Detection of bacterial gene expression in soil emerged in the early 1990s and provided information on bacterial responses in their original soil environments. As a key procedure in the detection, extraction of bacterial RNA from soil has attracted much interest, and many methods of soil RNA extraction have been reported in the past 20 years. In addition to various RT-PCR-based technologies, new technologies for gene expression analysis, such as microarrays and high-throughput sequencing technologies, have recently been applied to examine bacterial gene expression in soil. These technologies are driving improvements in RNA extraction protocols. In this mini-review, progress in the extraction of bacterial RNA from soil is summarized with emphasis on the major difficulties in the development of methodologies and corresponding strategies to overcome them.
A total of 828 isolates of fluorescent pseudomonads (FPs) were obtained from the leaves (305 isolates) and roots (523 isolates) of potato plants grown in different geographical locations in Japan, and 16S rRNA gene sequences of 776 isolates were successfully determined by direct PCR sequencing. Clustering analysis (≥99% identity) identified 13 and 26 operational taxonomic units (OTUs) for leaf- and root-associated FPs, respectively, and 29 OTUs were identified in the phytosphere of potato plants. Among them, 7 and 9 OTUs showed a significantly biased distribution to the leaves and roots, respectively. Phylogenetic analysis revealed that 3 dominant OTUs for leaf-associated FPs were grouped in a cluster of leaf-associated pathogens, such as Pseudomonas cichorii and Pseudomonas viridiflava. In contrast, 4 OTUs were located in a cluster of saprophytic pseudomonads. Among them, 3 OTUs showed high similarity to Pseudomonas koreensis and Pseudomonas vancouverensis, both of which have been reported to be beneficial for biological control or plant growth promotion. These data provide key information for efficient surveying and utilization of beneficial FPs in agricultural practices.
Aerobic aromatic-hydrocarbon-degrading bacteria from a semi-anaerobic microbial microcosm that exhibited apparent complete dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were isolated through enrichment and plating culture procedures with dibenzofuran as the model substrate. By 16S rRNA gene sequence comparisons, these dibenzofuran-degrading isolates were identified as being members of the phyla Actinobacteria, Firmicutes, and Proteobacteria, among which those of the genera Paenibacillus and Rhizobium were most abundant. All of the isolates utilized naphthalene as the sole carbon and energy source and degraded dibenzofuran metabolically or co-metabolically; however, they hardly attacked monochlorinated dibenzofuran and dibenzo-p-dioxin. By PCR cloning and sequencing, genes predicted to encode aromatic-ring-hydroxylating dioxygenase (AhDO) were detected in all test isolates. Real-time quantitative PCR assays with specific primer sets detected approximately 105 copies of the AhDO large subunit genes g−1 wet wt in the microcosm from which the isolates were obtained. This order of the copy number corresponded to approximately 1% of the 16S rRNA gene copies from “Dehalococcoides” and its relatives present as potent dechlorinators. These results suggest that aerobic AhDO-containing bacteria co-exist and play a role in the oxidative degradation of less chlorinated and completely dechlorinated products in the PCDD/F-dechlorinating process, thereby achieving the apparent complete dechlorination of PCDD/Fs.
Ciliates play important roles as prey and predators in ecosystems. Changes in the ciliate community can affect the composition and population of microfauna and microflora in ecosystems. To investigate the structure of ciliate communities, we developed a nested PCR-DGGE method, which combines a universal eukaryotic-specific primer set in the first PCR step with a ciliate-specific primer set in the second PCR step, to amplify 18S rRNA genes from ciliates. The 300 bp DGGE fragments generated more bands on the gel than the 600 bp DGGE fragments. Prior to bead beating, DNA extraction of ciliates from soil samples was optimized with a combination of freeze-thaw cycles and ultrasonication. We applied this nested PCR-DGGE method to agricultural soils amended with 0, 120, 300, and 600 t ha−1 year−1 of livestock slurry. The results from the DGGE profiles and principal component analysis (PCA) revealed that the supplement of slurry to soils influenced the ciliate communities. From phylogenetic analysis, 108 DGGE bands were assigned to six classes, which included Spirotrichea and Colpodea, of the subphylum Intramacronucleata, and one class of the subphylum Postciliodesmatophora. These results indicated that a wide variety of taxonomic groups were detected by DGGE profiling. Thus, the nested PCR-DGGE method described here could clearly differentiate between ciliate communities within soil samples and allowed for the phylogenetic identification of these ciliates at the class level.
