Structure and Species Composition of Mercury-Reducing Biofilms

Mercury-reducing biofilms from packed-bed bioreactors treating nonsterile industrial effluents were shown to consist of a monolayer of bacteria by scanning electron microscopy. Droplets of several micrometers in diameter which accumulated outside of the bacterial cells were identified as elemental mercury by electron-dispersive X-ray analysis. The monospecies biofilms of Pseudomonas putida Spi3 initially present were invaded by additional strains, which were identified to the species level by thermogradient gel electrophoresis (TGGE) and 16S rDNA sequencing. TGGE community fingerprints of the biofilms showed that they were composed of the effluent bacteria and did not contain uncultivable microorganisms. Of the 13 effluent bacterial strains, 2 were not mercury resistant, while all the others had resistance levels similar to or higher than the inoculant strain.     

Diversity and seasonal variability of beta-Proteobacteria in biofilms of polluted rivers: analysis by temperature gradient gel electrophoresis and cloning

The beta-subgroup of the Proteobacteria has been shown to be important in aquatic habitats and was investigated in depth here by molecular 16S rRNA techniques in biofilms of the Elbe River and its polluted tributary, the Spittelwasser River. The bacterial 16S rRNA genes were cloned from each site, screened for beta-proteobacterial clones and sequenced. River biofilm clones from both rivers grouped into 9 clusters (RBFs). RBFs 1, 2, and 3 fell into the recently described betaI cluster of cosmopolitan freshwater bacteria, where they represented new species related to Rhodoferax, Aquaspirillum, and Hydrogenophaga: RBFs 4 to 7 affiliated with Aquabacterium commune, Ideonella dechloratans, and Sphaerotilus natans, respectively. The two remaining RBFs were uncultivated clusters, one of them being distantly related to Gallionella ferruginea. Seasonal changes in the relative intensity of the beta-proteobacterial 16S rRNA genes of biofilms harvested monthly for 1 year were determined by specific amplification and separation by temperature gradient gel electrophoresis (TGGE). Bands were identified by comparison of clones to community fingerprints by TGGE. Eight of 13 identified bands were shared by both habitats but showed different relative abundance and seasonal variability in the two rivers, probably caused by differences in temperature and pollutants. The data indicate new not-yet-cultivated clusters of river biofilm organisms, some of them probably distributed globally. They confirm the importance of certain known freshwater genera in river biofilms. The high phylogenetic resolution obtained by clone library analysis combined with the high temporal resolution obtained by TGGE suggest that the observed microdiversity in the river biofilm clone libraries might be caused by phylogenetically closely related microbial populations which are adapted to ecological parameters.     

Mercury-resistant bacterial strains Pseudomonas and Psychrobacter spp. isolated from sediments of Orbetello Lagoon (Italy) and their possible use in bioremediation processes

This study was aimed to isolate Hg-resistant bacteria from contaminated sediments of the Orbetello Lagoon in Italy and to assess their possible use as biofilms in bioremediation processes. Enrichment cultures prepared from contaminated sediments in the presence of 0.05 mM of mercury and under aerobic conditions allowed the isolation of five heterotrophic bacterial strains. 16S rDNA gene sequencing assigned the isolated strains to the genera Pseudomonas and Psychrobacter. For the first time mercury-resistant bacterial strains belonging to the genus Psychrobacter were evidenced. Minimum inhibitory concentrations in the presence of HgCl₂ and of CH₃HgCl showed high levels of resistance. EC50 values for the isolated bacterial strains in the presence of HgCl₂ and of CH₃HgCl confirmed the adaptation to the metal. Hg-resistant strains ORHg1, ORHg4 and ORHg5 showed the capacity to volatilize inorganic and organic mercury to elemental mercury, and formed biofilms on pumice particles, and were shown to play a role in the removal of mercury from sediment leachates. This study reports isolation and characterization of new Hg-resistant bacterial strains and provides novel insight into their possible use in bioremediation processes of mercury polluted sediments.     

