Showing papers in "Microbes and Environments in 2009"

Endophytic bacteria in the rice plant.

Abstract: Endophytic bacteria are defined as bacteria detected inside surface-sterilized plants or extracted from inside plants and having no visibly harmful effects on the plants. Various kinds of endophytic bacteria, such as Pantoea, Methylobacterium, Azospirillum, Herbaspirillum, Burkholderia and Rhizobium etc., have been found inside rice plants. This minireview summarizes and discusses recent studies of endophytic bacteria residing in rice plants, focusing on flora, origin, movement, and interaction with plants/other microbes and referring to endophytes in other plants. The findings concerning bacterial flora obtained by cultural and non-cultural methods are also compared and discussed. Some attempts to apply endophytes to the rice plant and the resultant effects are introduced. The future perspective to deepen the study of endophytes in terms of both application and basic science is considered.

Cultivation of uncultured chloroflexi subphyla: significance and ecophysiology of formerly uncultured chloroflexi 'subphylum i' with natural and biotechnological relevance.

Abstract: Cultivation-independent molecular surveys have shown members of the bacterial phylum Chloroflexi to be ubiquitous in various natural and artificial ecosystems. Among the subphylum-level taxa of the Chloroflexi known to date, the formerly uncultured 'subphylum I' had well been recognized as a typical group that contains a number of environmental gene clones with no culture representatives. In order to reveal their ecophysiology, attempts were made over the past decade to domesticate them into laboratory cultures, and significant advances have been made in cultivating strains belonging to the group. The microorganisms characterized so far include seven species in six genera, i.e., Anaerolinea, Levilinea, Leptolinea, Bellilinea, Longilinea, and Caldilinea, and were proposed to represent two classes, Anaerolineae and Caldilineae, providing solid insights into the phenotypic and genetic properties common to the group. Another subphylum-level uncultured group of the Chloroflexi, i.e., the class Ktedonobacteria, has also been represented recently by a cultured strain. In addition to the results from these tangible cultures, data obtained from functional analyses of uncultured Chloroflexi populations by assessing substrate uptake patterns are accumulating at an encouraging rate. In this review, recent findings on the ecological significance and possible ecophysiological roles of 'Chloroflexi subphylum I' are discussed based on findings from both the characteristics of the cultured Chloroflexi and molecular-based analyses.

Endosymbiotic bacteria in insects: their diversity and culturability.

Abstract: Many animals and plants possess symbiotic microorganisms inside their body, wherein intimate interactions occur between the partners. The Insecta, often rated as the most diverse animal group, show various types of endosymbiotic associations, ranging from obligate mutualism to facultative parasitism. Although technological advancements in culture-independent molecular techniques, such as quantitative PCR, molecular phylogeny and in situ hybridization, as well as genomic and metagenomic analyses, have allowed us to directly observe endosymbiotic associations in vivo, the molecular mechanisms underlying insect-microbe interactions are not well understood, because most of these insect endosymbionts are neither culturable nor genetically manipulatable. However, recent studies have succeeded in the isolation of several facultative symbionts by using insect cell lines or axenic media, revolutionizing studies of insect endosymbiosis. This article reviews the amazing diversity of bacterial endosymbiosis in insects, focusing on several model systems with culturable endosymbionts, which provide a new perspective towards understanding how intimate symbiotic associations may have evolved and how they are maintained within insects.

Rice seeds as sources of endophytic bacteria.

Abstract: Endophytic bacteria are considered to originate from the external environment. To examine the hypothesis that rice (Oryza sativa, cultivar Kinuhikari) seeds are a source of endophytic bacteria, we isolated endophytic bacteria from the shoots, remains of the seeds, and roots of rice seedlings that were aseptically cultivated in vitro from surface-disinfected seeds. Of the various bacterial strains isolated, the closest relatives, identified by 16S rRNA gene sequencing, were: Bacillus firmus, B. fusiformis, B. pumilus, Caulobacter crescentus, Kocuria palustris, Micrococcus luteus, Methylobacterium fujisawaense, Me. radiotolerans, and Pantoea ananatis. The latter three species have been detected frequently inside both rice seedlings and mature rice plants. These results indicate that rice seeds are an important source of endophytic bacteria. The bacteria that colonize the seed interior appear to infect the subsequent generation via rice seeds and become the dominant endophytic species in the mature plant.

