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Database searches indicated that the genome of Bacillus subtilis contains three different genes encoding RNase H homologues. The ypdQ gene encodes an RNase HI homologue with 132 amino acid residues, whereas the rnh and ysgB genes encode RNase HII homologues with 255 and 313 amino acid residues, respectively. RNases HI and HII show no significant sequence similarity. These genes were individually expressed in Escherichia coli; the recombinant proteins were purified, and their enzymatic properties were compared with those of E. coli RNases HI and HII. We found that the ypdQ gene product showed no RNase H activity. The 2.2 kb pair genomic DNA containing this gene did not suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that this gene product shows no RNase H activity in vivo as well. In contrast, the rnh (rnhB) gene product (RNase HII) showed a preference for Mn2+, as did E. coli RNase HII, whereas the ysgB (rnhC) gene product (RNase HIII) exhibited a Mg2+-dependent RNase H activity. Oligomeric substrates digested with these enzymes indicate similar recognition of these substrates by B. subtilis and E. coli RNases HII. Likewise, B. subtilis RNase HIII and E. coli RNase HI have generated similar products. These results suggest that B. subtilis RNases HII and HIII may be functionally similar to E. coli RNases HII and HI, respectively. We propose that Mn2+-dependent RNase HII is universally present in various organisms and Mg2+-dependent RNase HIII, which may have evolved from RNase HII, functions as a substitute for RNase HI.

A biosurfactant termed arthrofactin produced by Arthrobacter species strain MIS38 was purified and chemically characterized as 3-hydroxydecanoyl-D-leucyl-D-asparagyl-D-threonyl-D-leucyl-D-leucyl-D-seryl-Lleucyl-D-seryl-L-isoleucyl-L-isoleucyl-L-asparagyl lactone. Surface activity of arthrofactin was examined, with surfactin as a control. Critical micelle concentration values of arthrofactin and surfactin were around 1.0 X 10 -5 M and 7.0 x 10-5 M at 25°C, respectively. Arthrofactin was found to be five to seven times more effective than surfactin. The minimum surface tension value of arthrofactin was 24 mN/m at a concentration higher than the critical micelle concentration. According to the oil displacement assay, arthrofactin was a better oil remover than synthetic surfactants, such as Triton X-100 and sodium dodecyl sulfate. Arthrofactin is one of the most effective lipopeptide biosurfactants.Biosurfactants are surface active substances derived from living organisms, mainly from microorganisms (2, 5). The biological function of biosurfactants is thought to be participation in the solubilization of hydrophobic substances (hydrocarbons, lipids, and sterols, etc.), promoting enhanced cell assimilation. Emulsification which increases the surface area between two immiscible phases, results in small oil drops in water. It is generally concluded that microorganisms growing on water-insoluble hydrocarbons benefit from the presence of a surfactant (6,8,13). Biosurfactants are usually complex lipids, with more chemically complicated structures than synthetic surfactants. Lipopeptide biosurfactants are structurally more heterogeneous than glycolipid types.The minimum surface tension and critical micelle concentration are parameters used to measure the efficiency of surfactant systems. The best-known lipopeptide biosurfactant is surfactin, which lowers the surface tension of 0.1 M NaHCO3 from 71.6 to 27.0 mN/m (1). Recently, some strains of Bacillus licheniformis were shown to produce quite similar biosurfactant to surfactin (9,15). Arthrobacter species have been reported to produce extracellular glycolipids (12,20), none of which lowers the surface tension of water to 30 mN/m. We describe the chemical structure of a new lipopeptide-type biosurfactant. MATERIALS AND METHODSScreening of biosurfactant-producing bacteria. Biosurfactant-producing strains were selected as described previously (16), except that the cultivation temperature was 30°C. A slimy colony (strain MIS38) surrounded by a large halo on an oil-agar plate was obtained and used for further experiments.Production and purification of biosurfactant. Strain MIS38 was cultivated in 3 liters of L broth (1% Bacto tryptone, 0.5% yeast extract, 0.5% NaCl [pH 7.2]) at 30°C for 48 h. The culture was centrifuged (10,000 x g for 10 min), and the supernatant fluid was concentrated by ultrafiltration (exclusion molecular size, 10 kDa).The concentrated biosurfactant was extracted three times * Corresponding author.with an equal volume of hexane. After evaporation, the biosur...

Summary A hyperthermophilic archaeal strain, KOD1, isolated from a solfatara on Kodakara Island, Japan, has previously been reported as Pyrococcus sp. KOD1. However, a detailed phylogenetic tree, made possible by the recent accumulation of 16S rRNA sequences of various species in the order Thermococcales, indicated that strain KOD1 is a member of the genus Thermococcus. We performed DNA-DNA hybridization tests against species that displayed high similarity in terms of 16S ribosomal DNA sequences, including Thermococcus peptonophilus and Thermococcus stetteri. Hybridization results and differences in growth characteristics and substrate utilization differentiated strain KOD1 from T. peptonophilus and T. stetteri at the species level. Our results indicate that strain KOD1 represents a new species of Thermococcus, which we designate as Thermococcus kodakaraensis KOD1 sp. nov.

Lipopeptide biosurfactants (LPBSs) consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive) and Pseudomonas (Gram-negative) have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs). Production of active-form NRPSs requires not only transcriptional induction and translation but also post-translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.

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