Natural or engineered microbial populations often show variations over time. These variations may be due to environmental fluctuations or intrinsic factors. Thus, studying the dynamics of microbial diversity for different communities living in a spatially homogeneous landscape is of interest. As a model ecosystem, nitrifying biofilm communities were grown in a two litre inverse turbulent bed reactor (ITBR) containing an estimated 200 million small particles (about 150 μm in diameter). Each particulate biofilm is considered as a distinct community growing in the neighborhood of other similar particles, in a homogeneous and well-controlled environmental context. A molecular approach was adopted to test how microbial community structures might evolve: either in synchrony, converging or diverging. The shape of biofilm was observed by microscopy for each particle. The biomass content was evaluated by quantitative PCR and showed similar values for each particle. The microbial community structure was evaluated by Capillary Electrophoresis-Single Strand Conformation Polymorphism (CE-SSCP) fingerprinting and showed extraordinary homogeneity between particles, even though transitory community structures were observed when reactor operating conditions were modified. This homogeneity was observed for the Bacteria primer set but, more interestingly, was also observed when minor non-nitrifying bacteria making up the biofilm, representing about 5% and 10% of total cells, were targeted.
A number of molecular ecological studies have revealed complex and unique microbial communities in various terrestrial plant roots; however, little is known about the microbial communities of aquatic plant roots in spite of their potential use for water quality improvement in aquatic environments (e.g. floating treatment wetland system). Here, we report the microbial communities inhabiting the roots of emerged plants, reed (Phragmites australis) and Japanese loosestrife (Lythrum anceps), collected from a floating treatment wetland in a pond by both culture-independent and culture-dependent approaches. Culture-independent analysis based on 16S rRNA gene sequences revealed that the microbial compositions between the two aquatic plant roots were clearly different (e.g. the predominant microbe was Betaproteobacteria for reed and Alphaproteobacteria for Japanese loosestrife). In comparisons of microbial communities between the plant roots and pond water taken from near the plants, the microbial diversity in the plant roots (e.g. 4.40–4.26 Shannon-Weiner index) were higher than that of pond water (e.g. 3.15 Shannon-Weiner index). Furthermore, the plant roots harbored 2.5–3.5 times more phylogenetically novel clone phylotypes than pond water. The culture-dependent approach also revealed differences in the microbial composition and diversity among the two plant roots and pond water. More importantly, compared to pond water, we succeeded in isolating approximately two times more novel isolate phylotypes, including a bacterium of candidate phylum OP10 (recently named Armatimonadetes) from the plant roots. These findings suggest that aquatic plants roots are significant sources for a variety of novel organisms.
We investigated the prevalence of qnr and aac(6′)-Ib-cr genes in water-borne environmental bacteria and in clinical isolates of Enterobacteriaceae, as well as the subtypes of qnr. Environmental bacteria were isolated from surface water samples obtained from 10 different locations in Hangzhou City, and clinical isolates of Citrobacter freundii were isolated from several hospitals in four cities in China. qnrA, qnrB, qnrS, and aac(6′)-Ib-cr genes were screened using PCR, and the genotypes were analyzed by DNA sequencing. Ten of the 78 Gram-negative bacilli isolated from water samples were C. freundii and 80% of these isolates carried the qnrB gene. qnrS1 and aac(6′)-Ib-cr genes were detected in two Escherichia coli isolates and qnrS2 was detected in one species, Aeromonas punctata. The qnr and aac(6′)-Ib-cr genes were present in 75 (72.8%) and 12 (11.6%) of 103 clinical isolates of C. freundii, respectively. Of the clinical C. freundii isolates with the qnr gene, 65 isolates (63.1%) carried qnrB, but only three (2.9%) and one (1.0%) carried qnrA1 and qnrS2, respectively, while five isolates carried both qnrA1 and qnrB, and one isolate carried both qnrS1 and qnrB. The qnrB9 gene was the dominant qnrB subtype, followed by qnrB8 and qnrB6. Southern hybridization studies indicated that the qnr genes are located on different plasmids. Plasmids isolated from both environmental and clinical C. freundii isolates appeared to be homogenous.
