Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments.

A range of autotrophic and heterotrophic enrichment cultures were established to determine the cultural bacterial diversity present in samples obtained from the acidic runoff of a chalcocite overburden heap and from laboratory-scale (1- to 4-liter) batch and continuous bioreactors which were being used for the commercial assessment of the bioleachability of zinc sulfide ore concentrates. Strains identified as Thiobacillus ferrooxidans, Thiobacillus thiooxidans, "Leptospirillum ferrooxidans," and Acidiphilium cryptum were isolated from both the natural site and the batch bioreactor, but only "L. ferrooxidans," a moderately thermophilic strain of T. thiooxidans, and a moderately thermophilic iron-oxidizing bacterium could be recovered from the continuous bioreactor running under steady-state conditions. Sequence analysis of the 16S rRNA genes of 33 representative strains revealed that all of the strains were closely related to strains which have been sequenced previously and also confirmed the phylogenetic diversity of bacteria present in bioleaching environments.     

16S rDNA analysis reveals phylogenetic diversity among the polysaccharolytic clostridia

Abstract Small subunit rDNA sequences were determined for 13 mesophilic, polysaccharolytic, mainly cellulolytic species of the genus Clostridium and one cellulolytic Eubacterium specues. Sequences were compared to those of 36 representatives of mesophilic and thermophilic clostridia, including those of nine thermophilic polysaccharolytic species published previously. The majority of strains group with 23S rRNA clusters I and III, while the others group with the thermophilic polysaccharolytic clostridia, i.e. C. stercorarium, C. thermolacticum and C. thermocellum . Lack of close genetic relationships between the various polysaccharolytic species is unexpected and may indicate that these biotechnologically important organisms differ with respect to the enzymology of polysaccharolytic degradation as well.     

Nucleic acid hybridization studies on Microbacterium, Curtobacterium, Agromyces and related taxa

Thirty strains of Agromyces, Arthrobacter, Curtobacterium, Brevibacterium, Corynebacterium and Microbacterium, exhibiting the rare peptidoglycan of group B, were subjected to extensive nucleic acid hybridization studies. The DNA homology values indicate that Corynebacterium insidiosum DSM 20157 is genetically identical with Corynebacterium michiganense DSM 20134. Corynebacterium sepedonicum NCPPB 378 and Corynebacterium nebraskense DSM 20400 are closely related to Corynebacterium michiganense DSM 20134. Corynebacterium betae DSM 20141, Corynebacterium oortii ATCC 25283 and Corynebacterium poinsettiae ATCC 9682 are genetically identical with Corynebacterium flaccumfaciens DSM 20129. In addition, Curtobacterium citreum ATCC 15828, Curtobacterium luteum ATCC 15830 and Curtobacterium pusillum ATCC 19096 share a high degree of relatedness to Corynebacterium flaccumfaciens DSM 20129. All other described species are more distantly related to each other. DNa-rRNA cistron similarity studies reveal that all corynebacterium with a peptidoglycan group B are members of one homogeneous cluster for which the rank of a genus is suggested.     

Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes.

The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.     

Taxonomic and phylogenetic studies on a new taxon of budding, hyphal Proteobacteria, Hirschia baltica gen. nov., sp. nov

Four strains of budding, hyphal bacteria, which had very similar chemotaxonomic properties, were isolated from the Baltic Sea. The results of DNA-DNA hybridization experiments, indicated that three of the new isolates were closely related, while the fourth was only moderately related to the other three. Sequence signature and higher-order structural detail analyses of the 16S rRNA of strain IFAM 1418T (T = type strain) indicated that this isolate is related to the alpha subclass of the class Proteobacteria. Although our isolates resemble members of the genera Hyphomicrobium and Hyphomonas in morphology, assignment to either of these genera was excluded on the basis of their markedly lower DNA guanine-plus-cytosine contents. We propose that these organisms should be placed in a new genus, Hirschia baltica is the type species of this genus, and the type strain of H. baltica is strain IFAM 1418 (= DSM 5838).     

Rapid cataloging of ribonuclease T1 resistant oligonucleotides from ribosomal RNAs for phylogenetic studies

A rapid and simple method of oligonucleotide cataloging for phylogenetic studies is presented. It involves in vitro 5'-32P-labelling of RNase T1 - resistant oligonucleotides of ribosomal 16S RNA and finger-printing by high voltage electrophoresis and gradient thinlayer chromatography. Oligonucleotide sequences are established by the mobility shift method, using controlled alkali cleavage, high voltage electrophoresis and homochromatography. These procedures facilitate in particular the analysis of long RNase T1 - resistant oligonucleotides. Oligonucleotide catalogs are established fo three Actinomycetes, namely Oerskovia turbata, Actinoplanes philippinensis and Ampullariella regularis. These catalogs are equivalent to those obtained by methods which were described by Sanger and Woese.     

DNA-DNA homology studies among strains of Arthrobacter and Brevibacterium

Sixteen named strains of Arthrobacter and two strains of Brevibacterium were investigated by nucleic acid hybridisation. The Arthrobacter strains show homology values ranging between 11 and 55% to the type strain A. globiformis DSM 20124 (ATCC 8010), indicating only a low to moderate relationship. Two strains of A. globiformis, DSM 20124 and DSM 20125, exhibit only poor relationship to one another (30%). Among all the Arthrobacter strains the homology data range between 10 to 70% demonstrating separate status of almost all species. Only A. polychromogenes DSM 20136 was found to be a subspecies of A. oxydans DSM 20119. The type strain of A. citreus, DSM 20133 shows a remarkable lack of homology to four other strains of A. citreus, deposited as ATCC 15170, ATCC 17775, ATCC 21040 and ATCC 21348 (11–13%) which themselves can be separated into two groups according to the homology data (24–31%). Each of the two strains of Brevibacterium share high genetic relatedness with one of these A. citreus groups (71 and 73%, respectively). According to the DNA-DNA homology data, most of the species of Arthrobacter can actually be ranged taxonomically as species.Abbreviation DSM German Collection of Microorganisms, Menzinger Strasse 67, D-8000 Munich 19, FRG - ATCC American Type Culture Collection, Rockville, Maryland, U.S.A. - CCM Czechoslovak Collection of Microorganisms, J. E. Purkyne University, Tr. Obracu miru 10, Brno, CSSR - NCIB National Collection of Industrial Bacteria Aberdeen, Scotland