Bacterial Community Composition in Central European Running Waters Examined by Temperature Gradient Gel Electrophoresis and Sequence Analysis of 16S rRNA Genes

The bacterial community composition in small streams and a river in central Germany was examined by temperature gradient gel electrophoresis (TGGE) with PCR products of 16S rRNA gene fragments and sequence analysis. Complex TGGE band patterns suggested high levels of diversity of bacterial species in all habitats of these environments. Cluster analyses demonstrated distinct differences among the communities in stream and spring water, sandy sediments, biofilms on stones, degrading leaves, and soil. The differences between stream water and sediment were more significant than those between sites within the same habitat along the stretch from the stream source to the mouth. TGGE data from an entire stream course suggest that, in the upper reach of the stream, a special suspended bacterial community is already established and changes only slightly downstream. The bacterial communities in water and sediment in an acidic headwater with a pH below 5 were highly similar to each other but deviated distinctly from the communities at the other sites. As ascertained by nucleotide sequence analysis, stream water communities were dominated by Betaproteobacteria (one-third of the total bacteria), whereas sediment communities were composed mainly of Betaproteobacteria and members of the Fibrobacteres/Acidobacteria group (each accounting for about 25% of bacteria). Sequences obtained from bacteria from water samples indicated the presence of typical cosmopolitan freshwater organisms. TGGE bands shared between stream and soil samples, as well as sequences found in bacteria from stream samples that were related to those of soil bacteria, demonstrated the occurrence of some species in both stream and soil habitats. Changes in bacterial community composition were correlated with geographic distance along a stream, but in comparisons of different streams and rivers, community composition was correlated only with environmental conditions.     

Specificity in the settlement -- modifying response of bacterial biofilms towards zoospores of the marine alga Enteromorpha

Previous studies have shown that the rate of settlement of zoospores of the green alga Enteromorpha is stimulated by mixed microbial biofilms and that the number of zoospores settling is positively correlated with the number of bacteria in the biofilm. In the present study the specificity of this relationship has been investigated. Ninety-nine strains of marine bacteria were isolated from natural biofilms on rocks and the surface of Enteromorpha plants. Isolates were screened by denaturing gradient gel electrophoresis (DGGE) to eliminate replicates and 16S rDNA sequencing identified a total of 37 unique strains. Phylogenetic analysis revealed that the isolated bacterial strains belonged to three groups gamma-Proteobacteria (28 strains), Cytophaga-Flavobacteria-Bacteroid (CFB) group (six strains) and alpha-Proteobacteria (one strain). Two strains were unassigned, showing < 93% sequence similarity with the CFB group. The main genera of gamma-Proteobacteria were Pseudoalteromonas (14 strains), Vibrio (five strains), Shewanella (five strains), Halomonas (three strains) and Pseudomonas (one strain). Spore settlement experiments were conducted on single-species biofilms, developed for different times on glass slides. The effect of correcting spore settlement values for biofilm density was evaluated. Results showed that the effect of bacterial strains on spore settlement was strain- but not taxon-specific and activity varied with the age of the biofilm. However, most of the strains belonging to genera Vibrio and Shewanella showed stimulation. Pseudoalteromonas strains showed a range of effects including settlement-inhibiting, paralysing and lysing activities. Spatial analysis of bacterial density in the presence and absence of spores revealed a range of different types of association between spores and bacteria. Overall, the spatial association between spores and bacteria appears to be independent of the overall quantitative influence of bacterial cells on spore settlement.     

Diversity and Seasonal Variability of β-Proteobacteria in Biofilms of Polluted Rivers: Analysis by Temperature Gradient Gel Electrophoresis and Cloning

The β-subgroup of the Proteobacteria has been shown to be important in aquatic habitats and was investigated in depth here by molecular 16S rRNA techniques in biofilms of the Elbe River and its polluted tributary, the Spittelwasser River. The bacterial 16S rRNA genes were cloned from each site, screened for β-proteobacterial clones and sequenced. River biofilm clones from both rivers grouped into 9 clusters (RBFs). RBFs 1, 2, and 3 fell into the recently described βI cluster of cosmopolitan freshwater bacteria, where they represented new species related to Rhodoferax, Aquaspirillum, and Hydrogenophaga. RBFs 4 to 7 affiliated with Aquabacterium commune, Ideonella dechloratans, and Sphaerotilus natans, respectively. The two remaining RBFs were uncultivated clusters, one of them being distantly related to Gallionella ferruginea. Seasonal changes in the relative intensity of the β-proteobacterial 16S rRNA genes of biofilms harvested monthly for 1 year were determined by specific amplification and separation by temperature gradient gel electrophoresis (TGGE). Bands were identified by comparison of clones to community fingerprints by TGGE. Eight of 13 identified bands were shared by both habitats but showed different relative abundance and seasonal variability in the two rivers, probably caused by differences in temperature and pollutants. The data indicate new not-yet-cultivated clusters of river biofilm organisms, some of them probably distributed globally. They confirm the importance of certain known freshwater genera in river biofilms. The high phylogenetic resolution obtained by clone library analysis combined with the high temporal resolution obtained by TGGE suggest that the observed microdiversity in the river biofilm clone libraries might be caused by phylogenetically closely related microbial populations which are adapted to ecological parameters.     

