms (PAOs) was almost 50% of the total cell number in anoxic-1 tank, and these results indicate that this process has a high content of denitrifying phosphorus accumulating organisms (dPAOs). In contrast with FISH, 16S rDNA analysis showed that dominant groups were the Cytophaga-Flavobacterium group and high G+C% gram-positive bacteria, which were determined as PAOs in anoxic-1 tank. The beta subclass Proteobacteria did not accumulate a large amount of polyphosphate. The overall results indicate that high G+C% gram-positive bacteria and the Cytophaga-Flavobacterium group might play a key role as dPAOs in this process.

Incidence of a new sex-ratio-distorting endosymbiotic bacterium among arthropods.

Many intracellular micro-organisms are now known to cause reproductive abnormalities and other phenomena in their hosts. The endosymbiont Wolbachia is the best known of these reproductive manipulators owing to its extremely high incidence among arthropods and the diverse host effects it has been implicated as causing. However, recent evidence suggests that another intracellular bacterium, a Cytophaga-like organism (CLO), may also induce several reproductive effects in its hosts. Here, we present the first survey of arthropod hosts for infection by the CLO. We use a sensitive hemi-nested polymerase chain reaction method to screen 223 species from 20 arthropod orders for infection by the CLO and Wolbachia. The results indicate that, although not as prevalent as Wolbachia, the CLO infects a significant number of arthropod hosts (ca. 7.2%). In addition, double infections of the CLO and Wolbachia were found in individuals of seven arthropod species. Sequencing analysis of the 16S rDNA region of the CLO indicates evidence for horizontal transmission of the CLO strains. We discuss these results with reference to future studies on host effects induced by intracellular micro-organisms.

Extra- and intra-cellular lytic effects of Cytophaga sp. LR2 on the red microalgae Rhodella reticulata.

AIMS: To evaluate the lytic activities of crude enzymes from Cytophaga sp. LR2 on Rhodella reticulata cells and isolated algal polysaccharide. METHODS AND RESULTS: The Cytophaga compartment was separated after centrifugation in a cell suspension for 30 min at 18,000 g. The extracellular enzyme was obtained from the supernatant and the intracellular from the pelleted cells after sonication and removal of debris. Algal cells were incubated with extra- or intracellular preparations and sowed onto agar medium. The suppressive effect of the extracellular enzyme on colony-forming units was found to be almost twice as high. The result was still more pronounced when treated cells had been shocked osmotically before seeding. Saccharolytic activity was evaluated by changes in the reducing sugars in the media. Concerning isolated algal polysaccharide, the reducing power of the two bacterial preparates was relatively low. A combined fraction showed the highest lytic activity. Using native and SDS electrophoresis some relation between the prevalence of the extra and intracellular protein patterns was registered. Two of the common components' molecular weight masses of 50 and 21 kDa were found to be reproducible in native- and SDS-containing gel. CONCLUSIONS: Cytophaga sp. LR2 produce extra- and intracellular enzymes active in destroying Rhodella cultures. The agents excreted in the medium are more effective.We suppose that two or three different classes of enzymes are involved in the lysis process. The comparative electrophoresis in this case shows the protein components with predictable functions. SIGNIFICANCE AND IMPACT OF THE STUDY: Combining different simple and reproducible approaches to identify the lytic capability of Cytophaga sp. LR2 on R. reticulata.

Contribution of cytophaga-like bacteria to the potential of turnover of carbon, nitrogen, and phosphorus by bacteria in the rhizosphere of barley (Hordeum vulgare L.).

The functional potential of bacteria isolated from the rhizosphere of barley (Hordeum vulgare L.) in May, July, and August and cultivated on nutrient-rich substrate (1/10 TSBA) and nutrient-poor substrate (cold soil extract agar) was determined. There was no significant difference in numbers of CFU when counted on nutrient rich or poor substrate. Bacterial numbers increased approximately 3-fold in the rhizosphere soil from May to August but was unchanged in bulk soil over the same period. A total of 4474 randomly isolated bacteria were screened for enzymatic activities involved in carbon turnover (amylase, cellulase, mannanase, xylanase, and chitinase), nitrogen turnover (protease, nitrate and nitrite reductase), and phosphate turnover (phosphatase). In the rhizosphere soil, bacteria carrying C and P turnover enzymes were not stimulated by the growing plant whereas protease and nitrate and nitrite reductase were stimulated by the growing plant. No changes were observed in the bulk soil. Two taxonomic groups were followed: Cytophaga-like bacteria (CLB) and fluorescent pseudomonads, the latter being abundant in the rhizosphere and important contributors to the cycling of organic matter in soil. Unexpectedly in the spring samples, CLB were around 25% of all bacteria isolated, whereas fluorescent pseudomonads made up less than 10%. The relative proportion of these bacterial groups then decreased during the plant growth season but at all times showing a clear rhizosphere effect. Furthermore, up to 70% of the isolates carrying enzymes involved in the turnover of carbon, in the May sample, were identified as CLB, indicating the importance of this group in early colonization of the rhizosphere. The fluorescent pseudomonad group contributed less than 3%.