Ecology and taxonomy of chitinoclasticCytophaga and related chitin-degrading bacteria isolated from an estuary

A total of 103 strains of estuarine, Chitinoclastic bacteria isolated from water, and sediment samples collected from the upper Chesapeake Bay, including 17 freshwater and 11 seawater isolates, were subjected to numerical taxonomy analysis. The isolates included 44 yellow-orange pigmented strains classified asCytophaga-like bacteria (CLB) of theCytophagaceae. Salt requirement of the strains ranged from tolerance to 1% NaCl to an absolute requirement for NaCl, with 1% NaCl satisfying this requirement. The largest phenon consisted of facultatively anaerobic, oligo-nitrophilic, and flexirubin pigment-producing freshwater and estuarine isolates, and included reference strains of bothCytophaga johnsonae Stanier andCytophaga aquatilis Strohl and Tait. Other phena, containing a smaller number of strains, comprised marine and estuarine isolates which did not produce flexirubin pigments, and required organic nitrogen for growth and for production of chitinolytic enzymes. Salt-requiring, flexirubin pigment-producing, chitin-degrading strains were, on occasion, isolated from estuarine samples and represented phena found in estuaries. Most of theCytophaga isolates, as well as chitin-degrading species not of the genusCytophaga that were isolated from Chesapeake Bay, clustered in phena representing previously described species of aerobic, zymogenic, chitinoclastic bacteria. When the frequency of occurrence of features related to environmental parameters, viz., pH, salinity, temperature range of growth, and growth on media lacking organic nitrogen, was calculated, ecological groupings of strains in the 2 major phena of CLB could be distinguished among the estuarine, chitin-degrading bacteria.     

Growth and Thallus Morphogenesis of Ulva mutabilis (Chlorophyta) Depends on A Combination of Two Bacterial Species Excreting Regulatory Factors

Axenic Ulva mutabilis gametes develop parthenogenetically into callus‐like colonies consisting of undifferentiated cells without normal cell walls. From the accompanying microbial flora of established laboratory strains of U. mutabilis with normal morphology, a Roseobacter, a Sulfitobacter, and a Halomonas species were isolated. Each of these microbe species alone induced the development of the Ulva gametes into thalli composed of differentiated cells with characteristic deficiencies. Typical traits of these thalli were: an enhanced rate of cell division not followed by cell expansion, the presence of unusual cell wall protrusions, and the absence of differentiated rhizoid cells. The addition of a Cytophaga species, also derived from the same microbial flora, to either one of the three other strains resulted in the development of normal fast growing thalli with the typical morphology of the algal strain used. These effects are mediated by specific regulatory factors that are excreted into the environment by the bacteria and could be also isolated from the bacterial cell extracts. In contrast with the Cytophaga‐factor, the regulatory factor of the three other bacterial species was also found intracellularly in other bacterial strains not associated with Ulva, but in this case it was not excreted. Functionally, the Roseobacter‐, Sulfitobacter‐, and Halomonas‐factors resemble a cytokinin, while the Cytophaga‐factor acts similar to auxin. Neither factor could be replaced by known phytohormones. The Roseobacter species exhibits a specific chemotactic affinity to the rhizoid cells of U. mutabilis and seems to cooperate with the Cytophaga strain and the alga by chemical communication forming a symbiotic tripartite community.     

Sequence and Expression Analyses of Cytophaga-Like Hydrolases in a Western Arctic Metagenomic Library and the Sargasso Sea

