An Extracytoplasmic Function Sigma Factor Controls Cellulose Utilization by Regulating the Expression of an Outer Membrane Protein in Cytophaga hutchinsonii.

The common soil cellulolytic bacterium known as Cytophaga hutchinsonii makes use of a unique but poorly understood strategy in order to utilize cellulose. While several genes have been identified as being an active part of the utilization of cellulose, the mechanism(s) by which C. hutchinsonii both (i) senses its environment and (ii) regulates the expression of those genes are not as yet known. In this study, we identified and characterized the gene CHU_3097 encoding an extracytoplasmic function (ECF) σ factor (σcel1), the disruption of which compromised C. hutchinsonii cellulose assimilation to a large degree. The σcel1 and its putative partner anti-σcel1, encoded by the CHU_3096 gene found immediately downstream from CHU_3097, copurified in vitro The σcel1 was discovered to be associated with inner membrane when cells were cultured on glucose and yet was partially released from the membrane in response to cellulose. This release was found to occur on glucose when the anti-σcel1 was absent. Transcriptome analyses found a σcel1-regulated, cellulose-responsive gene regulon, within which an outer membrane protein encoding the gene CHU_1276, essential for cellulose utilization, was discovered to be significantly downregulated by CHU_3097 disruption. The expression of CHU_1276 almost fully restored cellulose utilization to the CHU_3097 mutant, demonstrating that CHU_1276 represents a critical regulatory target of σcel1 In this way, our study provided insights into the role of an ECF σ factor in coordinating the cellulolytic response of C. hutchinsoniiIMPORTANCE The common cellulolytic bacterium Cytophaga hutchinsonii uses a unique but poorly understood strategy in order to make use of cellulose. Throughout the process of cellulosic biomass breakdown, outer membrane proteins are thought to play key roles; this is evidenced by CHU_1276, which is required for the utilization of cellulose. However, the regulatory mechanism of its expression is not yet known. We found and characterized an extracytoplasmic function σ factor that is involved in coordinating the cellulolytic response of C. hutchinsonii by directly regulating the expression of CHU_1276 This study makes a contribution to our understanding of the regulatory mechanism used by C. hutchinsonii in order to adjust its genetic programs and so deal with novel environmental cues.

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.

Outer membrane proteins related to SusC and SusD are not required for Cytophaga hutchinsonii cellulose utilization.

Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, employs a novel collection of cell-associated proteins to digest crystalline cellulose. Other Bacteroidetes rely on cell surface proteins related to the starch utilization system (Sus) proteins SusC and SusD to bind oligosaccharides and import them across the outer membrane for further digestion. These bacteria typically produce dozens of SusC-like porins and SusD-like oligosaccharide-binding proteins to facilitate utilization of diverse polysaccharides. C. hutchinsonii specializes in cellulose digestion and its genome has only two susC-like genes and two susD-like genes. Single and multiple gene deletions were constructed to determine if the susC-like and susD-like genes have roles in cellulose utilization. A mutant lacking all susC-like and all susD-like genes digested cellulose and grew on cellulose as well as wild-type cells. Further, recombinantly expressed SusD-like proteins CHU_0547 and CHU_0554 failed to bind cellulose or ß-glucan hemicellulosic polysaccharides. The results suggest that the Bacteroidetes Sus paradigm for polysaccharide utilization may not apply to the cellulolytic bacterium C. hutchinsonii.

Identification and characterization of a novel locus in Cytophaga hutchinsonii involved in colony spreading and cellulose digestion.

Cytophaga hutchinsonii, an aerobic cellulolytic soil bacterium, is capable of degrading crystalline cellulose and gliding over surface rapidly. The involved mechanisms, however, are largely unknown. Here, we used the mariner-based transposon HimarEm1 to screen for C. hutchinsonii mutants deficient in utilizing filter paper as the sole carbon source. A novel gene locus, chu_1719, encoding a hypothetical protein was identified, whose inactivation resulted in a compromised growth of C. hutchinsonii on filter paper. Further analysis revealed that disruption of chu_1719 suppressed colony spreading but had no significant effect on Avicel degradation in liquid medium. Carboxymethylcellulase (CMCase) activity of the mutant membrane proteins was reduced by about 40% as compared with the wild-type strain. Moreover, profiles of cellulose-adsorbed outer membrane proteins were significantly different between the mutant and wild-type (WT) strains. These results suggest that chu_1719 plays an important role in controlling the spreading motility and cellulose utilization probably by affecting the appropriate production of membrane proteins in C. hutchinsonii.

