Moderate Halophile Research Articles

Deciphering the streamlined genome of Streptomyces xiamenensis 318 as the producer of the anti-fibrotic drug candidate xiamenmycin.

Streptomyces xiamenensis 318, a moderate halophile isolated from a mangrove sediment, produces the anti-fibrotic compound xiamenmycin. The whole genome sequence of strain 318 was obtained through long-read single-molecule real-time (SMRT) sequencing, high-throughput Illumina HiSeq and 454 pyrosequencing technologies. The assembled genome comprises a linear chromosome as a single contig of 5,961,401-bp, which is considerably smaller than other reported complete genomes of the genus Streptomyces. Based on the antiSMASH pipeline, a total of 21 gene clusters were predicted to be involved in secondary metabolism. The gene cluster responsible for the biosynthesis of xiamenmycin resides in a strain-specific 61,387-bp genomic island belonging to the left-arm region. A core metabolic network consisting of 104 reactions that supports xiamenmycin biosynthesis was constructed to illustrate the necessary precursors derived from the central metabolic pathway. In accordance with the finding of a putative ikarugamycin gene cluster in the genome, the targeted chemical profiling of polycyclic tetramate macrolactams (PTMs) resulted in the identification of ikarugamycin. A successful genome mining for bioactive molecules with different skeletons suggests that the naturally minimized genome of S. xiamenensis 318 could be used as a blueprint for constructing a chassis cell with versatile biosynthetic capabilities for the production of secondary metabolites.

Screening of Strong 1-Aminocyclopropane-1-Carboxylate Deaminase Producing Bacteria for Improving the Salinity Tolerance of Cowpea

Aim: To isolate rhizobacteria containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and evaluate the ability of selected bacteria for improving the growth of cowpea seedlings under salt stress conditions. Methods: This study isolates salt-tolerant rhizobacteria which have strong ability to produce ACC deaminase and the phytohormone indol-3-acetic acid (IAA). Inoculation experiments with selected bacteria strains were used to verify the plant growth promoting activity of bacteria under salt stress conditions. Result: Two isolates belong to Enterobacter cloacae and one isolate belongs to Pseudomonas sp. have been identified. Those rhizobacteria were found to be highly salt-tolerant at salinity level up to 10% NaCl. The selected bacterial strains were also capable to produce and secrete large amounts of ACC deaminase and the phytohormone IAA into the growth medium. Cowpea plants inoculated with ST3 strain revealed a significant increase in shoot length and shoot fresh weight over uninoculated control at the salinity level of 1.5% NaCl. Conclusion: Three rhizobacterial strains belonging to the genera Enterobacter and Pseudomonas have been isolated. All three bacterial strains were identified as moderate halophiles and they can produce high levels of ACC deaminase and IAA. The strain Pseudomonas sp. ST3 showed the possible ability to promote the growth of cowpea under salt stress conditions.

Physiology and Molecular Phylogeny of Bacteria Isolated from Alkaline Distillery Lime.

This paper presents the results of the research on the number, taxonomic composition, and biochemical properties of bacterial strains isolated from the alkaline Solvay distillery lime, deposited at the repository in Janikowo (central Poland). Fifteen strains out of 17 were facultative alkaliphiles and moderate halophiles, and two were alkalitolerants and moderate halophiles. The number of aerobic bacteria cultured in alkaline lime was approximately 10(5) CFU ml(-1), and the total number of bacteria was 10(7) cells g(-1). According to 16S rRNA gene sequence analysis, nine strains belonged to the genus Bacillus, six to the genus Halomonas, one to the genus Planococcus, and one to the genus Microcella. Strains that hydrolyse starch and protein were the most numerous. Esterase (C4) and esterase lipase (C8) were detected in the majority of bacterial strains. Twelve strains exhibited α-glucosidase activity and nine, naphtol-AS-BI-phosphohydrolase activity. The present study proves that alkaliphilic bacteria of this type may constitute a source of potentially useful extremozymes.

Identification of N(α)-acetyl-α-lysine as a probable thermolyte and its accumulation mechanism in Salinicoccus halodurans H3B36.

