Actinomycetes are considered an inexhaustible source of chemically diverse secondary metabolites. In addition to the genera Streptomyces, Micromonospora, and Actinoplanes, coryneform and nocardioform bacteria have been used in screening programs for new compounds of medical and biotechnological importance (5, 8).In the course of a program aimed at isolating actinomycetes that have potential for producing novel bioactive compounds, we isolated a large number of strains from different soils. In about 65 of these strains diaminobutyric acid (DAB) was the diagnostic diamino acid in the peptidoglycan. These strains had morphological and chemotaxonomic characteristics that placed them near the genera Agromyces and Clavibacter.In this paper we describe the isolation and characterization of two strains which differed markedly from the members of the genera Agromyces (27), Clavibacter, and Rathayibacter (26) that have been described. On the basis of our morphological, physiological, and biochemical data, as well as the results of our 16s ribosomal DNA (rDNA) analysis, we concluded that these strains belong to a new genus and species, for which we propose the name Agrococcus jenensis. These strains have been deposited in the German Collection of Microorganisms and MATERIALS AND METHODSBacterial strains and cultural conditions. Strain 2002-39/lT was isolated from a sample of frozen compost soil obtained near Jena, Germany, at a depth of about 10 cm. Isolation of this organism involved dilution plating on nutrient agar containing 2% peptone, a pancreatic digest (meat, fish), 0.5% NaCI, and 1.2% agar. Strain ST54 was isolated from the sandstone surface of the Alte Pinakothek building in Munich, Germany. The Agromyces and Cluvibacter type strains which we used in this study are listed in Table 1. General laboratory cultivation was performed on solid medium or in liquid rich (R) medium (25) containing 1% Bacto Peptone (Difco Laboratories), 0.5% yeast extract, 0.5% Casamino Acids, 0.2% meat extract, 0.5% malt extract, 0.2% glycerol, 0.1% MgSO, -7H,O, and 0.005% Tween 80 (pH 7.2) at 28°C.Morphological and physiological characteristics. Cell morphology was determined by examining cultures of different ages by phase-contrast microscopy. Colony morphology was studied by using a stereomicroscope. For scanning electron microscopy an 18-h-old culture of strain 2002-39/lT on an agar plate was suspended in a phosphate-buffered salt solution. The cells were fixed with 0 5 % glutaraldehyde, washed, and dehydrated in a series containing increasing concentrations of ethanol. After sputter coating with gold-palladium, the cells were observed with a Zeiss model 962 scanning electron microscope.

Many crude oil constituents are biodegradable in the presence of oxygen; however, a substantial anaerobic degradation has never been demonstrated. An unusually low content of n-alkanes in oils of certain deposits is commonly attributed to selective utilization of these hydrocarbons by aerobic microorganisms. On the other hand, oil wells and production fluids were shown to harbour anaerobic sulphate-reducing bacteria, but their actual electron donors and carbon sources were unknown. On the basis of nutritional properties of various bacterial isolates it was assumed that fatty acids and H2 are potential electron donors for sulphate reduction in situ. Here we demonstrate that hydrocarbons in crude oil are used directly by sulphate-reducing bacteria growing under strictly anoxic conditions. A moderately thermophilic pure culture selectively utilizes n-alkanes in oil for sulphate reduction to sulphide. In addition, a mesophilic sulphate-reducing enrichment culture is shown to oxidize alkylbenzenes in oil. Thus, sulphate-reducing bacteria utilizing aliphatic and aromatic hydrocarbons as electron donors may present a significant source of sulphide in oil deposits and oil production plants.

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Natural relationships, improvement of anaerobic growth on hydrocarbons, and properties that may provide clues to an understanding of oxygen-independent alkane metabolism were studied with two mesophilic sulfate-reducing bacteria, strains Hxd3 and Pnd3. Strain Hxd3 had been formerly isolated from an oil tank; strain Pnd3 was isolated from marine sediment. Strains Hxd3 and Pnd3 grew under strictly anoxic conditions on n-alkanes in the range of C12-C20 and C14-C17, respectively, reducing sulfate to sulfide. Both strains shared 90% 16 S rRNA sequence similarity and clustered with classified species of completely oxidizing, sulfate-reducing bacteria within the delta-subclass of Proteobacteria. Anaerobic growth on alkanes was stimulated by alpha-cyclodextrin, which served as a non-degradable carrier for the hydrophobic substrate. Cells of strain Hxd3 grown on hydrocarbons and alpha-cyclodextrin were used to study the composition of cellular fatty acids and in vivo activities. When strain Hxd3 was grown on hexadecane (C16H34), cellular fatty acids with C-odd chains were dominant. Vice versa, cultures grown on heptadecane (C17H36) contained mainly fatty acids with C-even chains. In contrast, during growth on 1-alkenes or fatty acids, a C-even substrate yielded C-even fatty acids, and a C-odd substrate yielded C-odd fatty acids. These results suggest that anaerobic degradation of alkanes by strain Hxd3 does not occur via a desaturation to the corresponding 1-alkenes, a hypothetical reaction formerly discussed in the literature. Rather an alteration of the carbon chain by a C-odd carbon unit is likely to occur during activation; one hypothetical reaction is a terminal addition of a C1 unit. In contrast, fatty acid analyses of strain Pnd3 after growth on alkanes did not indicate an alteration of the carbon chain by a C-odd carbon unit, suggesting that the initial reaction differed from that in strain Hxd3. When hexadecane-grown cells of strain Hxd3 were resuspended in medium with 1-hexadecene, an adaptation period of 2 days was observed. Also this result is not in favor of an anaerobic alkane degradation via the corresponding 1-alkene.

A re-evaluation of the taxonomy of Paracoccus denitrificans and a proposal for the combination Paracoccus pantotrophus comb. nov.Frederick A. Rainey,' Donovan P. Kelly Comparison of both 16s rRNA coding sequences and DNA-DNA hybridization of ten strains of a-subclass of Proteobacteria currently classified as strains of Paracoccus denitrificans has shown that they fall into two groups which are distinct from each other a t the species level. Comparison with published data on the cytochrome c profiles and other 16s rRNA coding sequences in the literature has confirmed these observations and enabled several other strains also to be assigned to these two groups. Group A comprises strains ATCC 1774IT (the type strain of P. denitrificans), LMD 22.2IT, DSM 413T, ATCC 19367, ATCC 13543, DSM 1404, DSM 1405, Pd 1222 (a genetic modification of DSM 4133 and NCIMB 8944. Group B comprises ATCC 35512T (the original type strain of Thiosphaera pantotropha), LMD 82.5T, LMD 92.63, DSM 65, LMG 4218, IAM 12479, JCM 6892, DSM 11072, DSM I1073 and DSM 11104. In light of these findings, it is proposed that: (1) strains of group A are retained as P. denitrificans, with ATCC 1774IT as the type strain of the type species; and (2) all strains of group B are assigned to the new species combination Paracoccus pantotrophus comb. nov., with strain ATCC 35512T as the type strain. Comparative 16s rRNA sequence analysis and DNA-DNA hybridization of strains of Paracoccus versutus confirm that this species is distinct from both P. denitrificans and P. pantotrophus, but that its nearest phylogenetic neighbour is P. pantotrophus.