Seasonal growth potential of rare lake water bacteria suggest their disproportional contribution to carbon fluxes.

We studied the seasonal growth potential of opportunistic bacterial populations in Lake Zurich (Switzerland) by a series of grazer-free dilution culture assays. Pronounced shifts in the composition of the bacterial assemblages were observed within one doubling of total cell numbers, from initially abundant Actinobacteria to other fast-growing microbial lineages. Small populations with growth potentials far above community average were detected throughout the year with striking seasonal differences in their respective taxonomic affiliations. Members of Cytophaga-Flavobacteria (CF) were disproportionally proliferating only during phytoplankton blooms in spring and summer, while Beta- and Gammaproteobacteria showed superior growth at all other occasions. Growth rates of Alphaproteobacteria and esp. Sphingomonadaceae were significantly correlated to water temperatures and were far above community average in summer. Within the genus Flavobacterium, two species-like populations showed a tendency for fast growth in most experiments, while four others were exclusively proliferating either during a spring or during a summer phytoplankton bloom. Their high growth potentials but low in situ abundances hint at a tight control by bacterivorous grazers and at a consequently accelerated carbon flux to higher trophic levels.

Spatio-temporal patterns of major bacterial groups in alpine waters.

Glacial alpine landscapes are undergoing rapid transformation due to changes in climate. The loss of glacial ice mass has directly influenced hydrologic characteristics of alpine floodplains. Consequently, hyporheic sediment conditions are likely to change in the future as surface waters fed by glacial water (kryal) become groundwater dominated (krenal). Such environmental shifts may subsequently change bacterial community structure and thus potential ecosystem functioning. We quantitatively investigated the structure of major bacterial groups in glacial and groundwater-fed streams in three alpine floodplains during different hydrologic periods. Our results show the importance of several physico-chemical variables that reflect local geological characteristics as well as water source in structuring bacterial groups. For instance, Alpha-, Betaproteobacteria and Cytophaga-Flavobacteria were influenced by pH, conductivity and temperature as well as by inorganic and organic carbon compounds, whereas phosphorous compounds and nitrate showed specific influence on single bacterial groups. These results can be used to predict future bacterial group shifts, and potential ecosystem functioning, in alpine landscapes under environmental transformation.

[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.

Rapid successions affect microbial N-acetyl-glucosamine uptake patterns during a lacustrine spring phytoplankton bloom.

The vernal successions of phytoplankton, heterotrophic nanoflagellates (HNF) and viruses in temperate lakes result in alternating dominance of top-down and bottom-up factors on the bacterial community. This may lead to asynchronous blooms of bacteria with different life strategies and affect the channelling of particular components of the dissolved organic matter (DOM) through microbial food webs. We followed the dynamics of several bacterial populations and of other components of the microbial food web throughout the spring phytoplankton bloom period in a pre-alpine lake, and we assessed bacterial uptake patterns of two constituents of the labile DOM pool (N-acetyl-glucosamine [NAG] and leucine). There was a clear genotypic shift within the bacterial assemblage, from fast growing Cytophaga-Flavobacteria (CF) affiliated with Fluviicola and from Betaproteobacteria (BET) of the Limnohabitans cluster to more grazing resistant AcI Actinobacteria (ACT) and to filamentous morphotypes. This was paralleled by successive blooms of viruses and HNF. We also noted the transient rise of other CF (related to Cyclobacteriaceae and Sphingobacteriaceae) that are not detected by fluorescence in situ hybridization with the general CF probe. Both, the average uptake rates of leucine and the fractions of leucine incorporating bacteria were approximately five to sixfold higher than of NAG. However, the composition of the NAG-active community was much more prone to genotypic successions, in particular of bacteria with different life strategies: While 'opportunistically' growing BET and CF dominated NAG uptake in the initial period ruled by bottom-up factors, ACT constituted the major fraction of NAG active cells during the subsequent phase of high predation pressure. This indicates that some ACT could profit from a substrate that might in parts have originated from the grazing of protists on their bacterial competitors.