Salinicoccus halodurans H3B36 is a moderate halophile that was isolated from a 3.2-m-deep sediment sample in Qaidam Basin, China. Our results suggest that Nα-acetyl-α-lysine can accumulate and act as a probable thermolyte in this strain. The accumulation mechanism and biosynthetic pathway for this rare compatible solute were also elucidated. We confirmed that the de novo synthesis pathway of Nα-acetyl-α-lysine in this strain starts from aspartate and passes through lysine. Through RNA sequencing, we also found an 8-gene cluster (orf_1582–1589) and another gene (orf_2472) that might encode the biosynthesis of Nα-acetyl-α-lysine in S. halodurans H3B36. Orf_192, orf_193, and orf_1259 might participate in the transportation of precursors for generating Nα-acetyl-α-lysine under the heat stress. The transcriptome reported here also generated a global view of heat-induced changes and yielded clues for studying the regulation of Nα-acetyl-α-lysine accumulation. Heat stress triggered a global transcriptional disturbance and generated a series of actions to adapt the strain to heat stress. Furthermore, the transcriptomic results showed that the regulon of RpoN (orf_2534) may be critical to conferring heat stress tolerance and survival to S. halodurans.

Draft Genome Sequence of Yellow Pigmented Jeotgalibacillus alimentarius JY-13T, the First Halophile Strain of the Genus Jeotgalibacillus.

Jeotgalibacillus alimentarius JY-13T (=KCCM 80002T = JCM 10872T) is a moderate halophile. In 2001, this was the first strain of the newly proposed Jeotgalibacillus genus. The draft genome of J. alimentarius was found to consist of 32 contigs (N50, 315,125 bp) with a total size of 3,364,745 bp. This genome information will be helpful for studies on pigmentation as well as applications for this bacterium.

Nucleoside Diphosphate Kinase from Psychrophilic Pseudoalteromonas sp. AS-131 Isolated from Antarctic Ocean.

Nucleoside diphosphate kinase isolated from psychrophilic Pseudoalteromonas sp. AS-131 (ASNDK) was expressed in Escherichia coli and purified to homogeneity. Comparing to mesophilic NDK isolated from Pseudomonas aeruginosa, ASNDK exhibited highly elevated thermolability: E. coli expression at 37 °C as a denatured insoluble form, 30 °C lower optimum temperature of enzymatic activity, and greatly reduced heat stability with 38 °C lower Tm value, fourfold higher Km and reduced Kcat/Km by 0.4-fold upon reaction temperature increase from 20 to 37 °C. The subunit structure of ASNDK was suggested to be dimer, as in NDKs isolated from moderate halophiles.

Degradation of corn stover by fungal cellulase cocktail for production of polyhydroxyalkanoates by moderate halophile Paracoccus sp. LL1

Bioprocessing of lignocellulose as a renewable resource for fuels, chemicals or value added products is a necessity to fulfil demands of petroleum products. This study aims to convert corn stover to polyhydroxyalkanoates (PHA). Corn stover was hydrolyzed to crude sugars by an on-site prepared cellulase cocktail from co-culture of Trichoderma reesei and Aspergillus niger. The potent PHA producer, Paracoccus sp. LL1, was isolated from Lonar Lake, India and could accumulate PHA up to 72.4% of its dry cell weight. PHA production reached 9.71g/L from corn stover hydrolysate containing 40g/L sugar mixture. The PHA synthase gene (phaC) sequence of the isolate showed 79% identity with the phaC gene of Paracoccus seriniphilus (E71) strain from the NCBI database. The nature/type of PHA was found to be poly(3-hydroxybutyrate) by Fourier transform infrared spectroscopy.

Statistical Optimization of the Production of NaCl-Tolerant Proteases by a Moderate Halophile, Virgibacillus sp. SK37.

