Comparative 16S rDNA Analysis Research Articles

Isolation and Selection of Purple Non-Sulfur Bacteria for Nutrient Rich Biomass Production from Wastes

Purple nonsulfur bacteria (PNSB) are anoxygenic photosynthetic bacteria, which are able to photoheterotrophically grow in the presence of excessive nutrients. Hence, PNSB can convert organic components, in waste waters into nutrient rich biomass. This suggests the feasibility of converting wastes into valuable products. In this research, method for isolation of PNSB was optimized, and subsequently used for the isolation of PNSB from domestic waste and pond water samples taken in Hanoi. In addition, the effects of carbon and nitrogen sources and types of waste on biomass and nutrients (protein and carotenes) were also investigated. Four PNSB strains were isolated and based on comparative 16S rDNA analysis and their morphological characteristics, they were identified as Rhodobacter (Rb.) capsulatus, Rhodobacter (Rb.) sphaeroides, Rhodobacter (Rb.) sediminis and Rhodopseudomonas (Rp.) palustris. In terms of the effects of carbon and nitrogen sources on their biomass and nutrient production, glucose or maltose and ammonium chloride or urea were found to be more enhancive than starch and peptone, respectively. Among the studied strains, Rb. capsulatus MD1 and Rb. sphaeroides MD3 showed significantly higher biomass production (up to 0.7 g/l) when growing with various carbon and nitrogen sources, in comparison with the other strains. Moreover, MD1 and MD3 also produced at least four-fold more carotenoid and up to two-fold more protein in tofu processing wastewater compared with the other wastewater. The results suggest potential applications of the PNSB strains for efficient conversions of organic compounds in wastes into biomass of high nutritional values.

Microbacterium awajiense sp. nov., Microbacterium fluvii sp. nov. and Microbacterium pygmaeum sp. nov.

The taxonomic positions of three novel strains isolated from soil, driftwood and sediment samples collected in Japan were investigated based on the results of chemotaxonomic, phenotypic and genotypic characteristics. The strains that we examined were Gram-positive, catalase-positive bacteria with L-ornithine as a diagnostic diamino acid of peptidoglycan. The acyl type of peptidoglycan was N-glycolyl. The major menaquinones were MK-11, -12, -13 and/or -14. Mycolic acids were not detected. The G+C content of the DNA was 68 to 70 mol%. These morphological and chemotaxonomical characters and comparative 16S rDNA analysis of the three isolated strains revealed that they belong to the genus Microbacterium. DNA-DNA relatedness data revealed that the three isolates are three new species of the genus Microbacterium. On the basis of the polyphasic evidence, the isolates should be classified as novel species of the genus Microbacterium: Microbacterium awajiense sp. nov., Microbacterium fluvii sp. nov. and Microbacterium pygmaeum sp. nov. with the type strains YM13-414T (=MBIC08276T, DSM 18907T), YSL3-15T (=MBIC08277T, DSM 18908T) and KV-490T (=NRRL B-24469T, NBRC 101800T), respectively.

Isolation from poultry litter and characterization in high phosphate conditions of Staphylococcus spp. capable of growth

To isolate and characterize micro-organisms from poultry litter capable of growing under phosphate concentrations typical of poultry litter. Poultry litter extracts were plated onto brain-heart infusion medium (BHI) containing an additional 0.75 mol l(-1) phosphate (BHI-P). Colonies were screened for the presence of inclusion granules with five being selected for further study. All strains displayed identical biochemical characteristics consistent with Staphylococcus spp. and grouped with Staphylococcus spp. by comparative 16S rDNA analysis. Thus all five strains were identified as such. All strains displayed elevated intracellular phosphate levels when cultured in BHI-P broth (0.417-0.600 microg phosphate mg(-1) protein) vs BHI broth (0.075-0.093 microg phosphate mg(-1) protein). When grown using an austere semi-defined medium or BHI-P, Staph. sp. #7 displayed similar elevated intracellular phosphate levels compared with growth in BHI. Poultry litter contains novel Staphylococcus spp. capable of robust growth when exposed to phosphate levels comparable with that typically found in poultry litter. Data suggest intracellular phosphate levels in these strains increase in response to increasing phosphate in the medium or austere medium conditions. Intracellular phosphate did not reach levels comparable with known hyper-accumulating micro-organisms. These data suggest poultry litter possesses a resident microflora that thrives and accumulates intracellular phosphate in response to high phosphate conditions.

An abundance of Escherichia coli is harbored by the mucosa-associated bacterial flora of interleukin-2-deficient mice.