Seasonal patterns of the bacterioplankton community composition in a lake threatened by a pesticide disposal site.

UNLABELLED: BACKGROUND AIM AND SCOPE: The objective of the study was to determine the effects of ca. 35 years of pesticide contamination (pesticide dump-PD) of Lake Szelag Wielki (located in the north-eastern Poland) on changes in the microbial communities of aquatic ecosystems. In the years 2008-2009, analyses were carried out for seasonal changes in the quantity and composition of bacterioplankton in the lake examined, which is of high significance to the tourism and fishing industries and is located in the vicinity of an area subjected to reclamation after a pesticide dump. METHODS: Bacterioplankton composition was assayed by fluorescence in situ hybridisation technique for the contribution of major groups of the Bacteria domain: ά-, ß- and γ-Proteobacteria, Cytophaga-Flavobacterium and Actinobacteria as well as bacteria capable of degrading pesticides in an aquatic environment-Pseudomonas spp. Seasonal patterns of the total number of bacteria were determined by direct counting of 4',6-diamidino-2-phenylindole (DAPI)-stained cells. RESULTS: The percentage of the detected Eubacteria (EUB 338 probe) relative to all the DAPI-stained bacteria in Lake Szelag Wielki ranged from 46% to 63%. Bacteria capable of degrading pesticides in an aquatic environment-Pseudomonas spp.-were identified with a highly specific probe PEA 998. The highest mean values of this parameter reached 5.1%. In the spring, Pseudomonas spp. bacteria accounted for up to 80% of all Gamma-Proteobacteria microbes. CONCLUSION: The study showed that the qualitative and quantitative changes in the bacterioplankton of the lake can be characterised by tendencies which are typical of a eutrophic water reservoir. However, a higher contribution of microorganisms capable of degrading sparingly degradable, toxic compounds and pesticides was determined in bacterioplankton from the PD-contaminated lake, as compared to microbial communities of a lake not contaminated with pesticides.

Cellulose and cellodextrin utilization by the cellulolytic bacterium Cytophaga hutchisonii.

Cytophaga hutchinsonii is an abundant aerobic cellulolytic soil bacterium utilizing very few substrates as sole carbon and energy sources. In this study, growth of C. hutchinsonii on different substrates including crystalline cellulose, regenerated amorphous cellulose (RAC) as well as soluble sugars including cellodextrins was analyzed. Soluble sugars including glucose and cellodextrins were produced extracellularly when C. hutchinsonii was cultured on cellulose. Preferential use of cellulooligosaccharides as the carbon source by C. hutchinsonii was largely dependent on its inoculation status. Compared with glucose-grown cells, inoculation of cellobiose-grown cells led to a rapid assimilation of cellobiose or cellodextrins with longer-chain cellodextrins being hydrolyzed extracellularly to smaller oligomers during the culture. Further analysis of the distribution of cellulase activity revealed that, while the carboxymethylcellulase activity significantly induced by crystalline cellulose was highest in the outer membrane, the cellobiase activity was highest in the cytoplasmic membrane. These results suggest that membrane-bound cellulases may play an important role in cellulose solubilization by C. hutchinsonii and that metabolism of cello-oligosaccharides is a tightly coupled step in this process.

The uronic acids assay: a method for the determination of chemical activity on biofilm EPS.

In this work, the uronic acids assay was evaluated for its potential to function as a bioassay to screen for antagonistic activity against the production of microbial biofilm exopolysaccharide (EPS). The assay was first applied to biofilms produced in the presence of two universal disinfectants (sodium hypochlorite and sodium dodecyl sulfate) known to inhibit microbial growth and biofilm formation. The performance of the assay was then characterized through statistical assessment of threshold concentrations for disinfection efficiency and consistency relative to values reported in the literature. The assay was then evaluated for its utility in screening for enzymatic or chemical inhibitors of biofilm formation (eg glycosidases, halogenated furanones, and semi-crude fractions extracted from minimally fouled marine plants) and its ability to distinguish between true anti-biofilm activity and simple disinfection. Activity was characterized as (i) no effect, (ii) a true positive effect (ie increased biofilm EPS), (iii) anti-bacterial activity (ie decreased biofilm EPS and analogous decrease in planktonic growth), and (iv) anti-biofilm EPS activity (ie decreased biofilm EPS, without analogous decrease in planktonic growth). Results demonstrate that the uronic acids assay can augment existing biofilm characterization methods by providing a quantitative measure of biofilm EPS.

