Phylogeny, novel bacterial lineage and enzymatic potential of haloalkaliphilic bacteria from the saline coastal desert of Little Rann of Kutch, Gujarat, India.
ABSTRACT: This report describes cultivation-dependent diversity, phylogeny and enzymatic potential of the haloalkaliphilic bacteria isolated from the unvegetated desert soil of yet unexplored, saline desert of Little Rann of Kutch (LRK), India. The LRK is a unique ecosystem displaying a combination of Dry Rann and Wet Rann. A total of 25 bacteria were isolated and characterized on the basis of colony morphology, biochemical profile, sugar utilization, secretion of the extracellular enzymes and antibiotic sensitivity. Further, the identification and phylogenetic relatedness of 23 bacteria were established by the analysis of 16S rRNA gene sequences. The phylogenetic analysis indicated that the isolates belong to the phylum Firmicutes, comprising low G + C, Gram-positive bacteria, with different genera: Bacillus (~ 39%), Staphylococcus (~ 30%), Halobacillus (~ 13%), Virgibacillus (~ 13%), Oceanobacillus (~ 4%). Majority of the bacterial isolates produced proteases (30% isolates) followed by cellulases (24% isolates), CMCases (24% isolates) and amylases (20% isolates). Halobacillus, Virgibacillus and Bacillus predominantly produced hydrolases, while many produced multiple enzymes at high salinity and alkaline pH. Highest antibiotic resistance was observed against Ampicillin and Penicillin (32%) followed by Cefaclor (20%); Colistin, Cefoperazone and Cefotaxime (16%); Cefuroxime (12%); Gentamycin and Cefixime (8%); Erythromycin, Cefadroxil, Azithromycin, Co-trimoxazole, Amoxycillin, Norfloxacin, Cefpodoxime, Amikacin and Augmentin (4%). KJ1-10-99 and KJ1-10-93 representing < 97% of 16S rRNA gene sequence similarity belong to a novel lineage within the family Bacillaceae. Comparison of the phenogram and phylogram revealed the contradiction of the phenogram pattern and the phylogenetic placement of the isolates. The isolates belonging to same species have shown considerable phenotypic variation. The study on the cultivable haloalkaliphilic bacteria of an unexplored enigmatic niche reflects ecological and biotechnological significance.
Project description:Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3-20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.
Project description:Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications.
Project description:Microbes from hypersaline environments are useful in biotechnology as sources of novel enzymes and proteins. The current study aimed to characterize halophilic bacteria from the rhizosphere of halophytes (Salsola stocksii and Atriplex amnicola), non-rhizospheric, and brine lake-bank soils collected from Khewra Salt Mine and screening of these bacterial strains for industrially important enzymes. A total of 45 bacterial isolates from the rhizosphere of Salsola, 38 isolates from Atriplex, 24 isolates from non-rhizospheric, and 25 isolates from lake-bank soils were identified by using 16S rRNA gene analysis. Phylogenetic analysis showed that bacterial strains belonging to Bacillus, Halobacillus, and Kocuria were dominant in the rhizosphere of halophytes (Salsola and Atriplex), and Halobacillus and Halomonas were dominating genera from non-rhizospheric and lake-bank soils. Mostly identified strains were moderately halophilic bacteria with optimum growth at 1.5-3.0 M salt concentrations. Most of the bacterial exhibited lipase, protease, cellulase, amylase, gelatinase, and catalase activities. Halophilic and halotolerant Bacilli (AT2RP4, HL1RS13, NRS4HaP9, and LK3HaP7) identified in this study showed optimum lipase, protease, cellulase, and amylase activities at 1.0-1.5 M NaCl concentration, pH 7-8, and temperature 37 °C. These results indicated that halophilic and halotolerant bacteria can be used for bioconversion of organic compounds to useful products under extreme conditions.