Mice deficient in interleukin-2 are well suited for use as an animal model for inflammatory bowel disease. Raised under specific-pathogen-free conditions, interleukin-2-deficient mice develop an inflammatory bowel disease resembling ulcerative colitis in humans. The finding that colitis was attenuated when the mice were kept under germfree conditions implies that the resident intestinal flora is involved in the pathogenesis of colitis. The present study addresses the composition of the mucosa-associated bacterial flora in colon samples from interleukin-2-deficient mice that developed colitis. This was investigated by comparative 16S ribosomal DNA (rDNA) sequence analysis and fluorescence in situ hybridization using rRNA-targeted fluorescent probes to quantify the bacterial populations of the mucosa-associated flora. The investigations revealed distinct differences in the bacterial composition of the mucosa-associated flora between interleukin-2-deficient mice and healthy controls. Fluorescence in situ hybridization identified up to 10% of the mucosa-associated flora in interleukin-2-deficient mice as Escherichia coli, whereas no E. coli was detected in the mucosa from healthy wild-type mice. This finding was consistent with the results from comparative 16S rDNA analysis. About one-third of the clones analyzed from 16S rDNA libraries of interleukin-2-deficient mice represented Enterobacteriaceae, whereas none of the clones analyzed from the healthy controls harbored 16S rDNA from Enterobacteriaceae. The abundance of E. coli in the colonic mucosa of interleukin-2-deficient mice strongly suggests a participation in the pathogenesis of colitis in the interleukin-2-deficient mouse model for inflammatory bowel disease.

Balneomonas flocculans gen. nov., sp. nov., A New Cellulose-producing Member of the α-2 subclass of Proteobacteria

A new bacterial strain capable of producing cellulose was isolated from a hot spring. The isolate was Gram-negative, aerobic, and rod-shaped. The optimum temperature for growth was 40-45 degrees C. Methanol, glucose and other common carbohydrates were not utilized as sole growth substrates. Thiosulfate was not oxidized. The G+C content of the DNA was determined to be 64.0 mol%. Comparative 16S rDNA analysis indicated that Bosea thiooxidans and some strains of the genus Methylobacterium were the nearest relatives. The isolate can be distinguished from these relatives by its defectiveness in methanol utilization and thiosulfate oxidation. On the basis of its phenotypic properties and phylogeny, it is proposed that the isolate be designated Balneomonas flocculans gen. nov., sp. nov. The type strain is TFBT (= JCM 11936T, = KCTC 12101T, = IAM 15034T, = ATCC BAA-817T).

Polaromonas naphthalenivorans sp. nov., a naphthalene-degrading bacterium from naphthalene-contaminated sediment.

Strain CJ2T, capable of growth on naphthalene as a sole carbon and energy source, was isolated from coal-tar-contaminated freshwater sediment. The Gram reaction of strain CJ2T was negative. The cells were non-spore-forming, non-motile cocci (without flagella). The isolate was found to be an aerobic heterotroph capable of utilizing glucose and other simple sugars. Growth was observed between 4 and 25 degrees C (optimum, 20 degrees C) and between pH 6.0 and 9.0 (optimum, pH 7.0-7.5). The G+C content of the genomic DNA was 61.5 mol% and the major quinone was ubiquinone-8. The peptidoglycan of strain CJ2T was determined as belonging to type A1-gamma, meso-diaminopimelic acid. The major fatty acids of strain CJ2T were 16:1omega7c (67.0%), 16:0 (19.6%), 18:1omega7c (approximately 7.9%) and 10:0 3-OH (approximately 2.5%). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Mycolic acid and glycolipids could not be detected. Comparative 16S rDNA analysis indicated that strain CJ2T is related to the family Comamonadaceae and that the nearest phylogenetic relative was Polaromonas vacuolata 34-PT (97.1% similarity). On the basis of the physiological and molecular properties, the naphthalene-degrading isolate was designated Polaromonas naphthalenivorans sp. nov. The type strain is CJ2T (=ATCC BAA-779T=DSM 15660T).

Microbial diversity and functional characterization of sediments from reservoirs of different trophic state

Sediment samples from four reservoirs of different trophic state were compared with regard to chemical gradients in the pore water, composition of microbial communities and extracellular enzyme activities. The trophic state was clearly reflected by steep vertical concentration gradients of ammonium and alkalinity in the pore water. A high concentration of these parameters indicated a high microbial in situ activity in the more eutrophic reservoirs. However, the total number of bacteria in sediments seemed hardly to be influenced by the trophic conditions in the water column. Differences in the microbial composition of the sediments became evident by comparative 16S rDNA analysis of extracted DNA and by fluorescence in situ hybridization. Although a high proportion of the cells detectable with the EUB probe could not be identified at the subdomain level, members of the β- Proteobacteria constituted an important fraction in the sediments of the more eutrophic reservoirs, whereas γ-subgroup Proteobacteria were most frequently detected in sediment samples from the dystrophic Muldenberg reservoir. The assessment of extracellular enzyme activities (esterases, phosphatases, glucosidases and aminopeptidases, respectively) in sediment samples of the four reservoirs revealed specific patterns of metabolic potentials in accordance with the trophic state and characteristics of the catchment.