A small population of planktonic Flavobacteria with disproportionally high growth during the spring phytoplankton bloom in a prealpine lake.

Bacterioplankton growth in temperate Lake Zurich (Switzerland) was studied during the spring phytoplankton bloom by in situ techniques and short-term dilution bioassays. A peak of chlorophyll a (Chl a) concentrations was followed by a rise of bacterial cell numbers and leucine assimilation rates, of the proportions of cells incorporating 5-bromo-2-deoxyuridine (BrdU), and of community net growth rates in dilution cultures. Incorporation of BrdU was low in Betaproteobacteria (2 +/- 1%), indicating that these bacteria did not incorporate the tracer. Pronounced growth of Betaproteobacteria in the enrichments was only observed after the decline of the phytoplankton bloom. An initial peak in the proportions of BrdU-positive Actinobacteria (30%) preceded a distinct rise of their cell numbers during the period of the Chl a maximum. Cytophaga-Flavobacteria (CF) changed little in numbers, but featured high proportions of BrdU-positive cells (28 +/- 12%). Moreover, CF represented > 90% of all newly formed cells in dilution cultures before and during the phytoplankton bloom. One phylogenetic lineage of cultivable Flavobacteria (FLAV2) represented a small (0.5-1%) but highly active population in lake plankton. The growth rates of FLAV2 in dilution cultures doubled during the period of the Chl a maximum, indicating stimulation by phytoplankton exudates. Thus, CF, and specifically Flavobacteria, appeared to be substantially more important for carbon transfer in Lake Zurich spring bacterioplankton than was suggested by their standing stocks. The high in situ growth potential of these bacteria might have been counterbalanced by top-down control.

Quorum sensing by N-acylhomoserine lactones is not required for Aeromonas hydrophila during growth with organic particles in lake water microcosms.

It was investigated whether quorum sensing (QS) mediated by N-acylhomoserine lactones (AHLs) was important for heterotrophic bacteria from the littoral zone of the oligotrophic Lake Constance for growth with organic particles. More than 900 colonies from lake water microcosms with artificial organic aggregates consisting of autoclaved unicellular algae embedded in agarose beads were screened for AHL-production. AHL-producing bacteria of the genus Aeromonas enriched in the microcosms but AHLs could not be detected in any microcosm. To test for a potential function of AHL-mediated QS, growth experiments with the wild type and an AHL-deficient mutant of Aeromonas hydrophila in lake water microcosms were performed. Growth of both strains did not differ in single cultures and showed no mutual influence in co-cultures. In co-cultures with a competitor bacterium belonging to the Cytophaga-Flavobacterium group, growth of both A. hydrophila strains was reduced while growth of the competitor bacterium was not affected. Exogenous AHL-addition did not influence growth of the Aeromonas strains in any microcosm experiment. These results showed that AHL-mediated QS was not required for A. hydrophila during colonization and degradation of organic particles in lake water microcosms, suggesting that cell-cell signalling of heterotrophic bacteria in oligotrophic waters relies on novel signal molecules.

Fate and distribution of brevetoxin (PbTx) following lysis of Karenia brevis by algicidal bacteria, including analysis of open A-ring derivatives.

