Direct sequencing of double-stranded polymerase chain reaction-amplified 16S rDNA.

A number of different procedures have been developed for direct sequence analysis of PCR products. These methods rely on the cumbersome isolation of specific PCR products from agarose gels or the production of single-stranded template DNAs. In the approach presented here, we describe primers for the amplification of 16-S rDNA and a simple preparation of PCR product for sequencing.     

Red blood cell potassium types of angora goats (Capra hircus)

Two distinct K phenotypes with normal frequency distribution curves were found and identified as low (LK) and high (HK) potassium types in the red blood cells of 748 Angora goats. The division between the two types was arbitrarily assigned at 24.9 meq/l. Of the two KRBC level types; the LK type was predominating, its curve showed evidence of leptokurtosis and was negatively skewed. The HK type had a curve slightly mesokurtic and the population was normal with respect to skewness.     

Methanocorpusculaceae fam. nov., represented by Methanocorpusculum parvum,Methanocorpusculum sinense spec. nov. and Methanocorpusculum bavaricum spec. nov.

Two new methanogenic bacteria, Methanocorpusculum sinense spec. nov. strain DSM 4274 from a pilot plant for treatment of distillery wastewater in Chengdu (Province Sichuan, China), and Methanocorpusculum bavaricum spec. nov. strain DSM 4179, from a wastewater pond of the sugar factory in Regensburg (Bavaria, FRG) are described. Methanocorpusculum strains are weakly motile and form irregularly coccoid cells, about 1 μm in diameter. The cell envelope consists of a cytoplasmic membrane and a S-layer, composed of hexagonally arranged glycoprotein subunits with molecular weights of 90000 (Methanocorpusculum parvum), 92000 (M. sinense), and 94000 (M. bavaricum). The center-to-center spacings are 14.3 nm, 15.8 nm and 16.0 nm, respectively. Optimal growth of strains is obtained in the mesophilic temperature range and at a pH around 7. Methane is produced from H₂/CO₂, formate, 2-propanol/CO₂ and 2-butanol/CO₂ by M. parvum and M. bavaricum, whereas M. sinense can only utilize H₂/CO₂ and formate. Growth of M. sinense and M. bavaricum is dependent on the presence of clarified rumen fluid. The G+C content of the DNA of the three strains is ranging from 47.7–53.6 mol% as determined by different methods. A similar, but distinct polar lipid pattern indicates a close relationship between the three Methanocorpusculum species. The polyamine patterns of M. parvum, M. sinense and M. bavaricum are similar, but distinct from those of other methanogens and are characterized by a high concentration of the otherwise rare 1,3-diaminopropane. Quantitative comparison of the antigenic fingerprint of members of Methanocorpusculum revealed no antigenic relationship with any one of the reference methanogens tested. On the basis of the distant phylogenetic position of M. parvum and the data presented in this paper a new family, the Methanocorpusculaceae fam. nov., is defined.     

Microbial Diversity at a Hot,Shallow-Sea Hydrothermal Vent in the Southern Tyrrhenian Sea (Italy)

Microbial community thriving at shallow hydrothermal vent named Black Point, close to the Island of Panarea in the Southern Tyrrhenian Sea (Italy), was studied by microscopic, cultural and molecular methods. New strains of Bacillus and Geobacillus were isolated. Members of sulphur-oxidizing bacteria, belonging to the genera Halothiobacillus and Thiomicrospira were demonstrated by both culture-dependent and -independent methods. Genetic diversity of Bacteria was higher than that of Archaea. Bacterial 16S rRNA gene sequences from Black Point showed low levels of similarity with other yet deposited sequences, suggesting that new bacterial phylotypes are present in this site. Dominant populations of Bacteria belonged to uncultured members of Proteobacteria (mainly of the class Gammaproteobacteria), Firmicutes and Acidobacteriaceae. Primary production appeared to be supported by chemosynthetic and photosynthetic bacteria affiliated with members of Gammaproteobacteria. Most of the archaeal sequences matched those of yet-uncultivated Archaea, thus their role at this vent is not well understood.     