The objectives of this study are to optimize the conditions for providing high yield of NaCl-tolerant extracellular protease from Virgibacillus sp. SK37 based on a fish-based medium and to investigate the effects of the key factors (mass per volume ratios of dried anchovy, yeast extract and NaCl, and initial pH of the medium) on the secretion pattern of proteases. Based on the predicted response model, the optimized medium contained 1.81% of dried anchovy, 0.33% of yeast extract and 1.25% of NaCl at pH=7.8. Under these conditions, a 5.3-fold increase in protease production was achieved, compared with the broth containing only 1.2% of dried anchovy (5% of NaCl at pH=7). The cubic regression adequately described the protease production. Protease activity was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on the synthetic substrate (Suc-Ala-Ala-Pro-Phe-AMC). Proteases of molecular masses of 19, 34, 35 and 44 kDa were secreted in the presence of NaCl, whereas those of 22 and 42 kDa were the main proteases detected in the absence of NaCl. In addition, no secreted proteases were detected when initial pH of the medium was pH=6. The peptide mass fingerprint of the medium cultured with 10% NaCl showed a higher abundance of peptides with lower mass of 500-1000 m/z compared with the medium containing 0% NaCl, indicating the higher proteolytic activity of the high-salt medium. The Virgibacillus sp. SK37 proteases showed a marked preference towards Lys, Arg and Tyr in the presence of NaCl and towards Lys and Arg in the absence of NaCl.

Draft Genome Sequence of the Moderately Halophilic Methanotroph Methylohalobius crimeensis Strain 10Ki

Methylohalobius crimeensis strain 10Ki is a moderately halophilic aerobic methanotroph isolated from a hypersaline lake in the Crimean Peninsula, Ukraine. This organism has the highest salt tolerance of any cultured methanotroph. Here, we present a draft genome sequence of this bacterium.

Draft Genome Sequence of Erythrobacter vulgaris Strain O1, a Glycosyl Hydrolase-Producing Bacterium.

Erythrobacter vulgaris strain O1, a moderate halophile, was isolated from a beach in Johor, Malaysia. Here, we present the draft genome and suggest potential applications of this bacterium.

Beneficial effects of Ectothiorhodospira shaposhnikovii WF on larval cultivation of Litopenaeus vannamei.

To develop high quality probiotics for shrimp larviculture, the effects of a photosynthetic purple sulphur bacterium WF identified as Ectothiorhodospira shaposhnikovii on survival and development of Litopenaeus vannamei larvae were evaluated in vivo. The larvae exhibited a better survival rate after administration of strain WF compared to the probiotic Rhodopseudomonas palustris. To investigate the effect of dose and dosing frequency, strain WF was added to larvae, stages nauplius 6 to zoea 3, at three different doses and dosing frequencies. Larval treatment with strain WF twice at 10(6) cfu/ml exhibited significantly higher survival compared to the other doses and dosing frequencies as well as the control. The effect on water quality was assessed by applying strain WF to larvae, stages nauplius 6 to postlarvae 1, under conditions of zero water exchange and one-third water exchange. The larvae exhibited higher survival and faster growth when treated under conditions of zero water exchange. No significant difference was detected in the levels of three water quality parameters and in vibrio counts between these two conditions. Therefore, E. shaposhnikovii WF acts both as a bioremediation agent and nutrient source and can benefit shrimp larvae if given at an appropriate dose and dosing frequency. Strain WF, a moderate halophile, shows great promise as a water additive in improving water quality and providing nutrition for shrimp larviculture.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by a moderate halophile Yangia sp. ND199 using glycerol as a carbon source.

Yangia sp. ND199, a moderate halophile isolated from mangrove soil sample in Vietnam, was found to accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated carbon sources in a medium with 4.5% (w/v) NaCl. Cultivation with glycerol as carbon source and yeast extract as nitrogen source resulted in maximum cell dry weight of 5.7 g/l and PHBV content of 52.8 wt% (containing 2.9 mol% of 3HV) after 40 h. The 3HV content of the PHBV was the highest during initial stages of copolymer production and decreased with increase in the copolymer amount with time, but was not affected by changing the pH of the culture medium. Only homopolymer poly(3-hydroxybutyrate) was synthesized when monosodium glutamate was used as the nitrogen source. Fed-batch cultivation of Yangia sp. ND199 with glycerol and yeast extract gave PHBV content and productivity of 53.2 wt% and 0.44 g/l/h, respectively, which were reduced to 40.6 wt% and 0.25 g/l/h, respectively, with crude glycerol as carbon source. Both the copolymer content and productivity were improved to 56 wt% and 0.61 g/l/h, respectively, by using 1:1 mixture of crude glycerol and high fructose corn syrup. This is the first report of PHBV production by a wild-type halophilic bacterium using glycerol as carbon source.