Flavobacteriaceae (strain S03) and Cytophaga sp. (strain 41-DBG2) are algicidal bacteria active against the brevetoxin (PbTx)-producing, red tide dinoflagellate, Karenia brevis. Little is known about the fate of PbTx associated with K. brevis cells following attack by such bacteria. The fate and distribution of PbTx in K. brevis cultures exposed to these algicidal strains were thus examined by receptor binding assay and liquid chromatography/mass spectrometry (LC/MS) in three size fractions (>5, 0.22-5, <0.22microm) over a 2-week time course. In control cultures, brevetoxin concentrations in the >5microm particulate size fraction correlated with changes in cell density, whereas significant increases in dissolved (i.e., <0.22microm) toxin were observed in the later stages of culture growth. Exposure of K. brevis to either of the two algicidal bacteria tested caused cell lysis, coinciding with a rapid decline in the >5microm PbTX size fraction and a simultaneous release of dissolved toxin into the growth medium. Upon cell lysis, dissolved brevetoxin accounted for ca. 60% of total toxin and consisted of 51-82% open A-ring derivatives. Open A-ring PbTx-2 and PbTx-3 derivatives bound with lower affinity (approximately 22- and 57-fold, respectively) to voltage-gated sodium channels and were considerably less cytotoxic (86- and 142-fold, respectively) to N2A cells than their individual parent toxins (i.e., PbTx-2 and PbTx-3). These novel findings of changes in PbTx size-fractioned distribution and overall reduction in K. brevis toxicity following attack by algicidal bacteria improve our understanding of potential trophic transfer routes and the fate of PbTx during red tide events. Moreover, this information will be important to consider when evaluating the potential role of algicidal bacteria in harmful algal bloom (HAB) management strategies involving control of bloom populations.

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.

Characterization of a sulfide-oxidizing biofilm developed in a packed-column reactor

The potential of microbial mats to develop sulfide-oxidizing biofims was explored. A bioreactor specially designed for the treatment of sulfide-containing effluents was inoculated with a microbial-mat sample, and a complex microbial biofilm with sulfide-oxidation activity developed. The microbial composition of the biofilm was studied by pigment, microscopy, and 16S rRNA gene analyses. Purple sulfur bacteria and diatoms were observed by microscopy, chlorophyll a and bacteriochlorophyll a were detected in the pigment analysis, and high genetic diversity was found in the 16S rRNA gene library. Specialized anaerobic sulfur oxidizers (i.e., phototrophic purple and green sulfur bacteria) dominated the library. Aerobic phototrophs (diatoms) also developed and the oxygen produced allowed the growth of aerobic sulfide oxidizers, such as Thiomicrospira-like spp. Cyanobacteria, which are significant organisms in natural microbial mats, did not develop in the reactor but unexpected uncultured members from the Epsilonproteobacteria developed profusely. Moreover, a variety of more minor organisms, such as members of the Cytophaga-Flavobacterium-Bacteroides (CFB) and purple non-sulfur bacteria (Roseospirillum sp.), were also present. The results showed that a complex community with high genetic and metabolic diversity, including many uncultured organisms, can develop in a laboratory-scale reactor (AU)

No disponible

Diverse responses to UV-B radiation and repair mechanisms of bacteria isolated from high-altitude aquatic environments.

Acinetobacter johnsonii A2 isolated from the natural community of Laguna Azul (Andean Mountains at 4,560 m above sea level), Serratia marcescens MF42, Pseudomonas sp. strain MF8 isolated from the planktonic community, and Cytophaga sp. strain MF7 isolated from the benthic community from Laguna Pozuelos (Andean Puna at 3,600 m above sea level) were subjected to UV-B (3,931 J m-2) irradiation. In addition, a marine Pseudomonas putida strain, 2IDINH, and a second Acinetobacter johnsonii strain, ATCC 17909, were used as external controls. Resistance to UV-B and kinetic rates of light-dependent (UV-A [315 to 400 nm] and cool white light [400 to 700 nm]) and -independent reactivation following exposure were determined by measuring the survival (expressed as CFU) and accumulation of cyclobutane pyrimidine dimers (CPD). Significant differences in survival after UV-B irradiation were observed: Acinetobacter johnsonii A2, 48%; Acinetobacter johnsonii ATCC 17909, 20%; Pseudomonas sp. strain MF8, 40%; marine Pseudomonas putida strain 2IDINH, 12%; Cytophaga sp. strain MF7, 20%; and Serratia marcescens, 21%. Most bacteria exhibited little DNA damage (between 40 and 80 CPD/Mb), except for the benthic isolate Cytophaga sp. strain MF7 (400 CPD/Mb) and Acinetobacter johnsonii ATCC 17909 (160 CPD/Mb). The recovery strategies through dark and light repair were different in all strains. The most efficient in recovering were both Acinetobacter johnsonii A2 and Cytophaga sp. strain MF7; Serratia marcescens MF42 showed intermediate recovery, and in both Pseudomonas strains, recovery was essentially zero. The UV-B responses and recovery abilities of the different bacteria were consistent with the irradiation levels in their native environment.