Poly(ethylene terephthalate) polymer surfaces as a substrate for bacterial attachment and biofilm formation.

Abstract: Plastic debris causes extensive damage to the marine environment, largely due to its ability to resist degradation. Attachment on plastic surfaces is a key initiation process for their degradation. The tendency of environmental marine bacteria to adhere to poly(ethylene terephthalate) (PET) plastic surfaces as a model material was investigated. It was found that the overall number of heterotrophic bacteria in a sample of sea water taken from St. Kilda Beach, Melbourne, Australia, was significantly reduced after six months from 4.2-4.7×10(3) cfu mL(-1) to below detectable levels on both full-strength and oligotrophic marine agar plates. The extinction of oligotrophs after six months was detected in all samples. In contrast, the overall bacterial number recovered on full strength marine agar from the sample flasks with PET did not dramatically reduce. Heterotrophic bacteria recovered on full-strength marine agar plates six months after the commencement of the experiment were found to have suitable metabolic activity to survive in sea water while attaching to the PET plastic surface followed by the commencement of biofilm formation.

Comparative analysis of bacterial and archaeal communities in methanogenic sludge granules from upflow anaerobic sludge blanket reactors treating various food-processing, high-strength organic wastewaters.

Abstract: A comprehensive survey of bacterial and archaeal community structures within granular sludges taken from twelve different types of full-scale, food-processing wastewater-treating, upflow anaerobic sludge blanket (UASB) reactors was performed with a 16S rRNA gene-based clone library method. In total, 1,282 bacterial 16S rRNA gene clones and 722 archaeal clones were analyzed, and their identities were determined by phylogenetic analyses. Overall, clones belonging to the bacterial phyla Proteobacteria (the class Deltaproteobacteria in particular), Firmicutes, Spirochaetes, and Bacteroidetes were observed in abundance within the bacterial clone libraries examined, indicating common bacterial denominators in such treatment systems. Within the domain Archaea, clones affiliated with the classes Methanomicrobia and Methanobacteria were found to be abundant in the archaeal libraries. In relation to features of reactor performance (such as chemical oxygen demand removal, fatty acid accumulation, and sludge bulking), possible representative phylotypes likely to be associated with process failures, such as sludge bulking and the accumulation of propionate, were found in comparative analyses of the distribution of phylotypes in the sludge libraries.

Powdery mildew-infection changes bacterial community composition in the phyllosphere.

Abstract: To investigate changes in bacterial communities associated with a fungal foliar disease, epiphytic bacteria from powdery mildew-infected and uninfected leaves of cucumber and Japanese spindle were analyzed using both culture-dependent and -independent methods. Dilution plate counting suggested that powdery mildew-infected leaves likely accommodated larger populations of phyllosphere bacteria than uninfected leaves. Community-level physiological profiles (CLPP) also indicated that functional diversity, richness, and evenness of bacterial communities were significantly greater in the phyllosphere of powdery mildew-infected leaves. Genotype diversity and richness based on band patterns of denaturing gradient gel electrophoresis (DGGE) of the phyllosphere bacterial community were greater for leaves infected by powdery mildew. A principle component analysis of CLPP and DGGE patterns revealed a clear difference between infected and uninfected leaves of both plant species. These results suggest that powdery mildew-infection results in larger bacterial populations, and greater diversity and richness, and also changes the structure of the phyllosphere bacterial community. Furthermore, DNA sequences of the DGGE bands that showed greater intensity in the infected than uninfected leaves, differed between cucumber and Japanese spindle. This suggests that specific bacteria are associated with the plant species accompanying this fungal infection.

Phylogenetic analyses of bacterial communities developed in a cassette-electrode microbial fuel cell.