The first evidence of anaerobic CO oxidation coupled with H<Subscript>2</Subscript> production by a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent

From 24 samples of hydrothermal venting structures collected at the East Pacific Rise (13°N), 13 enrichments of coccoid cells were obtained which grew on CO, producing H₂ and CO₂ at 80°C. A hyperthermophilic archaeon capable of lithotrophic growth on CO coupled with equimolar production of H₂ was isolated. Based on its 16S rRNA sequence analysis, this organism was affiliated with the genus Thermococcus. Other strains of Thermococcales species (Pyrococcus furiosus, Thermococcus peptonophilus, T. profundus, T. chitonophagus, T. stetteri, T. gorgonarius, T. litoralis, and T. pacificus) were shown to be unable to grow on CO. Searches in sequence databases failed to reveal deposited sequences of genes related to CO metabolism in Thermococcales. Our work provides the first evidence of anaerobic CO oxidation coupled with H₂ production performed by an archaeon as well as the first documented case of lithotrophic growth of a Thermococcales representative.     

Molecular detection and isolation of facultatively methylotrophic bacteria, including Methylobacterium podarium sp. nov., from the human foot microflora

This is the first study to demonstrate that diverse methylotrophic bacteria occur in the human foot microflora. Polymerase chain reaction (PCR) amplification of DNA from the soles and toe clefts of feet of five subjects indicated Methylobacterium strains to be present in all cases. Polymerase chain reaction amplification also showed the gene for the alpha-subunit of methanol dehydrogenase (mxaF) to be present in all samples. Two types of mxaF were recovered, one closest to that of Methylobacterium extorquens and the other most similar to that of Hyphomicrobium methylovorum. Numerous methylotrophic strains able to grow on methylamine were isolated with ease from the feet of nine volunteers. These were found by 16S rRNA analysis to be most closely related to Methylobacterium species, Brevibacterium casei, Pseudomonas strain NZ099 and P. migulae. Three strains from two subjects were of a novel species, Methylobacterium podarium sp. nov. This facultatively methylotrophic, obligately aerobic, pink-pigmented, non-motile rod grew with a wide range of multicarbon and one-carbon compounds including citrate, xylose, mono-, di-, and trimethylamine, dimethylsulphide, methanethiol, dimethylsulphoxide, dimethylsulphone and methanol.     

Concurrent evolution of nitrogenase genes and 16S rRNA in Rhizobium species and other nitrogen fixing bacteria

It was known that nitrogenase genes and proteins are well conserved even though they are present in a large variety of phylogenetically diverse nitrogen fixing bacteria. This has lead to the speculation, among others, that nitrogen fixation (nif) genes were spread by lateral gene transfer relatively late in evolution. Here we report an attempt to test this hypothesis.We had previously established the complete nucleotide sequences of the three nitrogenase genes from Bradyrhizobium japonicum, and have now analyzed their homologies (or the amino acid sequence homologies of their gene products) with corresponding genes (and proteins) from other nitrogen fixing bacteria. There was a considerable sequence conservation which certainly reflects the strict structural requirements of the nitrogenase iron-sulfur proteins for catalytic functioning. Despite this, the sequences were divergent enough to classify them into an evolutionary scheme that was conceptually not different from the phylogenetic positions, based on 16S rRNA homology, of the species or genera harboring these genes. Only the relation of nif genes of slow-growing rhizobia (to which B. japonicum belongs) and fast-growing rhizobia was unexpectedly distant. We have, therefore, performed oligonucleotide cataloguing of their 16S rRNA, and found that there was indeed only a similarity of S _AB=0.53 between fast- and slowgrowing rhizobia.In conclusion, the results suggest that nif genes may have evolved to a large degree in a similar fashion as the bacteria which carry them. This interpretation would speak against the idea of a recent lateral distribution of nif genes among microorganisms.     

A biphasic approach to the determination of the phenotypic and genotypic diversity of some anaerobic,cellulolytic, thermophilic,rod-shaped bacteria

A biphasic approach, involving a numerical phenetic and a phylogenetic study, was used to determine diversity among some anaerobic, cellulolytic, thermophilic, rod-shaped bacteria. Ninety two characters were determined for 51 strains in the numerical taxonomy study, and partial 16S rDNA sequences from 16 isolates were compared. Both the phenetic and phylogenetic data indicate diversity within this group of organisms, and reveal the lack of similarity between sporogenous and asporogenous isolates. The results of the phylogenetic study demonstrate the lack of relationship of the majority of the strains studied to previously studied thermophilic bacteria. In general, good correlation exists between the two data sets, but discrepancies arise when strains with a high level of similarity are examined. The need for caution in the interpretation of data obtained from such a biphasic approach is discussed.