RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis.

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.

Salinicoccus salsiraiae sp. nov.: a new moderately halophilic gram-positive bacterium isolated from salted skate.

Two moderately halophilic low G + C Gram-positive bacteria were isolated from a sample of salted skate (Class Chondrychthyes, Genus Raja). Phylogenetic analysis of the 16S rRNA gene sequence of strains RH1(T) and RH4 showed that these organisms represented a novel species of the genus Salinicoccus. The new isolates formed pink-red colonies and flocculated in liquid media, with optimum growth in media containing 4% NaCl and pH of about 8.0. These organisms are aerobic but reduce nitrate to nitrite under anaerobic conditions. Acid is produced from several carbohydrates. Oxidase and catalase were detected. Menaquinone 6 was the major respiratory quinone. The major fatty acids of strains RH1(T) and RH4 were 15:0 anteiso and 15:0 iso. The G + C contents of DNA were 46.2 and 46.0 mol%, respectively. The peptidoglycan was of A3alpha L-Lys-Gly(5-6) type. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we suggest that strain RH1(T) (=LMG 22840 = CIP 108576) represents a new species of the genus Salinicoccus, for which we propose the name Salinicoccus salsiraiae.

Alkalibacterium iburiense sp. nov., an obligate alkaliphile that reduces an indigo dye.

Three indigo-reducing obligately alkaliphilic strains, M3(T), 41A and 41C, were isolated. The isolates grew at pH 9-12, but not at pH 7-8. They were Gram-positive, facultatively anaerobic, straight rod-shaped strains with peritrichous flagella. The isolates grew in 0-14% (w/v) NaCl, with optimum growth at 3-13%. They grew at temperatures between 10 and 45 degrees C, with optimum growth at around 30-37 degrees C. They did not hydrolyse starch or gelatin. DL-lactate was the major end-product from D-glucose. No quinones could be detected. The peptidoglycan type was A4beta, Orn-d-Asp. The major cellular fatty acids were C(16:0), C(16:1)7c and C(18:1)9c. The DNA G+C content was 42.6-43.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that the isolates belong to the genus Alkalibacterium. DNA-DNA hybridization revealed low similarity (less than 16%) of the isolates with respect to the two closest phylogenetically related strains, Alkalibacterium olivapovliticus and Alkalibacterium psychrotolerans. On the basis of phenotypic and chemotaxonomic characteristics, phylogenetic data and DNA-DNA relatedness, the isolates merit classification as a novel species of the genus Alkalibacterium, for which the name Alkalibacterium iburiense is proposed. The type strain is M3(T) (=JCM 12662(T)=NCIMB 14024(T)).

Identification of anaerobic nonsporeforming gram-positive bacilli by biochemical tests and gas-liquid chromatography.

There are many methods to identify anaerobic nonsporeforming bacilli: histological, bacteriological (biochemical test, microsystem API 20 A), serological, cell wall composition analysis, molecular methods and gas-liquid chromatography (GLC). A comparison between biochemical tests and gas-liquid chromatography was made in this study for the identification of this group of microorganisms. GLC conditions were established with the aid of reference strains. These conditions were then applied to ten strains which were previously identified by biochemical tests. Strains were grown in PYG broth and fermentation end products were analyzed, volatile and non volatile fatty acids. Their qualitative determination was made by comparing the retention time of known standards and the chromatographic pattern of reference strains. In addition, a semiquantitative analysis was made. The results of identification by biochemical tests were: five strains belonged to Actinomyces genus; three were Propionibacterium acnes; one Propionibacterium granulosum and one P. propionicum. By the GLC only seven strains were identified: four were Actinomyces and three P. acnes. Only six strains showed identification correlation by both biochemical tests and GLC. GLC is a presumptive identification method that can be used along with other complementary tests for a definitive identification at genus level.

Cryptobacterium curtum gen. nov., sp. nov., a new genus of gram-positive anaerobic rod isolated from human oral cavities.