High densities of Escherichia coli and enterococci are common in freshwaters on Oahu and other Hawaiian Islands. Soil along stream banks has long been suspected as the likely source of these bacteria; however, the extent of their occurrence and distribution in a wide range of soils remained unknown until the current investigation. Soil samples representing the seven major soil associations were collected on the island of Oahu and analyzed for fecal coliforms, E. coli, and enterococci by the most probable number method. Fecal coliforms, E. coli, and enterococci were found in most of the samples analyzed; log mean densities (MPN ± SE g soil−1) were 1.96±0.18, n=61; 1.21±0.17, n=57; and 2.99±0.12, n=62, respectively. Representative, presumptive cultures of E. coli and enterococci collected from the various soils were identified and further speciated using the API scheme; at least six species of Enterococcus, including Enterococcus faecalis and Enterococcus faecium, were identified. In mesocosm studies, E. coli and enterococci increased by 100-fold in 4 days, after mixing sewage-spiked soil (one part) with autoclaved soil (nine parts). E. coli remained metabolically active in the soil and readily responded to nutrients, as evidenced by increased dehydrogenase activity. Collectively, these findings indicate that populations of E. coli and enterococci are part of the natural soil microflora, potentially influencing the quality of nearby water bodies.
Small cyanobacteria (<2 μm, pico-cyanobacteria) are abundant in waters deeper than the oxic-anoxic zone in the halocline of a saline meromictic lake, Lake Suigetsu, Fukui, Japan. We have isolated 101 strains that were grouped into six groups by means of the phycobiliprotein composition and sequence homology of the intergenic spacer between the 16S and 23S rRNA genes. Significant growth was observed under weak green light (1.5 μmol m−2 s−1, approx. 460 to 600 nm), whereas the cells died under white light at even moderate intensities. The isolates grew in a wide range of salinities (0.2 to 3.2%). Tolerance to sulfide varied: four groups grew in medium containing sulfide, however, two groups did not. None of the isolates were capable of anoxygenic photosynthetic (PS-II independent photosynthetic) growth using sulfide as an electron donor. All groups were included within fresh and brackish water of Synechococcus/Cyanobium clade, but they were not monophyletic in the 16S rRNA gene-based phylogenetic tree. The physiological properties of pico-cyanobacteria showed that they had the ability to survive in unique physicochemical environments in the halocline of this saline meromictic lake.
Enterococcus are emerging as important putative pathogens resistant to chemicals that are widely released into the environment, and urban pigeons might act as a natural reservoir contributing to the spread of resistant strains. This study aimed to evaluate the occurrence of Enterococcus in pigeon feces and their antimicrobial and toxic metal susceptibility. Bacteria were isolated and identified from 150 fresh feces by phenotypic and genetic techniques. Antimicrobial and toxic metal susceptibility was determined by the agar dilution method, and the multiple antibiotic resistance index (MAR) was calculated. Out of 120 isolates, no resistance was observed against penicillin and vancomycin, but was observed against gentamicin (55.8%), chloramphenicol (21.7%), tetracycline (13.3%), ciprofloxacin (8.4%) and rifampin (2.5%). 18.3% presented a MAR index ≥0.2, ranging between 0.14 to 0.57, indicating resistance to more than one antimicrobial. All samples were tolerant to >1024 μg mL−1 zinc and chromium. Minimal inhibitory concentration (MIC) of 1,024 μg mL−1 was observed for copper (100%) and nickel (71.4%). Mercury inhibited 88.4% at 32 μg mL−1 and the MIC for cadmium ranged from 0.125–128 μg mL−1. Since pigeons were found to harbor drug-resistant Enterococcus, our data support that their presence in the urban environment may contribute to the spread of resistance, with an impact on public health.