Abstract: Quantitative and qualitative differences were analyzed between planktonic and anode-biofilm bacterial communities developed in a cassette-electrode microbial fuel cell treating starch, peptone, and fish extract. Quantitative analyses based on protein contents and rRNA-gene copy numbers indicated that planktonic microbes were over eight-times more abundant than anode-biofilm microbes. Clone-library analyses of PCR-amplified 16S rRNA gene fragments revealed the presence of bacteria affiliated with the phyla Bacteroidetes, Firmicutes, and Proteobacteria in these two communities. The most abundant sequence was affiliated with the family Porphyromonadaceae, and accounted for over 50% and 20% of all the sequences in the planktonic- and biofilm-microbe libraries, respectively.

Snow molds: A group of fungi that prevail under snow.

Abstract: Snow molds are a group of fungi that attack dormant plants under snow. In this paper, their survival strategies are illustrated with regard to adaptation to the unique environment under snow. Snow molds consist of diverse taxonomic groups and are divided into obligate and facultative fungi. Obligate snow molds exclusively prevail during winter with or without snow, whereas facultative snow molds can thrive even in the growing season of plants. Snow molds grow at low temperatures in habitats where antagonists are practically absent, and host plants deteriorate due to inhibited photosynthesis under snow. These features characterize snow molds as opportunistic parasites. The environment under snow represents a habitat where resources available are limited. There are two contrasting strategies for resource utilization, i.e., individualisms and collectivism. Freeze tolerance is also critical for them to survive freezing temperatures, and several mechanisms are illustrated. Finally, strategies to cope with annual fluctuations in snow cover are discussed in terms of predictability of the habitat.

Biogenic amines in rhizobia and legume root nodules.

Abstract: Root-nodule bacteria (rhizobia) are of great importance for nitrogen acquisition through symbiotic nitrogen fixation in a wide variety of leguminous plants. These bacteria differ from most other soil microorganisms by taking dual forms, i.e. a free-living form in soils and a symbiotic form inside of host legumes. Therefore, they should have a versatile strategy for survival, whether inhabiting soils or root nodules formed through rhizobia-legume interactions. Rhizobia generally contain large amounts of the biogenic amine homospermidine, an analog of spermidine which is an essential cellular component in most living systems. The external pH, salinity and a rapid change in osmolarity are thought to be significant environmental factors affecting the persistence of rhizobia. The present review describes the regulation of homospermidine biosynthesis in response to environmental stress and its possible functional role in rhizobia. Legume root nodules, an alternative habitat of rhizobia, usually contain a variety of biogenic amines besides homospermidine and the occurrence of some of these amines is closely associated with rhizobial infections. In the second half of this review, novel biogenic amines found in certain legume root nodules and the mechanism of their synthesis involving cooperation between the rhizobia and host legume cells are also described.

Characterization of active microbes in a full-scale anaerobic fluidized bed reactor treating phenolic wastewater.

Abstract: This study investigated the active microbial community in a full-scale granular activated carbon-anaerobic fluidized bed (GAC-AFB) reactor treating wastewater from the manufacturing of phenolic resin, using 16S rRNA-based molecular analyses. The results of cDNA from 16S rRNA revealed that Methanosaeta-related (83.9% of archaeal clones) and Syntrophorhabdaceae (formerly named Deltaproteobacteria group TA)-related (68.9% of bacterial clones) microorganisms were as the most predominant populations in the phenol-degrading GAC-AFB reactor. The high abundance of Syntrophorhabdaceae was supported by a terminal restriction fragment length polymorphism (T-RFLP) analysis, which showed that a Syntrophorhabdaceae-like fragment of 119 bp (~80% of total fragments) was the most predominant phylotype. Furthermore, fluorescence in situ hybridization (FISH) analyses suggested that Syntrophus- and Chloroflexi-like cells were also in high abundance in the GAC biofilm. A non-layered structure of microorganisms was found in the GAC biofilm, where Methanosaeta (thick filamentous), Syntrophorhabdaceae (oval-shaped), Syntrophus (small rods) and Chloroflexi (thin-filamentous) were randomly distributed with high abundance. These findings greatly improve our understanding of the diversity and distribution of microbial populations in a full-scale mesophilic bioreactor treating an actual phenol-containing waste stream.

Nitrous oxide emission and microbial community in the rhizosphere of nodulated soybeans during the late growth period.