Quantifying substrate uptake by individual cells of marine bacterioplankton by catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography.

Catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH) is increasingly being used to obtain qualitative information on substrate uptake by individual members of specific prokaryotic communities. Here we evaluated the potential for using this approach quantitatively by relating the measured silver grain area around cells taking up (3)H-labeled leucine to bulk leucine uptake measurements. The increase in the silver grain area over time around leucine-assimilating cells of coastal bacterial assemblages was linear during 4 to 6 h of incubation. By establishing standardized conditions for specific activity levels and concomitantly performing uptake measurements with the bulk community, MICRO-CARD-FISH can be used quantitatively to determine uptake rates on a single-cell level. Therefore, this approach allows comparisons of single-cell activities for bacterial communities obtained from different sites or growing under different ecological conditions.

Peptidoglycan from Bacillus cereus mediates commensalism with rhizosphere bacteria from the Cytophaga-Flavobacterium group.

Previous research in our laboratory revealed that the introduction of Bacillus cereus UW85 can increase the populations of bacteria from the Cytophaga-Flavobacterium (CF) group of the Bacteroidetes phylum in the soybean rhizosphere, suggesting that these rhizosphere microorganisms have a beneficial relationship (G. S. Gilbert, J. L. Parke, M. K. Clayton, and J. Handelsman, Ecology 74:840-854, 1993). In the present study, we determined the frequency at which CF bacteria coisolated with B. cereus strains from the soybean rhizosphere and the mechanism by which B. cereus stimulates the growth of CF rhizosphere strains in root exudate media. In three consecutive years of sampling, CF strains predominated among coisolates obtained with B. cereus isolates from field-grown soybean roots. In root exudate media, the presence of B. cereus was required for CF coisolate strains to reach high population density. However, rhizosphere isolates from the phylum Proteobacteria grew equally well in the presence and absence of B. cereus, and the presence of CF coisolates did not affect the growth of B. cereus. Peptidoglycan isolated from B. cereus cultures stimulated growth of the CF rhizosphere bacterium Flavobacterium johnsoniae, although culture supernatant from B. cereus grown in root exudate media did not. These results suggest B. cereus and CF rhizosphere bacteria have a commensal relationship in which peptidoglycan produced by B. cereus stimulates the growth of CF bacteria.

Incorporation of 3H-thymidine by different prokaryotic groups in relation to temperature and nutrients in a lacustrine ecosystem.

The incorporation of [3H-methyl] thymidine (3H-TdR) by Eubacteria, bacterial groups (alpha- and beta-Proteobacteria, Cytophaga-Flavobacter), and Archaea was measured according to temperature (7 and 17 degrees C) and nutrient levels (nitrogen, phosphorus, and carbon) in a lacustrine system (Sep, France). Short-term incubation was performed using a combination of microautoradiography and fluorescent in situ hybridization. Irrespective of the temperatures and nutrients studied, all the major phylogenetic bacterial groups assimilated 3H-TdR, and in most of the treatments studied, the proportion of beta-Proteobacteria taking up 3H-TdR was higher than those in the other bacterial groups. The proportion of Bacteria and different bacterial groups studied incorporating 3H-TdR were significantly increased, approximately 1.5-fold, by temperature except for alpha-Proteobacteria (7.6-fold). The nutrient effect was not the same for the different bacterial groups according to the temperatures studied. The proportions of alpha-Proteobacteria (at both temperatures) and Cytophaga-Flavobacter (at 7 degrees C) taking up 3H-TdR were significantly decreased and increased by adding N and P, respectively. Also, adding N, P, and C increased and decreased the percentage of beta-Proteobacteria incorporating 3H-TdR at 7 and 17 degrees C, respectively. The archaeal community showed a similar proportion of active cells (i.e., 3H-TdR) to the bacterial community, and uptake of 3H-TdR by Archaea was significantly increased (P < 0.05) by both temperature and nutrients. Thus, the assimilation of 3H-TdR by bacterial groups and Archaea in lacustrine system is significantly controlled by both temperature and nutrients.