Sequence analysis of environmental DNA promises to provide new insights into the ecology and biogeochemistry of uncultured marine microbes. In this study we used the Sargasso Sea Whole Genome Sequence (WGS) data set to search for hydrolases used by Cytophaga-like bacteria to degrade biopolymers such as polysaccharides and proteins. Analysis of the Sargasso WGS data for contigs bearing both the 16S rRNA genes of Cytophaga-like bacteria and hydrolase genes revealed a cellulase gene (celM) most similar to the gene found in Cytophaga hutchinsonii. A BLAST search of the entire Sargasso Sea WGS data set indicated that celM was the most abundant cellulase-like gene in the Sargasso Sea. However, the similarity between CelM-like cellulases and peptidases belonging to metalloprotease family M42 led us to question whether CelM is involved in the degradation of polysaccharides or proteins. PCR primers were designed for the celM genes in the Sargasso Sea WGS data set and used to identify celM in a fosmid library constructed with prokaryotic DNA from the western Arctic Ocean. Expression analysis of the Cytophaga-like Arctic CelM, which is 63% identical and 77% similar to CelM in C. hutchinsonii, indicated that there was peptidase activity, whereas cellulase activity was not detected. Our analysis suggests that the celM gene plays a role in the degradation of protein by Cytophaga-like bacteria. The abundance of peptidase genes in the Cytophaga-like fosmid clone provides further evidence for the importance of Cytophaga-like bacteria in the degradation of protein in high-molecular-weight dissolved organic matter.     

Grazing by protozoa as selection factor for activated sludge bacteria

In continuous culture enrichments that were inoculated with activated sludge and were fed with polymeric substrates, freely dispersed single-celled bacteria belonging to theCytophaga group dominated among the initial populations, irrespective of the activated sludge source. These populations were grazed by flagellated protozoa which after several days reached high cell densities. Other morphologic bacterial groups such as spiral-shaped or filamentous bacteria then became dominant. In defined mixed culture experiments with bacterial isolates from the enrichment cultures, it was shown that a grazing-resistantMicrocyclus strain outgrew aCytophaga strain in the presence of grazing protozoa. In contrast, theCytophaga strain competed successfully with theMicrocyclus strain and with other grazing-resistant strains under protozoa-free conditions. Furthermore, it was demonstrated that assumed grazing resistance factors such as floccing or filamentous growth were lost by some of the strains when they were grown for several generations in continuous culture under the same conditions, but in the absence of protozoa.     

Saprospira species—Natural predators

SomeSaprospira spp. from fresh waters have been shown to be predatory on other aquatic bacteria, particularly gliding bacteria. Some can be grown as well axenically but others so far have been cultured only on the host bacteria for which they show a degree of specificity. A marine strain resemblingSaprospira grandis, although capable of growth axenically, is also a predator on a marineCytophaga sp.S. grandis ATCC 23119 failed to grow on the base media or media overgrown with host cells of otherSaprospira spp.     

Deoxyribonucleic acid relatedness of some menaquinone-producing Flavobacterium and Cytophaga strains

Nine menaquinone-forming strains of the Flavobacterium-Cytophaga complex with DNA base compositions between 35 and 45 moles percent guanineplus-cytosine were investigated for genome sizes and DNA relatedness by DNA: DNA hybridization in vitro, using the optically recorded initial reassociation kinetics. Two strains representing C. hutchinsonii and C. marinoflava proved to be related on the 50 percent binding level, i.e. on a level of DNA relatedness commonly found within well-classified conventional genera of bacteria. Strains of C. johnsonae, F. heparinum, F. meningosepticum, F. odoratum, F. pectinovorum, and an unnamed Flavobacterium-Cytophaga strain were found to be interrelated, and linked to the genus Cytophaga, on the 30, or 20 percent binding levels, respectively. These findings indicate that the organisms in question are related to Cytophaga. They therefore should be transferred into the family Cytophagaceae.     