Growth and release of extracellular organic compounds by benthic diatoms depend on interactions with bacteria

Phototrophic epilithic biofilms harbour a distinct assemblage of heterotrophic bacteria, cyanobacteria and photoautotrophic algae. Secretion of extracellular polymeric substances (EPS) by these organisms and the physicochemical properties of the EPS are important factors for the development of the biofilms. We have isolated representative diatom and bacteria strains from epilithic biofilms of Lake Constance. By pairwise co-cultivating these strains we found that diatom growth and EPS secretion by diatoms may depend on the presence of individual bacteria. Similar results were obtained after addition of spent bacterial medium to diatom cultures, suggesting that soluble substances from bacteria have an impact on diatom physiology. While searching for putative bacterial signal substances, we found that concentrations of various dissolved free amino acids (DFAA) within the diatom cultures changed drastically during co-cultivation with bacteria. Further, the secretion of extracellular carbohydrates and proteins can be influenced by bacteria or their extracellular substances. We have performed mass spectrometric peptide mapping to identify proteins which are secreted when co-cultivating the diatom Phaeodactylum tricornutum Bohlin and Escherichia coli. The identified proteins are possibly involved in signalling, extracellular carbohydrate modification and uptake, protein and amino acid modification, and cell/cell aggregation of diatom and bacteria strains. Our data indicate that diatom-bacteria biofilms might be regulated by a complex network of chemical factors involving EPS, amino acid monomers and other substances. Thus interactions with bacteria can be considered as one of the main factors driving biofilm formation by benthic diatoms.     

Staphylococcus aureus - induced tumor necrosis factor - related apoptosis - inducing ligand expression mediates apoptosis and caspase-8 activation in infected osteoblasts

Background

Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up. The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, themerAgene coding for the mercuric reductase. We report on the development of a profiling method formerAand its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation.

Results

Based on an alignment of 30merAsequences from Gram negative bacteria, conserved primers were designed for amplification ofmerAfragments with an optimized PCR protocol. The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE), resulting in strain specific fingerprints for mercury resistant Gram negative isolates with differentmerAsequences. ThemerAprofiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community. One predominant new band of themerAcommunity profile was also detected in a biocatalyzer effluent isolate, which was identified asPseudomonas aeruginosa. The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing.

Conclusions

ThemerAprofiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation. In such a way, a predominant mercury reducingPs. aeruginosastrain was identified by its unique mercuric reductase gene.     

Molecular and ecological analyses of microbial community structures in biofilms of a full-scale Aerated Up-Flow Biobead process

Molecular and cultivation techniques were used to characterize the bacterial communities of biobead reactor biofilms in a sewage treatment plant to which an Aerated Up-Flow Biobead process was applied. With this biobead process, the monthly average values of various chemical parameters in the effluent were generally kept under the regulation limits of the effluent quality of the sewage treatment plant during the operation period. Most probable number (MPN) analysis revealed that the population of denitrifying bacteria was abundant in the biobead #1 reactor, denitrifying and nitrifying bacteria coexisted in the biobead #2 reactor, and nitrifying bacteria prevailed over denitrifying bacteria in the biobead #3 reactor. The results of the MPN test suggested that the biobead #2 reactor was a transition zone leading to acclimated nitrifying biofilms in the biobead #3 reactor. Phylogenetic analysis of 16S rDNA sequences cloned from biofilms showed that the biobead #1 reactor, which received a high organic loading rate, had much diverse microorganisms, whereas the biobead #2 and #3 reactors were dominated by the members of Proteobacteria. DGGE analysis with the ammonia monooxygenase (amoA) gene supported the observation from the MPN test that the biofilms of September were fully developed and specialized for nitrification in the biobead reactor #3. All of the DNA sequences of the amoA DGGE bands were very similar to the sequence of the amoA gene of Nitrosomonas species, the presence of which is typical in the biological aerated filters. The results of this study showed that organic and inorganic nutrients were efficiently removed by both denitrifying microbial populations in the anaerobic tank and heterotrophic and nitrifying bacterial biofilms well-formed in the three functional biobead reactors in the Aerated Up-Flow Biobead process.     