Molecular characterization of the microbial community structure in two activated sludge systems for the advanced treatment of domestic effluents

Although activated sludge systems with enhanced biological phosphorus removal (EBPR) represent state-of-the-art technology for phosphate removal from wastewater it is still unknown which species of bacteria are responsible for the EBPR process. The aim of this study was to compare the bacterial composition of activated sludge from two laboratory plants with different modes of operation, anoxic/oxic- (EBPR, no nitrification) and Phoredox-system (EBPR, nitrification and denitrification) with particular emphasis on microorganisms responsible for EBPR process. In addition to fluorescence in situ hybridization (FISH), we applied further rRNA-based molecular techniques like terminal restriction-fragment length polymorphism analysis and comparative 16S rDNA analysis to yield additional information and to verify the results from FISH analysis, like e.g. for the identification of polyphosphate accumulating organisms (PAO). Despite the different modes of operation only minor differences in the bacterial composition were detected by FISH analysis based on the probes used in this study. In contrast T-RFLP analysis yielded characteristic community fingerprints for each of the investigated plants and comparative 16S rDNA analysis indicated highly diverse microbial communities in both plants suggesting substantial differences in the microbial structure. The results obtained by FISH analysis with specific probes for PAOs support the presumption that not only one specific organism is responsible for the EBPR. In our case Tetrasphaera spp. dominated the PAO community, but other possible PAOs, like Microlunatus spp . and members of the Rhodocyclus group, were also detected.

Promicromonospora pachnodae sp. nov., a member of the (hemi)cellulolytic hindgut flora of larvae of the scarab beetle Pachnoda marginata.

Intestinal microorganisms play an important role in plant fiber degradation by larvae of the rose chafer Pachnoda marginata. In the hindgut of the larvae 2.5 to 7.4 x 10(8) bacteria per ml of gut content with xylanase or endoglucanase activity were found. Bacteria in the midgut were not (hemi)cellulolytic, but the alkaline environment in this part of the intestinal tract functions as a precellulolytic phase, solubilizing part of the lignocellulosic material. Accordingly, the degradation of lignocellulose-rich material in Pachnoda marginata larvae appeared to be a combination of a physico-chemical and microbiological process. A number of different facultative anaerobic and strictly anaerobic bacteria with (hemi)cellulolytic activity were isolated from the hindgut. A dominant (hemi)cellulolytic species was a Gram positive, irregular shaped, facultative aerobic bacterium. Further physiological identification placed the isolate in the genus Promicromonospora. Comparative 16S rDNA analysis and phenotypic features revealed that the isolate represented a new species for which the name Promicromonospora pachnodae is proposed. P. pachnodae produced xylanases and endoglucanases on several plant derived polymers, both under aerobic and anaerobic conditions.

Kytococcus schroeteri sp. nov., a novel Gram-positive actinobacterium isolated from a human clinical source.

A strain of a gram-positive, coccoid, yellow-pigmented bacterium was isolated from human blood. The bacterium was aerobic, non-encapsulated and non-motile. Phenotypically, the bacterium closely resembled Kytococcus sedentarius, but could be distinguished from this species by physiological tests and chemotaxonomic investigations. The peptidoglycan type is L-Lys-Glu2, variation A4alpha. The predominant menaquinones are MK-8 and MK-7. The major cellular fatty acids are iso-C17:1, iso-C17:0, iso-C15:0 and anteiso-C17:0. The strain contains catalase and does not produce acid from carbohydrates. The ability to hydrolyse Tween 80 and the lack of alpha-glucosidase activity are the most characteristic features. The results of comparative 16S rDNA analysis revealed that the strain represents a novel species within the genus Kytococcus, for which the name Kytococcus schroeteri sp. nov. is proposed. The type strain is strain Muenster 2000T (= DSM 13884T = CCM 4918T).

Caldimonas manganoxidans gen. nov., sp. nov., a poly(3-hydroxybutyrate)-degrading, manganese-oxidizing thermophile.