Abstract: We examined N2O emissions from the rhizosphere of field-grown soybeans during the late growth stage (99-117 days after sowing). Marked emissions were detected from the nodulated root systems of field-grown soybeans, whereas a non-nodulating soybean mutant showed no emission. Degraded nodules exclusively generated the N2O. A culture-independent analysis of microbial communities showed Bradyrhizobium sp., Acidvorax facilis, Salmonella enterica, Xanthomonas sp., Enterobacter cloacae, Pseudomonas putida, Fusarium sp., nematodes, and other protozoans to be more abundant in the degraded nodules, suggesting that some of these organisms participate in the N2O emission process in the soybean rhizosphere.

Seasonal Changes in Abundance of Ammonia-Oxidizing Archaea and Ammonia-Oxidizing Bacteria and Their Nitrification in Sand of an Eelgrass Zone

Abstract: Seasonal changes in the abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) within the sand of an eelgrass (Zostera marina) zone were examined by a quantitative PCR of both crenarchaeotal and betaproteobacterial ammonia monooxygenase alpha subunit (amoA) genes together with temperature and concentrations of ammonium, nitrite, and nitrate from May 2007 to June 2008 at Tanoura Bay, Shizuoka, Japan. The abundance of both amoAs in the sand between May and June 2007 and between January and March 2008 was 1.5 to 2 orders of magnitude higher than the 104 copies g-1 of estimated amoA between September and December. Archaeal amoA was more diverse than betaproteobacterial amoA. Betaproteobacterial amoA clone libraries were dominated by Nitrosospira-like sequence types. An incubation experiment was conducted with sands collected in February 2008 and community structure was analyzed based on reverse-transcribed amoAs. RNA was extracted from sand incubated for 12 days at 30°C, 17 days at 20°C, and 80 days at 10°C. Different amoA clones were detected from in situ sand and incubated sand. This study reveals clear evidence of seasonal change in the abundance of AOA and AOB within the sand of an eelgrass zone.

The influence of soil organic matter on DNA adsorptions on andosols.

Abstract: The influence of soil organic matter on DNA adsorption in andosols was investigated using various andosol samples including hydrogen peroxide (H2O2)-treated, heated (400°C), and slurry-added soils. Remarkably less DNA was adsorbed in the slurry-added soil than the original soil. The increase in soil organic matter with the addition of slurry had an obvious negative influence on the adsorption. The decrease in organic matter with H2O2 treatment slightly raised DNA adsorption per unit weight. Adsorption maxima estimated from a simple Langmuir equation were higher in the samples removed of organic matter by the H2O2 treatment and heating at 400°C than in the untreated soil, although surface area was greatly decreased by both treatments. There was no correlation between the total carbon (T-C) content and the estimated DNA adsorption maxima of any of the soil samples. These results suggest little contribution of soil organic matter to DNA adsorption in andosols.

Distribution of N-Acylhomoserine Lactone-Producing Fluorescent Pseudomonads in the Phyllosphere and Rhizosphere of Potato (Solanum tuberosum L.).

Abstract: Four hundred and fifty nine isolates of fluorescent pseudomonads were obtained from the leaves and roots of potato plants. Of these, 20 leaf isolates and 28 root isolates induced violacein production in two N-acylhomoserine lactone (AHL)-reporter strains-Chromobacterium violaceum CV026 and VIR24. VIR24 is a new reporter strain for long N-acyl-chain-homoserine lactones, which can not be detected by CV026. Thin-layer chromatography revealed that the isolates produced multiple AHL molecules. We compared the 16S rRNA gene sequences of these isolates with sequences from a known database, and examined phylogenetic relationships. The AHL-producing isolates generally separated into three groups. Group I was mostly composed of leaf isolates, and group III, root isolates. Group II comprised both leaf and root isolates. There was a correlation between the phylogenetic cluster and the AHL molecules produced and some phenotypic characteristics. Our study confirmed that AHL-producing fluorescent pseudomonads could be distinguished in the phyllosphere and rhizosphere of potato plants.

Nutrient-rich microhabitats within biofilms are synchronized with the external environment.