Probing of Microbial Biofilm Communities for Coadhesion Partners

Investigations of interbacterial adhesion in dental plaque development are currently limited by the lack of a convenient assay to screen the multitude of species present in oral biofilms. To overcome this limitation, we developed a solid-phase fluorescence-based screening method to detect and identify coadhesive partner organisms in mixed-species biofilms. The applicability of this method was demonstrated using coaggregating strains of type 2 fimbrial adhesin-bearing actinomyces and receptor polysaccharide (RPS)-bearing streptococci. Specific adhesin/receptor-mediated coadhesion was detected by overlaying bacterial strains immobilized to a nitrocellulose membrane with a suspended, fluorescein-labeled bacterial partner strain. Coadhesion was comparable regardless of which cell type was labeled and which was immobilized. Formaldehyde treatment of bacteria, either in suspension or immobilized on nitrocellulose, abolished actinomyces type 2 fimbrial adhesin but not streptococcal RPS function, thereby providing a simple method for assigning complementary adhesins and glycan receptors to members of a coadhering pair. The method''s broader applicability was shown by overlaying colony lifts of dental plaque biofilm cultures with fluorescein-labeled strains of type 2 fimbriated Actinomyces naeslundii or RPS-bearing Streptococcus oralis. Prominent coadhesion partners included not only streptococci and actinomyces, as expected, but also other bacteria not identified in previous coaggregation studies, such as adhesin- or receptor-bearing strains of Neisseria pharyngitis, Rothia dentocariosa, and Kingella oralis. The ability to comprehensively screen complex microbial communities for coadhesion partners of specific microorganisms opens a new approach in studies of dental plaque and other mixed-species biofilms.     

Induction of larval metamorphosis of the coral Acropora millepora by tetrabromopyrrole isolated from a Pseudoalteromonas bacterium

The induction of larval attachment and metamorphosis of benthic marine invertebrates is widely considered to rely on habitat specific cues. While microbial biofilms on marine hard substrates have received considerable attention as specific signals for a wide and phylogenetically diverse array of marine invertebrates, the presumed chemical settlement signals produced by the bacteria have to date not been characterized. Here we isolated and fully characterized the first chemical signal from bacteria that induced larval metamorphosis of acroporid coral larvae (Acropora millepora). The metamorphic cue was identified as tetrabromopyrrole (TBP) in four bacterial Pseudoalteromonas strains among a culture library of 225 isolates obtained from the crustose coralline algae Neogoniolithon fosliei and Hydrolithon onkodes. Coral planulae transformed into fully developed polyps within 6 h, but only a small proportion of these polyps attached to the substratum. The biofilm cell density of the four bacterial strains had no influence on the ratio of attached vs. non-attached polyps. Larval bioassays with ethanolic extracts of the bacterial isolates, as well as synthetic TBP resulted in consistent responses of coral planulae to various doses of TBP. The lowest bacterial density of one of the Pseudoalteromonas strains which induced metamorphosis was 7,000 cells mm(-2) in laboratory assays, which is on the order of 0.1-1% of the total numbers of bacteria typically found on such surfaces. These results, in which an actual cue from bacteria has been characterized for the first time, contribute significantly towards understanding the complex process of acroporid coral larval settlement mediated through epibiotic microbial biofilms on crustose coralline algae.     

Temperature Gradient Gel Electrophoresis Analysis of 16S rRNA from Human Fecal Samples Reveals Stable and Host-Specific Communities of Active Bacteria

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.     

The effectiveness of antibiotics on bacteria in biofilms

Bacteria can form different types of communities, united by common notion: biofilms. The aim of the present study was to determine the capacity of different antibiotics to penetrate into biofilms and act on unrelated bacteria. The study revealed that the formation of barriers between the community and the environment on artificial biofilms occurred in all strains of unrelated Gram-positive and Gram-negative bacteria used in this investigation. The capacity of antibiotics to penetrate into biofilms varied in different strains of the same species. For certain antibiotics similarity in their penetrating capacity was found to exist with respect to biofilms of unrelated bacteria. The penetration of antibiotics into mixed biofilms depended on the strain which determined its minimal value, so that the protection of one microorganism by another was thus observed. The method for the evaluation of the effectiveness of antibiotic penetration into bacterial biofilms, suitable for use in bacteriological laboratories, is proposed.     