Resistance of the fish-killing dinoflagellate Cochlodinium polykrikoides against algicidal bacteria isolated from the coastal sea of Japan

Noxious red tides of the dinoflagellate Cochlodinium polykrikoides tend to be long lasting and cause mass mortalities of cultured and natural fish and invertebrates along the western coast of Japan and the southern coast of Korea. In order to assess the tolerance of C. polykrikoides to attack by algicidal bacteria, the effects of algicidal bacteria strains on the growth of three C. polykrikoides strains were examined in laboratory culture experiments. Algicidal bacteria used were two strains of Cytophaga (J18/M01 and AA8-2, direct attack type and wide prey range), three strains of Alteromonas (S, K, D) and one strain of Pseudoalteromonas (R, indirect attack type), which were all isolated by using Chattonella antiqua as a prey organism. Neither Cytophaga strain showed any algicidal activity. In the cases of Alteromonas and Pseudoalteromonas, some cultures of C. polykrikoides were killed, but at least 10 days or more were required for the death of this dinoflagellate. C. polykrikoides survived in the presence of algicidal bacteria in concentrations up to 10⁶–10⁷ cells ml⁻¹, which is enough for other red tide microalgae to be killed. On the contrary, the algicidal effects of bacteria on C. antiqua were detected clearly within a few days. These results imply that C. polykrikoides is resistant to the six algicidal bacteria examined, which may reflect the capacity for mixotrophy. This resistance of C. polykrikoides to algicidal bacteria could provide a selective advantage for survival compared to other microalgae susceptible to attack by algicidal bacteria and hence prolong red tides caused by this harmful dinoflagellate.     

Occurrence of Cytophagas in Sewage Plants

With the application of plate count methods and of the KOH-flexirubin test, bacteria belonging to the Cytophaga group were proved to occur regularly in samples from biological sewage treatment facilities. Generally, the percentage of Cytophaga colonies of the total heterotrophic colonies was lowest in the inflow sewage water as compared with the values found in activated sludge, trickling filter, and effluent samples. During an observation period of 16 months, the highest percentages of cytophagas were found in winter samples from activated sludge and trickling filters. Furthermore, cytophagas were shown to have high percentages of the bacteria lytic to polymeric substrates such as cellulose, chitin, dextran, pectin, xylan, and gelatin. Thus, it is suggested that cytophagas may contribute to sewage purification, especially at cold temperatures and by polymer breakdown. Cytophaga strains isolated were shown to have gliding motility, flexirubin pigmentation, and a low guanine plus cytosine base ratio in common. The strains were roughly subdivided into a spreading, a nonspreading, and a cellulolytic group.     

Characterisation and differentiation of oligotrophic waters by culturable particle-attached and free-living bacterial communities

This study reports the development of a tool to characterise and differentiate northern Adriatic waters, particularly oligotrophic, high-salinity waters, based on the cellular fatty acids of culturable heterotrophic bacterioplankton. The growth abilities and population dominance were observed for particle-attached and free-living bacteria cultured in three types of media: Marine Broth, diluted Marine Broth (1:10) and R2 broth. Three groups of water layers were distinguished by hierarchical clustering analysis: eutrophic, oligotrophic and oligotrophic nutrient-selected. Significant differences between the resulting groups were tested by two-way ANOVA (with replication). Eutrophic layers were characterised by readily culturable particle-attached and free-living fractions of the bacterial community in all three media, all dominated by fast-growing γ-Proteobacteria. In contrast, oligotrophic water layers with low productivity had a much weaker culturability and a different population dominance for the free-living community, as compared to their attached or growth-arrested counterparts, for all media. The free-living bacteria from strictly oligotrophic environments demonstrated minimum culturability in Marine Broth, while those from selective oligotrophic environments were culturable and were dominated either by Cytophaga–Flavobacter complex, α-Proteobacteria or γ-Proteobacteria. The conclusive evidence regarding the selective and refractory nature of organic compounds in these waters demonstrates the dominant culturability of the Cytophaga–Flavobacter complex and α-Proteobacteria in free-living communities in all growth-media. The response of fatty acid dominance ratios depends significantly on the trophic state and fraction (p < 0.05), although the effect of the trophic state is completely different in attached and free fractions. Both fractions were tested separately, demonstrating a significant influence of the trophic state (p < 0.05), while the effect of the media on the fatty acid response was not significant (p > 0.05). An interaction between media and trophic status was present in the attached fraction (p < 0.05), yet this was not observed in the free fraction (p > 0.05), indicating that any systematic difference between trophic states was the same for each media tested. Accordingly, the free-living fraction of bacterioplankton is a more informative attribute and can be used solely as an indicator of the water layer trophic condition.     

