Bioprospecting potentials of moderately halophilic bacteria and the isolation of squalene producers from Kuwait sabkha.

Sabkhas in Kuwait are unique hypersaline marine environments under-explored for bacterial community composition and bioprospecting. The 16S rRNA sequence analysis of 46 isolates with distinct morphology from two Kuwait sabkhas recovered 11 genera. Phylum Firmicutes dominated these isolates, and Bacillus (32.6%) was recovered as the dominant genera, followed by Halococcus (17.4%). These isolates were moderately halophilic, and some of them showed tolerance and growth at extreme levels of salt (20%), pH (5 and/or 11), and temperature (55 °C). A higher percentage of isolates harbored protease (63.0), followed by DNase (41.3), amylase (41.3), and lipase (32.6). Selected isolates showed antimicrobial activity against E. faecalis and isolated Halomonas shengliensis, and Idiomarina piscisalsi harbored gene coding for dNDP-glucose 4,6-dehydratase (Glu 1), indicating their potential to produce biomolecules with deoxysugar moieties. Palmitic acid or oleic acid was the dominant fatty acid, and seven isolates had some polyunsaturated fatty acids (linolenic or γ-linolenic acid). Interestingly, six isolates belonging to Planococcus and Oceanobacillus genus produced squalene, a bioactive isoprenoid molecule. Their content increased 30-50% in the presence of Terbinafine. The potential bioactivities and extreme growth conditions make this untapped bacterial diversity a promising candidate for future bioprospecting studies.

Identification and characterization of the gene encoding an extracellular protease from haloarchaeon Halococcus salifodinae.

Extracellular proteases from haloarchaea (halolysins) can resist high salt conditions. In this study, the gene encoding a halolysin from Halococcus salifodinae was identified. The hlyA gene encoded an active halolysin with the classical Asp-His-Ser catalytic triad of serine proteases. Site-directed mutagenesis showed that the three cysteine residues in the catalytic domain were important for the extracellular proteolytic activity and displayed an additive effect on the activity. Truncation mutants of the C-terminal extension (CTE) domain displayed very low or almost no extracellular protease activity towards milk and small peptide substrates, indicating its importance for the function of HlyA. CTE can be functionally interchangeable among halolysins. Additionally, the HlyA expressing strain as a starter culture for fish sauce fermentation significantly increased the peptide release and total free amino acid content in fish sauce. This study enriches our knowledge of the key amino acid residues and domains of halolysins, and provides an opportunity for applications of halolysins in fish sauce fermentation.

The Effects of HZE Particles, γ and X-ray Radiation on the Survival and Genetic Integrity of Halobacterium salinarum NRC-1, Halococcus hamelinensis, and Halococcus morrhuae.

Three halophilic archaea, Halobacterium salinarum NRC-1, Halococcus hamelinensis, and Halococcus morrhuae, have been exposed to different regimes of simulated outer space ionizing radiation. Strains were exposed to high-energy heavy ion (HZE) particles, namely iron and argon ions, as well as to γ radiation (60Co) and X-rays, and the survival and the genetic integrity of the 16S rRNA gene were evaluated. Exposure to 1 kGy of argon or iron ions at the Heavy Ion Medical Accelerator in Chiba (HIMAC) facility at the National Institute for Radiological Sciences (NIRS) in Japan did not lead to a detectable loss in viability; only after exposure to 2 kGy of iron ions a decline in survival was observed. Furthermore, a delay in growth was manifested following exposure to 2 kGy iron ions. DNA integrity of the 16S rRNA was not compromised up to 1 kGy, with the exception of Hcc. hamelinensis following exposure to argon particles. All three strains showed a high resistance toward X-rays (exposed at the DLR in Cologne, Germany), where Hcc. hamelinensis and Hcc. morrhuae displayed better survival compared to Hbt. salinarum NRC-1. In all three organisms the DNA damage increased in a dose-dependent manner. To determine a biological endpoint for survival following exposure to γ radiation, strains were exposed to up to 112 kGy at the Beta-Gamma-Service GmbH (BGS) in Germany. Although all strains were incubated for up to 4 months, only Hcc. hamelinensis and Hcc. morrhuae recovered from 6 kGy of γ radiation. In comparison, Hbt. salinarum NRC-1 did not recover. The 16S rRNA gene integrity stayed remarkably well preserved up to 48 kGy for both halococci. This research presents novel data on the survival and genetic stability of three halophilic archaea following exposure to simulated outer space radiation. Key Words: Halophilic archaea-Radiation-Survival. Astrobiology 17, 110-117.

Halococcus sediminicola sp. nov., an extremely halophilic archaeon isolated from a marine sediment.

A novel, red-pigmented and coccoid haloarchaeon, designated strain CBA1101(T), was isolated from a marine sediment. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CBA1101(T) is most closely related to the genus Halococcus in the family Halobacteriaceae. Strain CBA1101(T) had a highest 16S rRNA gene sequence similarity of 98.4 % with Halococcus dombrowskii DSM 14522(T), followed by 93.7-98.3 % with sequences of other type strains in the genus Halococcus. The RNA polymerase subunit B' gene sequence similarity of strain CBA1101(T) with that of Halococcus qingdaonensis JCM 13587(T) is 89.5 % and lower with those of other members of the genus Halococcus. Strain CBA1101(T) was observed to grow at 25-40 °C, pH 6.0-9.0 and in the presence of 15-30 % (w/v) NaCl, with optimal growth at 35-40 °C, pH 7.0 and with 20 % NaCl. The cells of strain CBA1101(T) are Gram-negative and did not lyse in distilled water. The major polar lipids were identified as phosphatidylglyerol, phosphatidylglycerol phosphate methyl ester, sulfated diglycosyl diether, unidentified phospholipids and unidentified glycolipids. The genomic DNA G+C content was determined 66.0 mol%. The DNA-DNA hybridization experiment showed that there was less than 40 % relatedness between strain CBA1101(T) and the reference species in the genus Halococcus. Based on this polyphasic taxonomic analysis, strain CBA1101(T) is considered to represent a new species in the genus Halococcus, for which the name Halococcus sediminicola sp. nov. is proposed. The type strain is CBA1101(T) (=JCM 18965(T) = CECT 8275(T)).