Statistical Optimization and Secretion of NaCl-Tolerant Proteinases by a Moderate Halophile, Virgibacillus sp. SK37

Statistical Optimization and Secretion of NaCl-Tolerant Proteinases by a Moderate Halophile, Virgibacillus sp. SK37

Draft Genome Sequence of the Halophilic and Highly Halotolerant Gammaproteobacteria Strain MFB021.

We report the 4.25-Mbp first draft sequence of Gammaproteobacteria strain MFB021, a moderate halophile isolated from petroleum-contaminated soil in Cochin, India. The genome of the strain MFB021 was sequenced to understand the mechanism of hydrocarbon degradation and the halophilicity of the bacterium.

English

Moderate halophilic bacteria from Dowma al Jandal in extreme north Al Jouf were isolated using cultural-dependent methods. Halophiles from hypersaline environments possess a biotechnological potential. Polymerase chain reaction (PCR) amplification of the 16s rRNA gene and phylogentic analysis were used to identify strains. Culturing was done aerobically in chemically defined media (CDM). Salt concentration (15%) was used at pH 7.2. Universal bacterial primers were used to amplify 16SrDNA from chromosomal DNA isolated from the four distinct colonies. Four moderate halophilic bacterial isolates were analyzed and identified wih 16srDNA sequencing as Virgibacillus salarius, Bacillus subtilis, Bacillus sp., and Virgibacillus marismortui. Comparison of the 16srDNA sequence alignment to reference sequence data bases showed samples M1, M2, M3 and M4 have 95-99% homology. All of the four isolates had at least 95% similarity to the published sequences implying that they could be species within the described genera. Key words: Moderate halophiles, hypersaline lake, Bacillus sp., 16srDNA sequencing.

Characterization of lignocellulolytic activities from a moderate halophile strain of Aspergillus caesiellus isolated from a sugarcane bagasse fermentation.

A moderate halophile and thermotolerant fungal strain was isolated from a sugarcane bagasse fermentation in the presence of 2 M NaCl that was set in the laboratory. This strain was identified by polyphasic criteria as Aspergillus caesiellus. The fungus showed an optimal growth rate in media containing 1 M NaCl at 28°C and could grow in media added with up to 2 M NaCl. This strain was able to grow at 37 and 42°C, with or without NaCl. A. caesiellus H1 produced cellulases, xylanases, manganese peroxidase (MnP) and esterases. No laccase activity was detected in the conditions we tested. The cellulase activity was thermostable, halostable, and no differential expression of cellulases was observed in media with different salt concentrations. However, differential band patterns for cellulase and xylanase activities were detected in zymograms when the fungus was grown in different lignocellulosic substrates such as wheat straw, maize stover, agave fibres, sugarcane bagasse and sawdust. Optimal temperature and pH were similar to other cellulases previously described. These results support the potential of this fungus to degrade lignocellulosic materials and its possible use in biotechnological applications.

Halophilic Aspergillus penicillioides from athalassohaline, thalassohaline, and polyhaline environments.

Aspergillus penicillioides is a true halophile, present in diverse econiches – from the hypersaline athalassohaline, and thalassohaline environments, to polyhaline systems, and in different geographical locations. Twenty seven isolates from these environments, were seen to be moderate halophiles, euryhaline in nature. They had an obligate need of a low aw and were unable to grow on a regular defined medium such as Czapek Dox Agar, as well as on varied nutrient rich agar media such as Malt Extract, Potato Dextrose and Sabouraud Agar; however, growth was obtained on all these media when amended with 10% solar salt. In absence of added salt, the conidia either did not germinate, or when germinated, distortions and lysis were seen in the short mycelial forms; on media with salt, the mycelia and vesicles appeared normal.