A poly(3-hydroxybutyrate) (PHB)-degrading, gram-negative, aerobic bacterium, strain HS(T), was isolated from a hot spring and chemotaxonomically and phylogenetically characterized. The oxidase-positive, weakly catalase-positive, non-pigmented cells (0.6 x 2.6 microm) exhibited a single polar flagellum and accumulated PHB granules. Strain HS(T) was capable of manganese oxidation. Highest growth rate was attained at 50 degrees C. The optimum pH for growth was 7-8. The major respiratory quinone was ubiquinone-8 and major cellular fatty acids were C16:0, C16:1 and C18:1. The G+C content of the DNA was 66.2 mol%. Comparative 16S rDNA analysis indicated that strain HS(T) is related to the Rubrivivax subgroup and the family Comamonadaceae. The nearest phylogenetic relatives were Ideonella dechloratans (92.1% similarity), Leptothrix discophora (93.6%), Roseateles depolymerans (92.4%) and Rubrivivax gelatinosus (92.2%). On the basis of its phylogenetic and phenotypic properties, it is proposed that this isolate be designated Caldimonas manganoxidans gen. nov., sp. nov.; the type strain is HS(T) (= JCM 10698T = IFO 16448T = ATCC BAA-369T).

Sampling DNA from the rhizosphere of Brassica napus to investigate rhizobacterial community structure

We have compared, using DNA analysis, the classical `pull and shake' method for sampling rhizosphere soil with a model rhizosphere system in which the soil was sampled at increasing distances (0.5 mm) from the root mat of Brassica napus. These studies showed that more DNA was extracted from the soil slices closest to the roots. Whilst the gradient in DNA recovered cannot be attributed solely to differences in microbial biomass, it clearly illustrates the lack of spatial resolution characteristic of the `pull and shake' method of sampling from the rhizosphere. The DNA-rich fraction (rhizosphere soil) was compared with bulk soil DNA by PCR using actinomycete 16S rDNA primers and 16S rDNA sequencing. Phylogenetic analyses of 16S rDNA sequences showed that these primers were not specific for actinomycetes but also amplified Gram positive low G+C bacterial sequences. Comparative 16S rDNA analysis of these sequences showed an enrichment for Gram positive low G+C bacteria in the rhizosphere of Brassica napus.

The phylogenetic position of Serratia, Buttiauxella and some other genera of the family Enterobacteriaceae.

The phylogenetic relationships of the type strains of 38 species from 15 genera of the family Enterobacteriaceae were investigated by comparative 16S rDNA analysis. Several sequences of strains from the genera Citrobacter, Erwinia, Pantoea, Proteus, Rahnella and Serratia, analysed in this study, have been analysed previously. However, as the sequences of this study differ slightly from the published ones, they were included in the analysis. Of the 23 enterobacterial genera included in an overview dendrogram of relatedness, members of the genera Xenorhabdus, Photorhabdus, Proteus and Plesiomonas were used as a root. The other genera formed two groups which could be separated, although not exclusively, by signature nucleotides at positions 590-649 and 600-638. Group A contains species of Brenneria, Buttiauxella, Citrobacter, Escherichia, Erwinia, Klebsiella, Pantoea, Pectobacterium and Salmonella. All seven type strains of Buttiauxella share 16S rDNA similarities greater than 99%. Group B embraces two phylogenetically separate Serratia clusters, a lineage containing Yersinia species, Rahnella aquatica, Ewingella americana, and also the highly related pair Hafnia alvei and Obesumbacterium proteus.

Caldicellulosiruptor kristjanssonii sp. nov., a cellulolytic, extremely thermophilic, anaerobic bacterium.

A cellulolytic anaerobic bacterium, strain I77R1BT, was isolated from a biomat sample of an Icelandic, slightly alkaline, hot spring (78 degrees C). Strain I77R1BT was rod-shaped, non-spore-forming, non-motile and stained Gram-negative at all stages of growth. It grew at 45-82 degrees C, with an optimum growth temperature around 78 degrees C. At 70 degrees C, growth occurred at pH 5.8-8.0, with an optimum near pH 7.0. At the optimum temperature and pH, with 2 g cellobiose l-1 as substrate, strain I77R1BT had a generation time of 2 h. During growth on Avicel, strain I77R1BT produced acetate, hydrogen and carbon dioxide as major fermentation products together with small amounts of lactic acid and ethanol. The strain fermented many substrates, including cellulose, xylan, starch and pectin, but did not grow with casein peptone, pyruvate, D-ribose or yeast extract and did not reduce thiosulfate to H2S. The G+C ratio of the cellular DNA was 35 mol%. Comparative 16S rDNA analysis placed strain I77R1BT among species of Caldicellulosiruptor. The closest relative was Caldicellulosiruptor lactoaceticus. Hybridization of total DNA showed 42% hybridization to C. lactoaceticus and 22% hybridization to Caldicellulosiruptor saccharolyticus. A new species, Caldicellulosiruptor kristjanssonii sp. nov. (I77R1BT) is proposed.