Abstract: The nutrient ion concentrations in the interstitial waters of biofilms (BFs) formed on reed and stone surfaces were investigated in the northern and southern basins of Lake Biwa over several years. The following were observed for both types of BF: 1) Concentrations of ammonium, nitrate, nitrite, and phosphate ions were much (hundreds to thousands of times) higher in the BFs than in the surrounding lake water; 2) the concentration of ions, especially nitrate ions, in the BFs changed seasonally, being higher from winter to spring and lower from summer to autumn, synchronizing with the changes in the lake water; 3) dissolved-form N:P ratios were higher in the lake water than BFs; and 4) the bacterial flora of the BFs differed from that of the lake water, with smaller seasonal variations. The present study reveals for the first time that the inside of BFs in a natural environment is rich in nutrient ions and shows similar seasonal changes as the lake water. The BFs in an aquatic environment provide a microenvironment capable of sustaining a specific bacterial flora different from that in the surrounding lake water.

Nodulation-dependent communities of culturable bacterial endophytes from stems of field-grown soybeans.

Abstract: Endophytic bacteria (247 isolates) were randomly isolated from surface-sterilized stems of non-nodulated (Nod-), wild-type nodulated (Nod+), and hypernodulated (Nod++) soybeans (Glycine max [L.] Merr) on three agar media (R2A, nutrient agar, and potato dextrose agar). Their diversity was compared on the basis of 16S rRNA gene sequences. The phylogenetic composition depended on the soybean nodulation phenotype, although diversity indexes were not correlated with nodulation phenotype. The most abundant phylum throughout soybean lines tested was Proteobacteria (58-79%). Gammaproteobacteria was the dominant class (21-72%) with a group of Pseudomonas sp. significantly abundant in Nod+ soybeans. A high abundance of Alphaproteobacteria was observed in Nod- soybeans, which was explained by the increase in bacterial isolates of the families Rhizobiaceae and Sphingomonadaceae. A far greater abundance of Firmicutes was observed in Nod- and Nod++ mutant soybeans than in Nod+ soybeans. An impact of culture media on the diversity of isolated endophytic bacteria was also observed: The highest diversity indexes were obtained on the R2A medium, which enabled us to access Alphaproteobacteria and other phyla more frequently. The above results indicated that the extent of nodulation changes the phylogenetic composition of culturable bacterial endophytes in soybean stems.

Phylogenetic and Transcriptional Analyses of a Tetrachloroethene-Dechlorinating "Dehalococcoides" Enrichment Culture TUT2264 and Its Reductive-Dehalogenase Genes

Abstract: A dechlorinating microbial enrichment culture designated TUT2264 was cultured with tetrachloroethene and then characterized for tetrachloroethene-dechlorination by culture-dependent and -independent methods. The fourth-transferred TUT2264 culture completely dechlorinated tetrachloroethene and trichloroethene, and accumulated more trans-1,2-dichloroethene than cis-1,2-dichloroethene. A real-time PCR analysis revealed that "Dehalococcoides" cells made up only 0.3% of the total. Eight distinct reductive-dehalogenase-homologous genes (rdh) were detected with degenerate primers. Phylogenetic analyses revealed 5 of the 8 RdhAs to be very similar to RdhAs reported previously but not to share 100% identity. Transcriptional levels were quantified as the number of transcripts per rdhA by combining the reverse transcription real-time PCR and exogenous internal reference mRNA methods. TUT2264 responded to all the chloroethenes tested. rdhA4 was transcribed with all chloroethenes except vinyl chloride, whereas rdhA8 was only transcribed on tetrachloroethene. Furthermore, multiple rdhAs were induced to express by a single chloroethene as a growth-supporting or non-supporting substrate. These results suggested that Rdhs are multi-functional and rdhAs are a powerful tool to evaluate the potential of contaminated sites and isolates to dechlorinate chloroethenes.