Cloning and identification of Group 1 mrp operon encoding a novel monovalent cation/proton antiporter system from the moderate halophile Halomonas zhaodongensis.

The novel species Halomonas zhaodongensis NEAU-ST10-25(T) recently identified by our group is a moderate halophile which can grow at the range of 0-2.5 M NaCl (optimum 0.5 M) and pH 6-12 (optimum pH 9). To explore its halo-alkaline tolerant mechanism, genomic DNA was screened from NEAU-ST10-25(T) in this study for Na(+)(Li(+))/H(+) antiporter genes by selection in Escherichia coli KNabc lacking three major Na(+)(Li(+))/H(+) antiporters. One mrp operon could confer tolerance of E. coli KNabc to 0.8 M NaCl and 100 mM LiCl, and an alkaline pH. This operon was previously mainly designated mrp (also mnh, pha or sha) due to its multiple resistance and pH-related activity. Here, we will also use mrp to designate the homolog from H. zhaodongensis (Hz_mrp). Sequence analysis and protein alignment showed that Hz_mrp should belong to Group 1 mrp operons. Further phylogenetic analysis reveals that Hz_Mrp system should represent a novel sub-class of Group 1 Mrp systems. This was confirmed by a significant difference in pH-dependent activity profile or the specificity and affinity for the transported monovalent cations between Hz_Mrp system and all the known Mrp systems. Therefore, we propose that Hz_Mrp should be categorized as a novel Group 1 Mrp system.

From metagenomics to pure culture: isolation and characterization of the moderately halophilic bacterium Spiribacter salinus gen. nov., sp. nov.

Recent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dominated by both Euryarchaeota and halophilic bacteria, with a gammaproteobacterium closely related to the genera Alkalilimnicola and Arhodomonas being one of the most predominant microorganisms, making up to 15% of the total prokaryotic population. Here we used several strategies and culture media in order to isolate this organism in pure culture. We report the isolation and taxonomic characterization of this new, never before cultured microorganism, designated M19-40(T), isolated from a saltern located in Isla Cristina, Spain, using a medium with a mixture of 15% salts, yeast extract, and pyruvic acid as the carbon source. Morphologically small curved cells (young cultures) with a tendency to form long spiral cells in older cultures were observed in pure cultures. The organism is a Gram-negative, nonmotile bacterium that is strictly aerobic, non-endospore forming, heterotrophic, and moderately halophilic, and it is able to grow at 10 to 25% (wt/vol) NaCl, with optimal growth occurring at 15% (wt/vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that strain M19-40(T) has a low similarity with other previously described bacteria and shows the closest phylogenetic similarity with species of the genera Alkalilimnicola (94.9 to 94.5%), Alkalispirillum (94.3%), and Arhodomonas (93.9%) within the family Ectothiorhodospiraceae. The phenotypic, genotypic, and chemotaxonomic features of this new bacterium showed that it constitutes a new genus and species, for which the name Spiribacter salinus gen. nov., sp. nov., is proposed, with strain M19-40(T) (= CECT 8282(T) = IBRC-M 10768(T) = LMG 27464(T)) being the type strain.

Cys139Ser mutation in dimeric nucleoside diphosphate kinase generates catalytically competent monomer

Nucleoside diphosphate kinase from a moderate halophile Halomonas sp. 593 (HaNDK) is dimer, while NDK from different origins has been shown to assemble into hexamer, or tetramer. Similar to HaNDK, halophilic NDK from Chromohalobacter salexigens DSM3043 (CsNDK) formed dimeric structure. Cysteine139 conserved between HaNDK and CsNDK is located in the monomer/monomer interface. Substitution of Cys139 for Ser caused dissociation of dimeric CsNDK into monomer in Tris buffer, as determined by field flow fractionation technique. Circular dichroism (CD) profile of the mutant CsNDK was nearly identical to the wild type CsNDK: however, the mutant CsNDK became more susceptible to “endproteinase GluC” cleavage, which could be suppressed by an NDK substrate, ATP. The monomer was enzymatically active, although it is possible that active structure is dimer in the presence of substrate.