Isolation of Hydrocarbon-Oxidizing Psychroactive Bacteria from Oil-Polluted Soils

Microorganisms growing on a mineral medium with crude oil and its light fractions as the only source of carbon and energy have been isolated from samples of oil-polluted soils collected in the Usa district (Komi Republic, Russia). Hydrocarbon-oxidizing psychroactive bacteria of the genus Cytophaga, which are clearly capable of consuming crude oil hydrocarbons, have been identified for the first time. A method for cultivating microorganisms on porous plastic is proposed.     

Alkaliflexus imshenetskii gen. nov. sp. nov., a new alkaliphilic gliding carbohydrate-fermenting bacterium with propionate formation from a soda lake

Anaerobic saccharolytic bacteria thriving at high pH values were studied in a cellulose-degrading enrichment culture originating from the alkaline lake, Verkhneye Beloye (Central Asia). In situ hybridization of the enrichment culture with 16S rRNA-targeted probes revealed that abundant, long, thin, rod-shaped cells were related to Cytophaga. Bacteria of this type were isolated with cellobiose and five isolates were characterized. Isolates were thin, flexible, gliding rods. They formed a spherical cyst-like structure at one cell end during the late growth phase. The pH range for growth was 7.5–10.2, with an optimum around pH 8.5. Cultures produced a pinkish pigment tentatively identified as a carotenoid. Isolates did not degrade cellulose, indicating that they utilized soluble products formed by so far uncultured hydrolytic cellulose degraders. Besides cellobiose, the isolates utilized other carbohydrates, including xylose, maltose, xylan, starch, and pectin. The main organic fermentation products were propionate, acetate, and succinate. Oxygen, which was not used as electron acceptor, impaired growth. A representative isolate, strain Z-7010, with Marinilabilia salmonicolor as the closest relative, is described as a new genus and species, Alkaliflexus imshenetskii. This is the first cultivated alkaliphilic anaerobic member of the Cytophaga/Flavobacterium/Bacteroides phylum.Dedicated to Prof. Dr. Hans Günter Schlegel on the occasion of his 80th birthday.     

Identification of three proteins up-regulated by raw starch in Cytophaga sp.

Raw starch-digesting amylases (RSDAs) in many microorganisms convert starch granules into maltodextrins and simple sugars. We cloned and sequenced from Cytophaga sp. an RSDA with an excellent raw starch digestion activity. This RSDA was highly inducible by raw starch, but not by other sugars, suggesting that an unknown signal transduction mechanism is involved in the degradation of raw starch. We used a proteomic approach to investigate the effect of raw starch on protein expression in Cytophaga sp. Using MALDI–TOF MS protein analysis, we have identified three proteins up-regulated by raw starch, i.e., a 60-kDa chaperonin (cpn60), glutaminase, and pyruvate phosphate dikinase (PPDK). Subsequent time-course studies detected an increased expression of RSDA as well as the highest expression of PPDK occurring 6 h post-incubation with raw corn starch, implying that the latter enzyme may work along with RSDA on the digestion of raw starch. Finding these proteins up-regulated by raw starch may provide an insight into how Cytophaga sp. cells respond to raw starch stimulation.     

The lysis of Gram-negative Alcaligenes eutrophus by enzymes from Cytophaga

Summary The use of Cytophaga lysing enzymes was investigated for the liberation of poly--hydroxybutyrate (PHB) granules from the Gram-negative bacterium Alcaligenes eutrophus. Complete cell lysis was approached within a 60 minute period. Contrary to previous findings for the lysis of Gram-negative bacteria, prior removal of the outer membrane was not essential for enzymic lysis. The destabilisation of the outer membrane by the removal of divalent cations resulted in no significant improvement in the disruption process.     