Recycling of the nitrogenous waste uric acid (UA) of wood-feeding termites by their gut bacteria is one of the significant aspects of symbiosis for the conservation of nitrogen sources. Diverse anaerobic UA-degrading bacteria comprising 16 species were isolated from the gut of eight termite species, and were assigned to Clostridia, Enterobacteriaceae, and low G+C Gram-positive cocci. UA-degrading Clostridia had never been isolated from termite guts. UA-degrading ability was sporadically distributed among phylogenetically various culturable anaerobic bacteria from termite guts. A strain of Clostridium sp., which was commonly isolated from three termite species and represented a probable new species in cluster XIVa of clostridia, utilized UA as a nitrogen source but not as a sole carbon and energy source. This feature is in clear contrast to that of well-studied purinolytic clostridia or previously isolated UA degraders from termite guts, which also utilize UA as a sole carbon and energy source. Ammonia is the major nitrogenous product of UA degradation. Various purines stimulated the growth of this strain when added to an otherwise growth-limiting, nitrogen poor medium. The bacterial species involved the recycling of UA nitrogen in the gut microbial community of termites are more diverse in terms of both taxonomy and nutritional physiology than previously recognized.
Fish farm sediments receive a large amount of organic matter from uneaten food and fecal material. This nutrient enrichment, or organic pollution, causes the accumulation of sulphide in the sediment from the action of sulphate-reducing bacteria (SRB). We investigated the effect of organic enrichment around coastal fish farms comparing the SRB community structure in these sediments. Sediment samples with different levels of organic pollution classified based upon the contents of acid-volatile sulphide and chemical oxygen demand were collected at three stations on the coast of western Japan. The SRB community composition was assessed using PCR amplification, cloning, sequencing and phylogenetic analysis of the dissimilatory sulphite reductase b-subunit gene (dsrB) fragments using directly extracted sediment DNA. Sequencing of the cloned PCR products of dsrB showed the existence of different SRB groups in the sediments. The majority of dsrB sequences were associated with the families Desulfobacteraceae and Desulfobulbaceae. Clones related to the phylum Firmicutes were also detected from all sediment samples. Statistical comparison of sequences revealed that community compositions of SRB from polluted sediments significantly differed from those of moderately polluted sediments and unpolluted sediments (LIBSHUFF, p<0.05), showing a different distribution of SRB in the fish farm sediments. There is evidence showing that the organic enrichment of sediments influences the composition of SRB communities in sediments at marine fish farms.
Microbial functional diversity in four soils sampled in the vicinity of Wanda Glacier, Antarctic Peninsula, was determined using Biolog EcoPlates at 5°C and 25°C. Comparisons of the patterns of substrate utilization and the diversity index showed differences in community composition, reflecting the heterogeneous distribution of microorganisms in this environment. Differences in microbial diversity may be related to soil chemical properties. Higher incubation temperature influenced the overall microbial diversity, reducing richness due to the selection of psychrotrophic microorganisms. To our knowledge, this is the first study with microbial communities from Wanda Glacier and contributes to understanding the microbial diversity of Antarctic environments.
Pyrosequence targeting of the 16S rRNA gene has been adopted for microbial communities associated with field-grown plants. To examine phylogenetic drifts according to read length and bioinformatic tools, original and chopped sequences (250–570 bp) covering the V1–V4 regions of 16S rRNA genes were compared using pyrosequence and Sanger reads of rice root microbiomes. The phylogenetic assignment at genus level depended on read length, especially in the genus Bradyrhizobium, which is one of the ecologically important bacterial genera associated with plants. We discuss the methodology of phylogenetic assignments of plant-associated bacteria by 16S rRNA pyrosequence.
Geologically and chemically distinct aquifers were screened for the presence of two genes coding for key enzymes of the reverse tricarboxylic acid (rTCA) cycle in autotrophic bacteria, 2-oxoglutarate : ferredoxin oxidoreductase (oorA) and the beta subunit of ATP citrate lyase enzymes (aclB). From 42 samples investigated, aclB genes were detected in two and oorA genes in six samples retrieved from polluted and sulfidic aquifers. aclB genes were represented by a single phylotype of almost identical sequences closely affiliated with chemolithoautotrophic Sulfurimonas species. In contrast, sequences analysis of oorA genes revealed diverse phylotypes mainly related to sequences from cultivation-independent studies.
Volume 26, no. 4, Page 271–281, 2011 Page 278, Acknowledgements, ‘This review was partially supported by the Czech Ministry of Education (1M0554) and by the Ministry of Industry and Trade (FR-TI3/564)’.