Efficacy of a Marine Bacterial Nuclease against Biofilm Forming Microorganisms Isolated from Chronic Rhinosinusitis

Background

The persistent colonization of paranasal sinus mucosa by microbial biofilms is a major factor in the pathogenesis of chronic rhinosinusitis (CRS). Control of microorganisms within biofilms is hampered by the presence of viscous extracellular polymers of host or microbial origin, including nucleic acids. The aim of this study was to investigate the role of extracellular DNA in biofilm formation by bacteria associated with CRS.

Methods/Principal Findings

Obstructive mucin was collected from patients during functional endoscopic sinus surgery. Examination of the mucous by transmission electron microscopy revealed an acellular matrix punctuated occasionally with host cells in varying states of degradation. Bacteria were observed in biofilms on mucosal biopsies, and between two and six different species were isolated from each of 20 different patient samples. In total, 16 different bacterial genera were isolated, of which the most commonly identified organisms were coagulase-negative staphylococci, Staphylococcus aureus and α-haemolytic streptococci. Twenty-four fresh clinical isolates were selected for investigation of biofilm formation in vitro using a microplate model system. Biofilms formed by 14 strains, including all 9 extracellular nuclease-producing bacteria, were significantly disrupted by treatment with a novel bacterial deoxyribonuclease, NucB, isolated from a marine strain of Bacillus licheniformis. Extracellular biofilm matrix was observed in untreated samples but not in those treated with NucB and extracellular DNA was purified from in vitro biofilms.

Conclusion/Significance

Our data demonstrate that bacteria associated with CRS form robust biofilms which can be reduced by treatment with matrix-degrading enzymes such as NucB. The dispersal of bacterial biofilms with NucB may offer an additional therapeutic target for CRS sufferers.     

Characterization of marine bacteria highly resistant to mercury exhibiting multiple resistances to toxic chemicals

Several strains of bacteria unusually highly resistant to mercury were isolated from seawater and marine sediment samples and identified by 16S rDNA sequencing and were also characterized by a battery of biochemical and morphological tests. The bacterial isolates were identified to belong to the genera Pseudomonas, Alcaligenes, Brevibacterium and Bacillus. Many of the chosen isolates were tested for growth in the presence of different heavy metals and a variety of xenobiotics. Growth curves of all six bacteria highly resistant to mercury examined for growth at different concentrations of Hg exhibited prolonged lag phase, during which time necessary physiological adaptations to toxic milieu were undergone. All the strains tested for antibiotic resistance showed little to no effect of antibiotics on their normal growth. Results of this study demonstrate the occurrence of diverse groups of marine prokaryotes capable of high tolerance to mercury with a potential to degrade a variety of toxic heavy metals and xenobiotics.     

The isolation and initial characterization of mercury resistant chemolithotrophic thermophilic bacteria from mercury rich geothermal springs

Mercury rich geothermal springs are likely environments where mercury resistance is critical to microbial life and where microbe-mercury interactions may have evolved. Eleven facultative thermophilic and chemolithoautotrophic, thiosulfate oxidizing bacteria were isolated from thiosulfate enrichments of biofilms from mercury rich hot sulfidic springs in Mount Amiata, Italy. Some strains were highly resistant to mercury (≥200 μM HgCl₂) regardless of its presence or absence during primary enrichments, and three reduced ionic mercury to its elemental form. The gene encoding for the mercuric reductase enzyme (MerA), was amplified by PCR from seven strains. However, one highly resistant strain did not reduce mercury nor carried merA, suggesting an alternative resistance mechanism. All strains were members of the order Bacillales and were most closely related to previously described thermophiles belonging to the Firmicutes. Phylogenetic analyses clustered the MerA of the isolates in two supported novel nodes within the Firmicutes lineage and a comparison with the 16S rRNA gene tree suggested at least one case of horizontal gene transfer. Overall, the results show that the thermophilic thiosulfate oxidizing isolates were adapted to life in presence of mercury mostly, but not exclusively, by possessing MerA. These findings suggest that reduction of mercury by chemolithotrophic thermophilic bacteria may mobilize mercury from sulfur and iron deposits in geothermal environments.     