Screening of marine bacteria for fucoidanases

Twenty-five strains of epiphytic marine bacteria isolated from the brown algaeFucus evanescens andChorda filum and fifty-three bacteria isolated from the sea cucumberApostichopus japonicus were screened for fucoidanases using fucoidans prepared from the brown algaeF. evanescens, Laminaria cichorioides, andL japonica. Eighteen bacterial epiphytes and thirty-eight bacterial isolates from the sea cucumber were found to contain fucoidanases, which were able to hydrolyze either all of the fucoidans studied or some of them. Bacteria of the generaCytophaga andAlteromonas/Pseudoalteromonas exhibited the highest fucoidanase activities, which, however, did not exceed the activity of fucoidanases from the already known sources.     

Genetic and functional diversities of bacterial communities in the rhizosphere of <Emphasis Type="Italic">Arachis hypogaea</Emphasis>

Bioinoculants are environmentally friendly, energy efficient and economically viable resources in sustainable agriculture. Knowledge of the structure and activities of microbial population in the rhizosphere of a plant is essential to formulate an effective bioinoculant. In this study, the bacterial community present in the rhizosphere of an important oilseed legume, Arachis hypogaea (L.) was described with respect to adjoining bulk soil as a baseline control using a 16S rDNA based metagenomic approach. Significantly higher abundance of Gamma-proteobacteria, a prevalence of Bacillus and the Cytophaga-Flavobacteria group of Bacteroidetes and absence of the Rhizobiaceae family of Alpha-proteobacteria were the major features observed in the matured Arachis-rhizosphere. The functional characterization of the rhizosphere-competent bacteria was performed using culture-dependent determination of phenotypes. Most bacterial isolates from the groundnut-rhizosphere exhibited multiple biochemical activities associated with plant growth and disease control. Validation of the beneficial traits in candidate bioinoculants in pot-cultures and field trials is necessary before their targeted application in the groundnut production system.     

High-diversity biofilm for the oxidation of sulfide-containing effluents

In the present work, we describe for the first time the utilization of a complex microbial biofilm for the treatment of sulfide-containing effluents. A non-aerated packed-column reactor was inoculated with anoxic lake sediment and exposed to light. A biofilm developed in the column and showed a stable oxidation performance for several weeks. Microbial species composition was analyzed by microscopy, pigment analysis and a bacterial 16S rRNA gene clone library. Colorless sulfur bacteria, green algae and purple sulfur bacteria were observed microscopically. Pigment composition confirmed the presence of algae and purple sulfur bacteria. The clone library was dominated by alpha-Proteobacteria (mostly Rhodobacter group), followed by gamma-Proteobacteria (Chromatiaceae-like and Thiothrix-like aerobic sulfur oxidizers) and the Cytophaga-Flavobacterium-Bacteroides group. Plastid signatures from algae were also present and a few clones belonged to both the beta- (Rhodoferax sp., Thiobacillus sp.) and delta-Proteobacteria (Desulfocapsa sp.) and to the low G+C Gram-positive bacteria (Firmicutes group). The coexistence of aerobic, anaerobic, phototrophic and chemotrophic microorganisms in the biofilm, the species richness found within these metabolic groups (42 operational taxonomic units) and the microdiversity observed within some species could be very important for the long-term functioning and versatility of the reactor.     

The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosis

In an organism (strain C 1/2 from Dr. P. R. Hayes, Leeds) regarded as a typical representative of the genus Flavobacterium, flexirubin-type pigments have been identified. The Flavobacterium pigments contain structural elements of both, the pigments of the genus Flexibacter and the pigments of the genus Cytophaga. As flexirubin-type pigments seem to have a rather restricted distribution among bacteria, and have formerly proved to be useful chemosystematic markers for the flexibacteria, this new observation may indicate that there is a relatively close phylogenetic relationship between this type of flavobacteria and the Cytophaga-Flexibacter group.     