Project description:The aim of the study was to explore the halophile metabolome in building materials using untargeted metabolomics which allows for broad metabolome coverage. For this reason, we used high-performance liquid chromatography interfaced to high-resolution mass spectrometry (HPLC/HRMS). As an alternative to standard microscopy techniques, we introduced pioneering Coherent Anti-stokes Raman Scattering Microscopy (CARS) to non-invasively visualize microbial cells. Brick samples saturated with salt solution (KCl, NaCl (two salinity levels), MgSO4, Mg(NO3)2), were inoculated with the mixture of preselected halophilic microorganisms, i.e., bacteria: Halobacillus styriensis, Halobacillus naozhouensis, Halobacillus hunanensis, Staphylococcus succinus, Marinococcus halophilus, Virgibacillus halodenitryficans, and yeast: Sterigmatomyces halophilus and stored at 28°C and 80% relative humidity for a year. Metabolites were extracted directly from the brick samples and measured via HPLC/HRMS in both positive and negative ion modes. Overall, untargeted metabolomics allowed for discovering the interactions of halophilic microorganisms with buildings materials which together with CARS microscopy enabled us to elucidate the biodeterioration process caused by halophiles. We observed that halophile metabolome was differently affected by different salt solutions. Furthermore, we found indications for haloadaptive strategies and degradation of brick samples due to microbial pigment production as a salt stress response. Finally, we detected changes in lipid content related to changes in the structure of phospholipid bilayers and membrane fluidity.
Project description:BACKGROUND:Generally, extremophiles have been deemed to survive in the extreme environments to which they had adapted to grow. Recently many extremophiles have been isolated from places where they are not expected to grow. Alkaliphilic microorganisms have been isolated from acidic soil samples with pH 4.0, and thermophiles have been isolated from samples of low temperature. Numerous moderately halophilic microorganisms, defined as those that grow optimally in media containing 0.5-2.5 Molar (3-15%) NaCl, and halotolerant microorganisms that are able to grow in media without added NaCl and in the presence of high NaCl have been isolated from saline environments such as salterns, salt lakes and sea sands. It has tacitly been believed that habitats of halophiles able to grow in media containing more than 20% (3.4 M) are restricted to saline environments, and no reports have been published on the isolation of halophiles from ordinary garden soil samples. RESULTS:We demonstrated that many halophilic bacteria that are able to grow in the presence of 20% NaCl are inhabiting in non-saline environments such as ordinary garden soils, yards, fields and roadways in an area surrounding Tokyo, Japan. Analyses of partial 16S rRNA gene sequences of 176 isolates suggested that they were halophiles belonging to genera of the family Bacillaceae, Bacillus (11 isolates), Filobacillus (19 isolates), Gracilibacillus (6 isolates), Halobacillus (102 isolates), Lentibacillus (1 isolate), Paraliobacillus (5 isolates) and Virgibacillus (17 isolates). Sequences of 15 isolates showed similarities less than 92%, suggesting that they may represent novel taxa within the family Bacillaceae. CONCLUSION:The numbers of total bacteria of inland soil samples were in a range from 1.4 x 10(7)/g to 1.1 x 10(6)/g. One tenth of the total bacteria was occupied by endospore-forming bacteria. Only very few of the endospore-forming bacteria, roughly 1 out of 20,000, are halophilic bacteria. Most of the halophilic bacteria were surviving as endospores in the soil samples, in a range of less than 1 to about 500/g soil. Samples collected from seashore in a city confronting Tokyo Bay gave the total numbers of bacteria and endospores roughly 1000 time smaller than those of inland soil samples. Numbers of halophilic bacteria per gram, however, were almost the same as those of inland soil samples. A possible source of the halophilic endospore originating from Asian dust storms is discussed.
Project description:Several moderately halophilic gram-positive, spore-forming bacteria have been isolated by conventional enrichment cultures from damaged medieval wall paintings and building materials. Enrichment and isolation were monitored by denaturing gradient gel electrophoresis and fluorescent in situ hybridization. 16S ribosomal DNA analysis showed that the bacteria are most closely related to Halobacillus litoralis. DNA-DNA reassociation experiments identified the isolates as a population of hitherto unknown Halobacillus species.