Bacteriophage and associated polysaccharide depolymerases – novel tools for study of bacterial biofilms

Bacteriophage for three representative strains of Gram-negative biofilm bacteria have proved to be of widespread occurrence. Lytic bacteriophage have been isolated from local sewage for the bacterium 1·15, an exopolysaccharide (EPS)-producing pseudomonad found originally as a component of biofilms in a local river, and for two Enterobacter agglomerans strains from industrial biofilms. Representative examples of all three bacteriophage possess a relatively low burst size and on solid media, exhibit very large plaques surrounded by a wide halo (5–20 mm) indicative of polysaccharide depolymerase action. The bacteriophage are thus similar to other viruses for EPS-producing bacteria in inducing the synthesis of enzymes degrading the polymers which occlude the bacterial cell surface. In each preparation, the polysaccharase activity was associated both with sedimented phage particles and with the supernate of bacterial lysates. The enzymes have been partially purified and used to prepare polysaccharide digests in which the major products from each polysaccharide are the presumed repeat units of the polymers or oligomers of these. The soluble phage enzymes each degrade their substrate by acting as endo -glycanohydrolases. The phage and their associated enzymes thus provide very useful highly specific tools for studies of biofilms incorporating the bacterial host strains. Their potential applications in studies on bacterial biofilms are discussed.     

An outbreak of nonflocculating catabolic populations caused the breakdown of a phenol-digesting activated-sludge process.

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter-1 day-1 and then to 1.5 g liter-1 day-1. After the loading rate was increased to 1.5 g liter-1 day-1, nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter-1 day-1. A total of 41 bacterial strains with different repetitive extragenic palindromic sequence PCR patterns were isolated from the activated sludge under different phenol-loading conditions, and the 16S rDNA and gyrB fragments of these strains were PCR amplified and sequenced. Some bacterial isolates could be associated with major TGGE bands by comparing the 16S rDNA sequences. All of the bacterial strains affiliated with the R6 population had almost identical 16S rDNA sequences, while the gyrB phylogenetic analysis divided these strains into two physiologically divergent groups; both of these groups of strains could grow on phenol, while one group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which specific gyrB sequences were used as the primers showed that a population shift from R6F to R6T occurred following the increase in the phenol-loading rate to 1.5 g liter-1 day-1. The R10 population corresponded to nonflocculating phenol-degrading bacteria. Our results suggest that an outbreak of nonflocculating catabolic populations caused the breakdown of the activated-sludge process. This study also demonstrated the usefulness of gyrB-targeted fine population analyses in microbial ecology.     

Molecular analyses of the intestinal microbiota of chimpanzees in the wild and in captivity

Little information is available regarding the intestinal bacteria of chimpanzees in the wild, due to the technical difficulties of studying intestinal bacteria in the field. In this study, molecular-based bacterial analyses were performed to overcome this difficulty because polymerase chain reaction (PCR)-based methods, such as temperature gradient gel electrophoresis (TGGE) and amplified ribosomal DNA restriction analysis (ARDRA), of the bacterial 16S rRNA gene can be applied to ethanol-fixed fecal samples. The common presence of bacteria belonging to the Clostridium rRNA sub-group XIVa, such as Ruminococcus obeum and Eubacterium sp., was indicated for Bossou wild chimpanzees by ARDRA. TGGE on partial 16S rDNA followed by hierarchical clustering analysis showed a systematic difference in the composition of intestinal microbiota between wild and captive chimpanzees. However, several TGGE bands commonly shared by wild and captured chimpanzees were excised, and their sequences were obtained. They were suggested to be the Clostridium leptum subgroup bacteria, Lactobacillus gasseri-like bacterium, and Bifidobacterium pseudocatenulatum- or B. catenulatum-like bacterium. These may be considered as common intestinal bacteria for chimpanzees, and may be transmitted vertically over generations.     

Mercury-resistant bacteria from permafrost sediments and prospects for their use in comparative studies of mercury resistance determinants

Mercury-resistant bacteria were isolated from permafrost sediments of Kolyma lowland and Canada existing over five thousand to two million years. Their content was shown to vary within the range 0.001-2.9% and to depend on the amount of mercury in sampling sites (coefficient of correlation 0.75). A collection of mercury-resistant bacterial strains was created. In this collection, various representatives of both Gram-positive bacteria (Bacillus, Exiguobacterium, Micrococcus, Arthrobacter) and Gram-negative bacteria (Pseudomonas, Acinetobacter, Plesiomonas, Myxobacteriales) were identified. Most resistant bacteria were found to contain determinants homologous to mer-operons of contemporary bacteria. The isolated strains of paleobacteria are proposed to be used for a comparative structural study of contemporary and ancient plasmids and transposons carrying mercury resistance determinants.