Transient Transformation of Frankia by Fusion Marker Genes in Liquid Culture

Abstract: Frankia is a nitrogen-fixing actinobacterium that establishes root nodule symbiosis with actinorhizal plants. The molecular basis of the symbiosis is largely unknown because genetic manipulation of Frankia has not been feasible. In this study we made novel technical attempts to transform Frankia strain CcI3. We generated fusion marker genes consisting of a tetracycline resistance gene with a high codon usage similarity to Frankia's and promoters of the strain's translation initiation factor 3 gene. We flanked the fusion genes with genomic sequences from strain CcI3 in the expectation that they would be integrated into the targeted site by homologous recombination. We introduced the transformation constructs into Frankia cells by electroporation and selected transformants in liquid media. The growth of antibiotic resistant cells was dependent on the presence of construct DNA. PCR analysis of the genome and reverse transcription-PCR analysis confirmed that the marker genes were introduced into the cells. Integration of the marker genes into the chromosome by homologous recombination did occur, but at a low frequency. Most of the constructs were not integrated into the chromosome and existed as degraded molecules in the cells. Marker genes declined in the transformant population during maintenance, showing that the transformation was unstable.

Analysis of bacterial community diversity in anaerobic fluidized bed bioreactors treating 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) using 16S rRNA gene clone libraries.

Abstract: Clone libraries were used to evaluate the effects of 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) on bacterial populations within three anaerobic bioreactors. Prior to the addition of DNAN and MNA greater than 69% of the clones in each reactor were identified as a single Desulfuromonales species. However, after 60 days of treatment the Desulfuromonales distribution decreased to less than 13% of the distribution and a clone identified as a Levilinea sp. became the dominant organism at greater than 27% of the clone distribution in each reactor suggesting the species may play an important roll in the reduction of DNAN and MNA.

Response to environmental stress as a global phenomenon in biology: the example of microorganisms.

Abstract: Any modification of the environment that leads to a physiological, genetic, or epigenetic adaptive response in microorganisms may be considered as a stress. Historically, forms of stresses affecting biological structures were classified either as non-thermal, such as osmotic, oxidative, or acid stress or as thermal stress, hot or cold. Currently, the classification in biology is as abiotic, including physical and chemical stress, or biotic. The aim of this mini-review is to show, through the example of microorganisms, that the response to stress can be considered, in biology, as a global phenomenon, which can be extended to anthropogenic pressure.

Analysis of How a Biofilm Forms on the Surface of the Aquatic Macrophyte Phragmites australis.

Abstract: The process by which a biofilm forms on the surface of the aquatic macrophyte Phragmites australis was investigated over a period of about two months (from mid-May to late-July, 2008) in Lake Biwa. The biofilm formed relatively quickly, its wet weight per unit area after seven day being that of a mature biofilm. This speed can be attributed to the many active bacteria in the early stage of its formation and the extracellular polymeric substances (EPS) they produce. The EPS carried electric charges that attracted nutrient ions from surrounding lake water, which, by electrostatic interaction, reached a high concentration as early as day 7 of the formation process. This significantly affected the biofilm community, which differed greatly from that of the lake water even at the beginning of biofilm formation. Brown amorphous compounds (a complex of organic and inorganic substances), covered the biofilm in the second half of its formation process producing a different community structure from that initially. This study revealed a fast and dynamic process of biofilm formation on the reed surface of reed.

Phylogenetic diversity and symbiotic effectiveness of root-nodulating bacteria associated with cowpea in the South-West area of Japan

Abstract: The phylogenetic diversity of cowpea root-nodulating bacteria in the South-West of Japan was investigated using 60 isolates. Seeds of cowpea were aseptically sown in vermiculite and inoculated with a suspension of Cowpea Soil (CS) or Bean Soil (BS) or without a soil suspension as a control. CS and BS were collected from the Kyushu University's farm (Japan) at sites where cowpea and bean, respectively, have been cultivated previously. Based on an analysis of the 16S rRNA gene and the Internal Transcribed Spacer (ITS) sequence between the 16S and 23S rRNA genes, 56 isolates were assigned to the genus Bradyrhizobium, while one isolate was found to be closely related to the genus Ralstonia. The ITS-based phylogeny showed 53 isolates, 2 isolates, and 1 isolate, to be closely related to B. yuanmingense, B. elkanii and B. japonicum, respectively, suggesting that B. yuanmingense strains predominated in the soils. Among the isolates tested, B. yuanmingense TSC10 and TTC9 exhibited a greater symbiotic activity and could be considered efficient inoculants for cowpea.

