Vibrio harveyi strains are pathogenic to a wide range of marine fish and shellfish, having a significant negative economic impact on aquaculture worldwide. However, a reliable and rapid method of detecting V. harveyi has yet to be established. We aimed to construct an improved one-step detection method for V. harveyi. Reanalysis of 16S rRNA gene sequences of type strains of V. harveyi revealed a unique consensus region compared to related Vibrio species. Using a VHARF-VHARR primer set from this region, a V. harveyi-specific PCR-based detection method was established and could differentiate V. harveyi from related species such as V. campbellii, V. rotiferianus, and V. alginolyticus. Furthermore, the new method (optimal amplification with 20 core cycles of (94°C-30 s, 60°C-30 s, 72°C-30 s)) could be applied to the identification of V. harveyi strains of colonies. This PCR was able to detect V. harveyi grown on plates in the environment within 3 days without bacterial isolation, DNA extraction, or the assistance of biochemical tests. The specificity and rapidity of the detection is reliable enough to understand the ecology of V. harveyi in environments, and to predict outbreaks of mass mortality caused by V. harveyi in aquaculture.
The diversity of sulphate-reducing bacteria (SRB) was investigated in sediments along environmental gradients in the River Colne estuary, Essex, UK. DNA samples were collected from four sites; marine-dominated (Alresford Creek), brackish (the Hythe), predominantly freshwater (East Hill Bridge) and freshwater (the Weir) between September 2001 and May 2002. SRB community composition was assessed by PCR amplification, cloning and sequencing of part of the α subunit of dissimilatory sulphite reductase (dsrA) using directly extracted sediment DNA. The majority of the dsrA sequences were associated with members of the Desulfobacteraceae family, the Desulfobulbaceae family and a deeply branched group in the dsrA tree with no cultured representatives. There was some evidence of a salinity-related distribution within both the Desulfobacteraceae and Desulfobulbaceae groups. Clones related to Desulfotomaculum of the xenologues Firmicutes and a phylogenetically distinct Colne group 3 were detected only at the freshwater East Hill Bridge and Weir sites. Conversely, clones related to an uncultured group (Colne group 1) were found only at the marine and brackish sites. A statistical analysis of composition revealed that dsrA sequences from the marine-dominated Alresford Creek and the brackish site at the Hythe were not significantly different from each other (P>0.05), but were significantly different from those of the freshwater-dominated East Hill Bridge and the Weir (P<0.05). The sequences from East Hill Bridge and the Weir were not significantly different from each other (P>0.05). The data presented show a complex distribution of SRB along the estuary with some evidence to support the idea that salinity and sulphate concentrations are an important factor in determining SRB community structure.
Two laboratory-scale solid-phase denitrification (SPD) reactors, designated reactors A and B, for nitrogen removal were constructed by acclimating activated sludge with pellets and flakes of poly(3-hydoxybutyrate-co-3-hydroxyvalerate) (PHBV) as the sole added substrate under denitrifying conditions, respectively. The average denitrification rate in both reactors was 60 mg NO3--N g-1 (dry wt) h-1 under steady-state conditions, whereas washed sludge taken from the reactors showed an average denitrification rate of 20 mg NO3-N g-1 (dry wt) h-1 with fresh PHBV as the sole substrate. The difference in the denitrification rate between the two might be due to the bioavailability of intermediate metabolites as the substrate for denitrification, because acetate and 3-hydroxybutyrate were detected in the reactors. Most of the predominant denitrifiers isolated quantitatively by the plate-counting method using non-selective agar medium were unable to degrade PHBV and were identified as members of genera of the class Betaproteobacteria by studying 16S rRNA gene sequence information. nirS and nosZ gene clone library-based analyses of the microbial community from SPD reactor A showed that most of the nirS and nosZ clones proved to be derived from members of the family Comamonadaceae and other phylogenetic groups of the Betaproteobacteria. These results suggest that the efficiency of denitrification in the PHBV-SPD process is affected by the availability of intermediate metabolites as possible reducing-power sources as well as of the solid substrate, and that particular species of the Betaproteobacteria play the primary role in denitrification in this process.
The sigma (σ) factor is a subunit for the RNA polymerase (RNAP) holoenzyme and can confer promoter selectivity at a target gene. On the inactivation of all group 2 and 3 σ factor genes, expression at the level of the transcript and protein was examined under continuous white light at an exponential phase in a unicellular cyanobacterium, Synechocystis sp. strain PCC 6803. It was found that interference expression exists among group 1, 2, and 3 σ factor genes. Authentic echoes of the expression did not always reflect correlations between the transcript and protein levels. Linkages for the expression network of the σ factors were summarized and unique properties discussed.
A total of 350 tetracycline-resistant (Tcr) bacteria were isolated from livestock feces, farmyard manure (FYM), and soil in Japan. The isolates were classified into 28 established genera of the phyla Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria by studying partial 16S rRNA gene sequences. PCR analysis revealed that 249 of the 350 Tcr isolates contained at least 1 of 15 Tcr genes (tet genes): 140 isolates contained an efflux pump gene and 109 isolates contained a ribosomal protection protein (RPP) gene. The predominant fecal isolates were identified as members of the genera Enterococcus and Escherichia, carrying tet (M) and tet (H), respectively. The predominant soil isolates were identified as members of the genera Burkholderia and Afipia, carrying tet (Z) and tet (B), respectively. This study is the first to describe the presence of tet genes in members of the genera Bordetella, Bradyrhizobium, Burkholderia, Dyella, Flexibacter, Kurthia, Luteibacter, Lysobacter, "Nordella", Ochrobactrum, Pediococcus, Rhodopseudomonas, and Vagococcus. It has revealed that tet genes are distributed in much more diverse phylogenetic groups of bacteria in the environment than has been reported previously.
