Comparative 16S rRNA sequencing was used to infer the phylogenetic relationships among selected species of mycobacteria and related organisms. The phylogeny inferred reflects the traditional classification, with major branches of the phylogenetic tree in general correspondence to the four Runyon groups and with numerical classification analyses. All the mycobacterial species compared, with the exception of M. chitae, are closely related (average similarity values greater than 95%). The slow growers form a coherent line of descent, distinct from the rapid growers, within which the overt pathogens are clustered. The distant relationship between M. chitae and the remaining mycobacteria suggests that this organism is incorrectly classified with the mycobacteria. M. paratuberculosis 18 was indistinguishable from M. avium-M. intracelulare-M. scrofulaceum serovar 1 by this analysis.

Two tetrachlorethene (PCE)-dechlorinating populations, designated strains BB1 and BRS1, were isolated from pristine river sediment and chloroethene-contaminated aquifer material, respectively. PCE-to-cis-1,2-dichloroethene-dechlorinating activity could be transferred in defined basal salts medium with acetate as the electron donor and PCE as the electron acceptor. Taxonomic analysis based on 16S rRNA gene sequencing placed both isolates within the Desulfuromonas cluster in the ␦ subdivision of the Proteobacteria. PCE was dechlorinated at rates of at least 139 nmol min ؊1 mg of protein ؊1 at pH values between 7.0 and 7.5 and temperatures between 25 and 30°C. Dechlorination also occurred at 10°C. The electron donors that supported dechlorination included acetate, lactate, pyruvate, succinate, malate, and fumarate but not hydrogen, formate, ethanol, propionate, or sulfide. Growth occurred with malate or fumarate alone, whereas oxidation of the other electron donors depended strictly on the presence of fumarate, malate, ferric iron, sulfur, PCE, or TCE as an electron acceptor. Nitrate, sulfate, sulfite, thiosulfate, and other chlorinated compounds were not used as electron acceptors. Sulfite had a strong inhibitory effect on growth and dechlorination. Alternate electron acceptors (e.g., fumarate or ferric iron) did not inhibit PCE dechlorination and were consumed concomitantly. The putative fumarate, PCE, and ferric iron reductases were induced by their respective substrates and were not constitutively present. Sulfide was required for growth. Both strains tolerated high concentrations of PCE, and dechlorination occurred in the presence of free-phase PCE (dense non-aqueous-phase liquids). Repeated growth with acetate and fumarate as substrates yielded a BB1 variant that had lost the ability to dechlorinate PCE. Due to the 16S rRNA gene sequence differences with the closest relatives and the unique phenotypic characteristics, we propose that the new isolates are members of a new species, Desulfuromonas michiganensis, within the Desulfuromonas cluster of the Geobacteraceae.The first reported anthropogenic production of tetrachloroethene (PCE) dates back to 1821, when Faraday produced PCE by thermal decomposition of hexachloroethane (15). Starting at the beginning of the 20th century, increasing amounts of PCE and trichloroethene (TCE) were manufactured due to the extensive use of these compounds in industry, in the military, and in private households, mainly as nonflammable solvents (summarized in reference 9). This widespread use, along with careless handling and storage, made chlorinated ethenes abundant groundwater pollutants. Often, PCE and TCE contamination coincided with spills of other organic compounds, such as crude oil constituents, other solvents (often seen at industrial and military sites), or starch (at dry cleaning operations). Aerobic microorganisms rapidly degrade the nonchlorinated contaminants, thereby depleting oxygen. Under anaerobic conditions PCE and TCE can be used as alternate growth-supportin...

The microbial composition of ancient permafrost sediments from the Kolyma lowland of Northeast Eurasia was examined through culture and culture-independent approaches. These sediments have been continuously frozen for 5,000 to 2-3 million years. A total of 265 Bacteria 16S rRNA gene sequences were amplified from the permafrost total-community genomic DNA and screened by amplified ribosomal 16S rRNA restriction analysis. Members of three major lineages were found: gamma-Proteobacteria (mostly Xanthomonadaceae), Actinobacteria, and Firmicutes. We also determined partial 16S rRNA gene sequences of 49 isolates from a collection of 462 aerobes isolated from these sediments. The bacteria included Actinomycetales (Arthrobacter and Microbacteriaceae); followed by the Firmicutes (Exiguobacterium and Planomicrobium); the Bacteroidetes (Flavobacterium); the gamma-Proteobacteria (Psychrobacter); and the alpha-Proteobacteria (Sphingomonas). Both culture and culture-independent approaches showed the presence of high and low G+C Gram-positive bacteria and gamma-Proteobacteria. Some of the 16S rRNA gene sequences of environmental clones matched those of Arthrobacter isolates. Two-thirds of the isolates grew at -2.5 degrees C, indicating that they are psychroactive, and all are closely related to phylogenetic groups with strains from other cold environments, mostly commonly from Antarctica. The culturable and non-culturable microorganisms found in the terrestrial permafrost provide a prototype for possible life on the cryogenic planets of the Solar System.

Congenic strains of mice susceptible (B10A.Bcgs) or resistant (B10A.Bcgr) to BCG were established. Here we describe the model system which has been established to analyze the functional activities of macrophages in the two strains. We have immortalized bone marrow macrophages from B10A.Bcgs and B10A.Bcgr congenic strains of mice and derived cloned macrophage lines designated B10S and B10R, respectively. B10R and B10S cell lines exhibited surface markers and morphology typical of macrophages. B10S and B10R were similar in their phagocytic activity, in their level of c-fms, in their transforming growth factor beta (TGF beta) mRNAs expression, and in their expression of tumoricidal activity in response to interferon-gamma (IFN gamma) plus lipopolysaccharides (LPS). However, B10R macrophages expressed a higher level of la mRNA when activated with IFN gamma compared with B10S macrophages. Analysis of the bacteriostatic activity of the two cell lines revealed that B10R macrophages were much more active in inhibiting Mycobacterium smegmatis replication than B10S. To measure the intracellular destruction of bacilli, a bactericidal assay based on hybridization with an oligonucleotide probe specific for mycobacterial ribosomal RNA was designed. The results demonstrated that B10R macrophages were endowed with enhanced constitutive bactericidal activity as compared with B10S. In conclusion we have obtained macrophage lines from bone marrow of B10A.Bcgs and B10A.Bcgr mice that express to a similar extent functional and phenotypic characteristics of macrophages. However, we demonstrate that relative to B10S macrophages, the B10R macrophages have higher expression of la mRNA and that they are constitutively more active in expressing mycobactericidal activity.