Variation and phylogeny of Fusarium oxysporum isolates based on nucleotide sequences of polygalacturonase genes.

Abstract: The nucleotide sequences of two endopolygalacturonase genes (pg1 and pg5) and two exopolygalacturonase genes (pgx1 and pgx4), which encode members of a major family of secreted cell-wall-degrading enzymes (CWDEs), were compared to detect the extent of genetic variation among isolates of Fusarium oxysporum. The nucleotide variation rate in exons was 0.23-0.93%, higher than that in introns (0.01-0.64%) and untranslated regions (UTRs) (0.07-0.25%), resulting in 0.05-0.31% variation in amino acids. pgx1 exhibited the most genetic diversity. Phylogenetic analysis of the four genes, which reside on different chromosomes, revealed different evolutionary patterns for each. Our results suggest a biased evolution of the polygalacturonase genes of F. oxysporum, or alternatively, that the genes were acquired at different times during evolution.

Reductive Dechlorination of Polychlorinated Biphenyls and Dibenzo-p-Dioxins in an Enrichment Culture Containing Dehalobacter Species

Abstract: The dechlorination of polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins was examined in an enrichment culture (KFL culture) that contained two phylotypes of Dehalobacter, FTH1 and FTH2. The KFL culture dechlorinated 2,3,4,5-tetrachlorobiphenyl, 2,3,4-trichorobiphenyl (2,3,4-TriCB), and 1,2,3-trichlorodibenzo-p-dioxin (1,2,3-TriCDD). Quantitative real-time PCR targeting FTH1 and FTH2 revealed significant increases with the addition of PCBs and 1,2,3-TriCDD, suggesting halorespiring growth of the Dehalobacter species in the KFL culture. This study demonstrated the reductive dechlorination of PCBs and 1,2,3-TriCDD by Dehalobacter species in a sediment-free culture and a novel dechlorination pathway, the conversion of 2,3,4-TriCB to 4-monochlorobiphenyl via 3,4-dichlorobiphenyl.

Nitrogenase activity (acetylene reduction) of an iron-oxidizing leptospirillum strain cultured as a pioneer microbe from a recent volcanic deposit on miyake-jima, Japan.

Abstract: The genus Leptospirillum is known to dominate acid mine drainage and bioleaching systems. In this paper, we describe the isolation of iron-oxidizing bacteria closely related to Leptospirillum ferrooxidans from an acidic volcanic ash deposit on the island of Miyake (Miyake-jima), Japan. We further show the nitrogenase activity (acetylene reduction) for one (strain C2-1) of the isolates. The deposit harbored 1.2×10(8) total direct count (g dry weight)(-1) and 7.1×10(5) (most-probable-number, MPN) (g dry weight)(-1) of iron-oxidizer. A ferrous iron-limited, aerobic chemostat culture using Leptospirillum HH medium (pH 1.8) was performed to select and isolate the Leptospirillum group. Nine isolates were regarded as pure cultures based on uniform colony morphology on Fe(2+)-containing silica gel plates and absence of growth on 100-fold diluted nutrient broth plates. Six strains examined further shared 99.9-100% identity in 16S rRNA gene sequence with each other. Homology-based searches showed that all of the strains belonged to the Leptospirillum ferrooxidans clade. Strain C2-1 grown in ammonium sulfate-free Leptospirillum HH medium (pH 1.8) showed an initial rate of acetylene reduction of 58 μmol h(-1) (g cell carbon)(-1).