Biological control agents such as bacteria and ciliates were tested alone and in combination for their abilities to decrease densities of the warm-weather cyanobacterium, Microcystis aeruginosa, and the cold-weather centric diatom, Stephanodiscus hantzschii. The growth of Microcystis aeruginosa was effectively suppressed by the algicidal bacterium, Streptomyces neyagawensis, and the predator heterotrich ciliate, Stentor roeselii. However, co-treatment increased the algal density rather than decreasing it, suggesting that Streptomyces neyagawensis and Stentor roeselii have an antagonistic relationship. Additional experiments revealed that the density of Stephanodiscus hantzschii was effectively reduced by the bacterium, Pseudomonas putida, and by the abovementioned strain of Stentor roeselii. Co-treatment had a greater antialgal effect than treatment with either bio-agent alone, indicating that the two act synergistically. These results suggest the potential antialgal efficacy of treatments with multiple biological control agents, providing new insight that may be useful for the bioremediation of nuisance algal blooms in freshwater.
This study was done to determine microbial interactions between nitrogen-fixing bacteria (NFB) and different indigenous bacteria, both of which were isolated from the same samples of sago palm. Co-culture of NFB and indigenous bacteria, isolated using nutrient agar medium, into a nitrogen-free Rennie medium showed significantly higher nitrogen-fixing activity (NFA) than single inoculations of NFB did in almost all combinations. A reduced oxygen status also enhanced the NFA of NFB. All NFB preferred simple sugars as their substrates for NFA and showed extremely low levels of NFA in starch, hemicellulose, and pectin-containing media. NFA was markedly stimulated in the consortium of starch-degrading Bacillus sp. strain B1 and NFB, ranging from 0-1.5 to 150-270 nmol C2H4 culture-1 h-1. The consortium of hemicellulose-degrading Agrobacterium sp. strain HMC1 or Flexibacter sp. strain HMC2 and NFB also showed enhanced NFA, ranging from 0-0.1 to 16-38 nmol. In contrast, no enhanced NFA was detected in the consortium of pectin-degrading Burkholderia sp. strain BT1 or Paenibacillus sp. strain P1 and NFB. These results may indicate that beneficial microbial interactions occur in sago palm to enhance nitrogen-fixing activity through collaborative utilizations of starch, hemicellulose and their degradation products.
DNA fragments of bacterial chitinase genes were successfully amplified from DNA extracts of maize rhizosphere and bulk soil. The molecular diversity of the bacterial chitinase genes in these soil samples was then evaluated by employing terminal-restriction fragment length polymorphism and sequencing analyses. Our results suggest the presence of novel groups of bacterial chitinase in both bulk and rhizosphere soils. Significant differences were observed, however, between the amplified chitinase genes from rhizosphere and bulk soils, and dominant clones obtained from the maize rhizosphere showed a high degree of similarity to the chitinases of Streptomyces avermitilis and close relatives there of. The current findings thus indicate the usefulness of culture-independent methods in the assessment of molecular diversity among the chitinolytic bacterial community in soils.
A PCR primer set was designed to amplify the 18S rRNA gene from the phylum Ciliophora (ciliates). The pair amplified DNA from 5 organisms representing the major taxonomic groups of ciliates but not from 13 non-ciliate organisms. To test the ability of the primers to amplify ciliate rRNA genes from environmental samples, a clone library was constructed from an agricultural soil sample and analyzed. The rRNA gene sequences of all clones were affiliated with the Ciliophora. This newly developed PCR primer set is able to amplify ciliate rRNA genes from soil, and may be useful for the detection and identification of ciliates.
The population dynamics and acetate utilization kinetics of two strains of phototrophic purple nonsulfur (PPNS) bacteria, Rhodopseudomonas sp. strain TUT3630 and Rhodobacter sp. strain TUT3733, in a continuous co-culture system were investigated. The mixed populations were cultured stepwise at different concentrations of acetate under semi-aerobic conditions in the light and were monitored by 16S rRNA-targeted fluorescence in situ hybridization (FISH) and PCR-denaturing gradient gel electrophoresis (DGGE). Cells of Rhodobacter sp. strain TUT3733, having a low affinity for acetate, dominated when the acetate concentration in the feed ranged from 5 to 20 mM. On the other hand, the feeding of less than 1 mM of acetate resulted in an increase in cell numbers of Rhodopseudomonas sp. strain TUT3630 due to its high affinity for acetate. These results suggest that the affinity for acetate is one of the most important determinants for the competitiveness among different species of PPNS bacteria in the environment. Our results provide a plausible explanation for why Rhodobacter species proliferate as the major PPNS bacteria in wastewater environments containing high levels of lower fatty acids, and the reverse is the case in Rhodopseudomonas species.