Taxonomic characterization of Ketogulonigenium vulgare gen. nov., sp. nov. and Ketogulonigenium robustum sp. nov., which oxidize L-sorbose to 2-keto-L-gulonic acid.

Four bacterial strains that oxidize L-sorbose to 2-keto-L-gulonic acid, a key intermediate in the synthesis of vitamin C, were isolated from soils of geographically distinct locations. All were Gram-negative, facultatively anaerobic, chemoheterotrophic rods. Comparative analysis revealed nearly identical 16S rDNA sequences amongst them (99.7-100% identical) and identified them as members of the alpha-subclass of the Proteobacteria. Phylogenetic analysis identified the closest taxonomically defined genus as Roseobacter (92.1-92.8% identical). On the basis of phylogenetic, phenotypic and genotypic analyses, a new genus is proposed, Ketogulonigenium gen. nov. Based upon these analyses, we also propose the reclassification of strain DSM 4025TP, originally identified as Gluconobacter oxydans, to the genus Ketogulonigenium. Two species are proposed: the type species Ketogulonigenium vulgare gen. nov., sp. nov., consisting of strains 62A-12APP, 266-13BPP and the type strain K. vulgare DSM 4025TP, and Ketogulonigenium robustum gen. nov., sp. nov., consisting of the type strain K. robustum X6LTP (= NRRL B-21627 = KCTC 0858BP). The species affiliation of the fifth strain (291-19PP) remains unresolved.

Pseudomonas sp. strain 273, an aerobic alpha, omega-dichloroalkaneDegrading bacterium.

A gram-negative, aerobic bacterium was isolated from soil; this bacterium grew in 50% (vol/vol) suspensions of 1,10-dichlorodecane (1,10-DCD) as the sole source of carbon and energy. Phenotypic and small-subunit ribosomal RNA characterizations identified the organism, designated strain 273, as a member of the genus Pseudomonas. After induction with 1,10-DCD, Pseudomonas sp. strain 273 released stoichiometric amounts of chloride from C5 to C12 alpha, omega-dichloroalkanes in the presence of oxygen. No dehalogenation occurred under anaerobic conditions. The best substrates for dehalogenation and growth were C9 to C12 chloroalkanes. The isolate also grew with nonhalogenated aliphatic compounds, and decane-grown cells dechlorinated 1,10-DCD without a lag phase. In addition, cells grown on decane dechlorinated 1,10-DCD in the presence of chloramphenicol, indicating that the 1,10-DCD-dechlorinating enzyme system was also induced by decane. Other known alkane-degrading Pseudomonas species did not grow with 1,10-DCD as a carbon source. Dechlorination of 1,10-DCD was demonstrated in cell extracts of Pseudomonas sp. strain 273. Cell-free activity was strictly oxygen dependent, and NADH stimulated dechlorination, whereas EDTA had an inhibitory effect.

Identification of bacterial isolates obtained from intestinal contents associated with 12,000-year-old mastodon remains.

Mastodon (Mammut americanum) remains unearthed during excavation of ancient sediments usually consist only of skeletal material, due to postmortem decomposition of soft tissues by microorganisms. Two recent excavations of skeletal remains in anoxic sediments in Ohio and Michigan, however, have uncovered organic masses which appear to be remnants of the small and large intestines, respectively. Macrobotanical examinations of the composition of these masses revealed assemblages of plant material radiocarbon dated to approximately 11,500 years before the present and thought to be incompletely digested food remains from this extinct mammal. We attempted to cultivate and identify bacteria from the intestinal contents, bone-associated sediments, and sediments not in proximity to the remains using a variety of general and selective media. In all, 295 isolates were cultivated, and 38 individual taxa were identified by fatty acid-methyl ester (FAME) profiles and biochemical characteristics (API-20E). The taxonomic positions of selected enteric and obligately anaerobic bacteria were confirmed by 16S ribosomal DNA (rDNA) sequencing. Results indicate that the intestinal and bone-associated samples contained the greatest diversity of bacterial taxa and that members of the family Enterobacteriaceae represented 41% of all isolates and were predominant in the intestinal masses and sediments in proximity to the skeleton but were uncommon in the background sediments. Enterobacter cloacae was the most commonly identified isolate, and partial rDNA sequencing revealed that Rahnella aquatilis was the correct identity of strains suggested by FAME profiles to be Yersinia enterocolitica. No Bacteroides spp. or expected intestinal anaerobes were recovered. The only obligate anaerobes recovered were clostridia, and these were not recovered from the small intestinal masses. Microbiological evidence from this study supports other, macrobotanical data indicating the intestinal origin of these masses. Whether these organisms are direct descendants of the original intestinal microbiota, however, cannot be established.

Acetivibrio cellulolyticus and Bacteroides cellulosolvens are members of the greater clostridial assemblage.

The nucleotide sequences of the 16S rRNA genes of Acetivibrio cellulolyticus, A. cellulosolvens, and Bacteroides cellulosolvens were determined and shown to be affiliated with the low G + C members of Gram-positive bacteria. The sequences for A. celluolyticus and A. cellulosolvens were revealed to be identical, supporting the proposal by W.D. Murray [Int. J. Syst. Bacteriol. (1986) 36, 314-316] that A. cellulosolvens be correctly classified as A. cellulolyticus. The closest relative to A. cellulolyticus is Clostridium aldrichii, related at 98.5% sequence similarity. B. cellulosolvens and A. cellulolyticus are related at 94.4% sequence similarity.

Genetic resistance/susceptibility to mycobacteria: phenotypic expression in bone marrow derived macrophage lines.

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.

The division between fast- and slow-growing species corresponds to natural relationships among the mycobacteria.

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. intracellulare-M. scrofulaceum serovar 1 by this analysis.

Isolation of Campylobacter jejuni from an aborted caprine fetus.

Campylobacter jejuni was isolated in pure culture from the tissues and stomach contents of an aborted fetus from a mixed-breed Illinois goat herd. In the herd, 5 of 21 does aborted in late gestation. The does had diarrhea before or concurrent with the abortions. Does that did not abort were vaccinated with ovine C fetus bacterin and were given chlortetracycline orally at the rate of 75 mg/day for 2 weeks. Further abortions did not occur.