Multi-Probe-Fluorescence in situ Hybridization for the Rapid Enumeration of Viable Vibrio parahaemolyticus

Abstract: A one-step multi-probe FISH method of detecting viable Vibrio parahaemolyticus was developed. Three candidate regions, corresponding to Helix 440+441, Helix 588, and Helix 1241 in 16S rRNA, were selected for detection, the thermodynamic parameters (ΔG(overall)) of the probes were optimized, and VP437, VP612 and VP1253, whose fluorescence were 1.7 to 11.3 times that of ΔG(overall)-unadjusted sequences, were designed. The addition of competitive oligonucleotides to reactions with VP612 and VP1253 strengthened the specificity of the probes. The three probes were labeled with FITC, TAMRA, and Cy5, respectively, and using a mixture of the probes and six competitive oligonucleotides, one-step FISH was applied to the species-specific detection of V. parahaemolyticus including epidemic strains of O3:K6 and O4:K68 serotypes. V. alginolyticus, V. rotiferianus, and V. campbellii were not detected in the reaction. Microcolonies (30-80 μm in diameter) of V. parahaemolyticus were observed within 6 hours at 37°C on seawater agar plates in both fresh and heat-damaged V. parahaemolyticus. Viable bacterial counts based on the proposed method were significantly different from those measured with typical vibrio selective media (CHROMagar Vibrio and TCBS).

Nitrogen Fixation and Translocation in Young Sugarcane (Saccharum officinarum L.) Plants Associated with Endophytic Nitrogen-Fixing Bacteria.

Abstract: The tracer 15 N2 was used to investigate sites of N2 fixation and the possible translocation of the fixed N. Young sugarcane plants (Saccharum officinarum L.) from a stem cutting were exposed to 15 N2-labeled air in a 500 mL plastic cylinder. Plants fed 15 N2 for 7 days were grown in normal air for a further chase period. After 21 days, about half of the N originating in the stem cutting had been transported to the shoot and roots, suggesting that the cutting played a role in supplying N for growth. After 3 days of feeding, the percentage of N derived from 15 N2 was higher in the roots (2.22%) and stem cutting (0.271%) than the shoot (0.027%). Most of the fixed N was distributed in the 80% ethanolinsoluble fractions in each plant part, and the 15 N fixed either in the roots or in the stem cutting remained there and was not appreciably transported to the shoot. The results were quite different from the fate of fixed N in soybean nodules, which is rapidly transported from nodules to roots and shoots.

Analysis of ammonia monooxygenase and archaeal 16S rRNA gene fragments in nitrifying acid-sulfate soil microcosms.

Abstract: The present study describes the occurrence of a unique archaeal ammonia monooxygenase alpha subunit (amoA) gene in nitrifying acid-sulfate soil microcosms at pH 3.5. The soil was collected from an abandoned paddy field in Thailand. Microcosms were incubated in the dark at 30°C for 372 days with the following three treatments: addition of ammonium sulfate solution once a month (I) or once a week (II), and addition of only sterilized water (III). A quantitative PCR analysis revealed an increase in abundance of the archaeal amoA gene in microcosm soils in which nitrate concentrations increased after incubation. A phylogenetic analysis indicated a predominance of the novel gene, and a predominance of a betaproteobacterial amoA gene affiliated with the genus Nitrosospira. A 16S rRNA gene-based PCR assay revealed that crenarchaeotic Group I.1d was predominant among the Crenarchaeota in microcosms. These results suggest the presence of ammonia-oxidizing archaea corresponding to the unique amoA lineage in nitrifying acid-sulfate soil microcosms at pH 3.5.

Bacterial population dynamics in a reverse-osmosis water purification system determined by fluorescent staining and PCR-denaturing gradient gel electrophoresis.

Abstract: The bacterial population dynamics in an industrial scale reverse-osmosis (RO) water purification system were analyzed by fluorescent staining methods and denaturing gradient gel electrophoresis (DGGE). Bacterial numbers increased with storage in a tank, and bacterial diversity changed during the water purification process. A DNA sequence-based analysis of the major bands on the DGGE gel revealed that Simonsiella sp. (Betaproteobacteria) was abundant in the source water (activated sludge-treated waste effluent), while Bosea sp. and Rhizobium sp. (Alphaproteobacteria), which usually exist in an oligotrophic environment, became abundant during the water purification process. These results suggest the importance of microbiological monitoring by culture-independent methods for quality control in RO water purification systems. These methods could provide an early warning of impending problems and clarify critical steps in controlling specific bacteria contributing to the contamination of RO water systems.