Novel Eubacterium-like isolates, strains 12-3T and KV43-B, which were isolated from the periodontal pocket of an adult patient with periodontal disease and necrotic dental pulp, respectively, were studied taxonomically and phylogenetically. The morphological and differential biochemical characteristics of these organisms are also described in this paper. These organisms were Gram-positive, anaerobic, non-spore-forming, rod-shaped bacteria that were inert in most of the conventional biochemical tests and closely resembled members of asaccharolytic oral Eubacterium species. On the other hand, protein profiles of whole cells in SDS-PAGE and Western immunoblotting reaction analysis distinguished these isolates from strains of the previously described genus Eubacterium. The G+C content of the DNAs from the novel isolates was 50 and 51 mol%, respectively. The levels of DNA-DNA relatedness to other asaccharolytic oral Eubacterium species, including Eubacterium brachy, Eubacterium lentum, Eubacterium nodatum, Eubacterium timidum, Eubacterium saphenum, Eubacterium minutum and Eubacterium exiguum, was less than 11%. These organisms also exhibited a very low level of reassociation with the DNA of Eubacterium limosum, the type species of the genus Eubacterium. The results of 16S rDNA sequence comparisons revealed that these organisms represent a novel lineage distinct from all previously described genera of Gram-positive, rod-shaped bacteria. On the basis of our results, it is suggested that strains 12-3T and KV43-B should be classified in a new genus and species, for which the name Cryptobacterium curtum gen. nov., sp. nov. is proposed. The type strain of Cryptobacterium curtum is 12-3T (= ATCC 700683T).

Leucobacter komagatae gen. nov., sp. nov., a new aerobic gram-positive, nonsporulating rod with 2,4-diaminobutyric acid in the cell wall.

A new aerobic, gram-positive, nonsporulating rod-shaped organism is described: Strain IFO 15245T (T = type strain) has the following characteristics: the menaquinone contains a side chain with 11 isoprenyl units; the guanine-plus-cytosine content of the DNA is 66.2 mol%; 2,4-diaminobutyric acid, glutamic acid, alanine, glycine, and gamma-aminobutyric acid are present in the cell wall at a molar ratio of ca. 1:1:2:1:1; and glucose and galactose are also present in the cell wall. A comparison of partial 16S rRNA sequences revealed that IFO 15245T represents a distinct line of descent within the gram-positive bacteria with high guanine-plus-cytosine contents. The taxonomic characteristics of this organism are different from those of previously described aerobic, gram-positive, nonsporulating, rod-shaped bacteria. The name Leucobacter komagatae gen. nov., sp. nov., is proposed for this organism. The type strain is strain IFO 15245.

Additional tests to differentiate Arcanobacterium haemolyticum and Actinomyces pyogenes.

A commercially available biochemical test panel, commercially available diagnostic tablets and gas-liquid chromatography (GLC) of cellular fatty acids were used to find out whether Arcanobacterium haemolyticum and Actinomyces pyogenes could be further differentiated from each other. Xylitol and alpha-methyl-D-glucoside fermentation, Voges-Proskauer reaction and tributyrate hydrolysis were found to be useful additional tests which differentiated Arc. haemolyticum and A. pyogenes. GLC analysis revealed major differences in the cellular 16:0, 18:2(9,12) and 18:1(9) fatty acid composition of the two species. Especially the Voges-Proskauer test available as diagnostic tablets can be easily performed in clinical microbiology laboratories, in addition to the tests now used to differentiate Arc. haemolyticum from A. pyogenes.

Proposal of six new species in the genus Aureobacterium and transfer of Flavobacterium esteraromaticum Omelianski to the genus Aureobacterium as Aureobacterium esteraromaticum comb. nov.

Twelve strains placed in the genera Flavobacterium, Pseudomonas, and Aureobacterium, including soil isolates, were characterized taxonomically. On the basis of morphological, physiological, and chemotaxonomic data, as well as DNA-DNA hybridization data, we propose that 11 of these strains should be classified in the genus Aureobacterium as new combinations or new species, as follows: Aureobacterium esteraromaticum comb. nov. (type strain, IFO 3751 [= ATCC 8091]), Aureobacterium arabinogalactanolyticum sp. nov. (type strain, IFO 14344), Aureobacterium keratanolyticum sp. nov. (type strain, IFO 13309), Aureobacterium luteolum sp. nov. (type strain, IFO 15074 [= DMS 20143]), Aureobacterium schleiferi sp. nov. (type strain, IFO 15075 [= DMS 20489]), Aureobacterium terrae sp. nov. (type strain, IFO 15300), and Aureobacterium trichothecenolyticum sp. nov. (type strain, IFO 15077 [= JCM 1358]). Whereas the peptidoglycan type of members of this genus is considered to be B2beta, the new species A. keratanolyticum was shown to have a new peptidoglycan type, murein variation B2alpha. An emended description of the genus Aureobacterium is presented.