Studien zur Bewegungsmechanik der Bakterien und Spirochäten des Hochmoores

Zusammenfassung Die Bewegungen der im Schlamm der Hochmoore lebenden Bakterien wurden mit stärkster Vergrößerung im Dunkelfeld und zum Teil im Tuschepräparat untersucht. Detailierte Beobachtungen waren möglich über die Geißelbewegungen von Stäbchenbakterien, die Gleitbewegungen vonSaprospira, Cristispira, Spirulina, Cytophaga, Chlamydobakterien und die verschiedenen Bewegungsweisen einiger Spirochäten.Mit Hilfe von Modellversuchen wird gezeigt, da\ die meisten Bewegungsphänomene nicht durch Fibrillenkontraktionen, sondern durch einfache Schraubenrotationen erklärbar sind. Lage und Verhalten der rotierenden Schrauben werden für die Haupttypen der Bewegung angegeben und verschiedene Probleme der Bewegungsmechanik besprochen.

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Summary The movements of bacteria living in the mud of high-moores have been investigated with maximum magnification and daik-field illumination, partly in indian-ink preparations. Detailed observations of the flagellar movements of rod-shaped bacteria, of the gliding movements ofSaprospira, Cristispira, Spirulina, Cytophaga, Chlamydobacteria, and of various motions of Spirochaeta have been possible.It can been shown with the help of model-experiments that most phenomena of motion are to be explained not by the contraction of fibrils but by simple rotations of helices. The situation and behaviour of the rotating helices are described of the main types of movements and the different problems of the mechanism of the particular movements are discussed.

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Herrn Prof. Dr.Lothar Geitler hochachtungsvoll zugeeignet.     

Isolamento di Schizomiceti cellulositici,Attinomiceti, Eumiceti dall' intestino delle Termiti e ricerche sulla attivita' cellulositica degli Attinomiceti

Riassunto Gli AA. sono riusciti ad isolare, per la prima volta, Schizomiceti cellulositici del genere Cytophaga ed Attinomiceti del genere Actinomyces, dall'intestino delle Termiti. Sono altresí isolate alcune specie fungine, come era già noto. Gli Attinomiceti isolati non dimostrano di possedera capacità di attaccare la cellulosa, analogamente a quanto accade per altri ceppi di diversa provenienza degli stessi microrganismi.Gli AA. confrontano e discutono i loro resultati con quelli di altri AA. tendenti ad attribuire ai Protozoi simbionti delle Termiti una attività cellulositica, che ad essi non sembra sufficientemente provata. Gli AA. ritengono che tale attività si debba, invece, attribuire ai Batteri simbionti dei Protozoi e a quelli aderenti alle pareti dell'intestino delle Termiti stesse.

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Summary From the intestinal apparatus of the Termites were isolated, for the first time, cellulositic bacteria of the genus Cytophaga, in addition to ray-fungi of the genus Actinomyces and species of true fungi previously known. The cellulositic activity of Actinomyces has not been, instead, experimentally demostrated.The results obtained are discussed in relation to the presumed cellulositic activity of Protozoa symbiotic of the Termite ants.

     

Florida reef sponges harbor coral disease-associated microbes

Sponges can filter large volumes of seawater and accumulate highly diverse and abundant microbial communities within their tissue. Culture-independent techniques such as fluorescent in situ hybridization (FISH), 16S small subunit (SSU) rRNA gene analyses, and transmission electron microscopy (TEM) were applied to characterize the presence and distribution of microbes within sponges abundant on south Florida reefs. This study found that coral disease-associated bacteria (CDAB) are harbored within Agelas tubulata and Amphimedon compressa. FISH probes detected several potential bacterial pathogens such as Aurantimonas coralicida, Cytophaga sp., Desulfovibrio spp, Serratia marcescans, and Vibrio mediterranei within A. compressa and A. tubulata host sponges. Spatial differences in the distribution of targeted bacteria were seen within sponge hosts. Transmission electron microscopy of A. compressa indicated there was a higher concentration of bacteria in the choanosome compared to the ectosome. These observed spatial distributions support the presence of internal sponge niches, which could play a role in the location of the CDAB within the sponges.