Project description:Soda lake sediments usually contain high concentrations of sulfide indicating active sulfate reduction. Monitoring of sulfate-reducing bacteria (SRB) in soda lakes demonstrated a dominance of two groups of culturable SRB belonging to the order Desulfovibrionales specialized in utilization of inorganic electron donors, such as formate, H(2) and thiosulfate. The most interesting physiological trait of the novel haloalkaliphilic SRB isolates was their ability to grow lithotrophically by dismutation of thiosulfate and sulfite. All isolates were obligately alkaliphilic with a pH optimum at 9.5-10 and moderately salt tolerant. Among the fifteen newly isolated strains, four belonged to the genus Desulfonatronum and the others to the genus Desulfonatronovibrio. None of the isolates were closely related to previously described species of these genera. On the basis of phylogenetic, genotypic and phenotypic characterization of the novel soda lake SRB isolates, two novel species each in the genera Desulfonatronum and Desulfonatronovibrio are proposed.
Project description:The isolation of soil bacteria from extreme environments represents a major challenge, but also an opportunity to characterize the metabolic potential of soil bacteria that could promote the growth of plants inhabiting these harsh conditions. The aim of this study was to isolate and identify bacteria from two Chilean desert environments and characterize the beneficial traits for plants through a biochemical approach. By means of different culture strategies, we obtained 39 bacterial soil isolates from the Coppermine Peninsula (Antarctica) and 32 from Lejía Lake shore soil (Atacama Desert). The results obtained from the taxonomic classification and phylogenetic analysis based on 16S rDNA sequences indicated that the isolates belonged to four phyla (Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes), and that the most represented genus at both sites was Pseudomonas. Regarding biochemical characterization, all strains displayed in vitro PGP capabilities, but these were in different proportions that grouped them according to their site of origin. This study contributes with microbial isolates from natural extreme environments with biotechnological potentials in improving plant growth under cold stress.
Project description:BACKGROUND:Biosynthetic gene clusters produce a wide range of metabolites with activities that are of interest to the pharmaceutical industry. Specific interest is shown towards those metabolites that exhibit antimicrobial activities against multidrug-resistant bacteria that have become a global health threat. Genera of the phylum Firmicutes are frequently identified as sources of such metabolites, but the biosynthetic potential of its Virgibacillus genus is not known. Here, we used comparative genomic analysis to determine whether Virgibacillus strains isolated from the Red Sea mangrove mud in Rabigh Harbor Lagoon, Saudi Arabia, may be an attractive source of such novel antimicrobial agents. RESULTS:A comparative genomics analysis based on Virgibacillus dokdonensis Bac330, Virgibacillus sp. Bac332 and Virgibacillus halodenitrificans Bac324 (isolated from the Red Sea) and six other previously reported Virgibacillus strains was performed. Orthology analysis was used to determine the core genomes as well as the accessory genome of the nine Virgibacillus strains. The analysis shows that the Red Sea strain Virgibacillus sp. Bac332 has the highest number of unique genes and genomic islands compared to other genomes included in this study. Focusing on biosynthetic gene clusters, we show how marine isolates, including those from the Red Sea, are more enriched with nonribosomal peptides compared to the other Virgibacillus species. We also found that most nonribosomal peptide synthases identified in the Virgibacillus strains are part of genomic regions that are potentially horizontally transferred. CONCLUSIONS:The Red Sea Virgibacillus strains have a large number of biosynthetic genes in clusters that are not assigned to known products, indicating significant potential for the discovery of novel bioactive compounds. Also, having more modular synthetase units suggests that these strains are good candidates for experimental characterization of previously identified bioactive compounds as well. Future efforts will be directed towards establishing the properties of the potentially novel compounds encoded by the Red Sea specific trans-AT PKS/NRPS cluster and the type III PKS/NRPS cluster.