Haloalkaliphilic Bacterium Research Articles

Cloning, heterologous expression and structural characterization of an alkaline serine protease from sea water haloalkaliphilic bacterium

Cloning and functional attributes of a serine protease gene from haloalkaliphilic bacteria are described. The protease gene of ∼1,600 bp amplified from the genomic DNA was cloned into TA vector followed by the sub-cloning into pUC19 for expression. Growth of the organism and gene expression was studied at 30 and 37 °C in the presence of 0.5–2.0 mM IPTG. Sequencing of the gene and homology search of the sequence revealed that the gene encoded an extracellular alkaline serine protease belonging to superfamily subtilisin-like hydrolases. The amino acid sequence alignment resulted from the BLAST search of the subtilisin exhibited high sequence homology with the Bacillus subtilis ssp. subtilis strain 168 and subtilisins of other Bacillus sp., B. subtilis and B. mojavensis. The deduced amino acid sequence exhibited a mature protease of a 419 amino acid, single-chained monomeric peptide with the large number of the positively charged amino acids suggesting its hydrophilic nature.

Biochemical and structural characterization of a detergent-stable serine alkaline protease from seawater haloalkaliphilic bacteria

An extracellular protease from a newly isolated seawater haloalkaliphilic bacterium, haloalkaliphilic bacteria Ve2-20-91 [HM047794], was purified and characterized. The enzyme is a monomer with a 37.2kDa estimated molecular weight. It catalyzed reactions in the pH range 8–11 and performed optimally at pH 10. While maximal activity occurred at 50°C, the temperature profile shifted from 50 to 80°C in 1–3M NaCl. The enzyme's thermal stability was probed using circular dichroism (CD) spectroscopy with NaCl at 50 and 70°C. The changes in the enzyme's secondary structure were also analyzed using Fourier transform infrared spectroscopy (FTIR). The N-terminal amino acid sequence GKDGPPGLCGFFGCI exhibited low homology with other bacterial proteases, which highlights the enzyme's novelty. The enzyme was labile in anionic surfactant (1% w/v SDS) but showed stability in non-ionic surfactants (Tween 20, Tween 80 and Triton X-100 all 1% v/v), commercial detergents, and oxidizing and reducing agents. The enzyme's excellent stability in commercial detergents highlights its potential as a detergent additive.

Screening for genes coding for putative antitumor compounds, antimicrobial and enzymatic activities from haloalkalitolerant and haloalkaliphilic bacteria strains of Algerian Sahara Soils.

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.

Ability of a haloalkaliphilic bacterium isolated from Soap Lake, Washington to generate electricity at pH 11.0 and 7% salinity

A variety of anaerobic bacteria have been shown to transfer electrons obtained from organic compound oxidation to the surface of electrodes in microbial fuel cells (MFCs) to produce current. Initial enrichments for iron (III) reducing bacteria were set up with sediments from the haloalkaline environment of Soap Lake, Washington, in batch cultures and subsequent transfers resulted in a culture that grew optimally at 7.0% salinity and pH 11.0. The culture was used to inoculate the anode chamber of a MFC with formate as the electron source. Current densities up to 12.5 mA/m2 were achieved by this bacterium. Cyclic voltammetry experiments demonstrated that an electron mediator, methylene blue, was required to transfer electrons to the anode. Scanning electron microscopic imaging of the electrode surface did not reveal heavy colonization of bacteria, providing evidence that the bacterium may be using an indirect mode of electron transfer to generate current. Molecular characterization of the 16S rRNA gene and restriction fragment length profiles (RFLP) analysis showed that the MFC enriched for a single bacterial species with a 99% similarity to the 16S rRNA gene of Halanaerobium hydrogeniformans. Though modest, electricity production was achieved by a haloalkaliphilic bacterium at pH 11.0 and 7.0% salinity.

Disproportionation of elemental sulfur by haloalkaliphilic bacteria from soda lakes

Microbial disproportionation of elemental sulfur to sulfide and sulfate is a poorly characterized part of the anoxic sulfur cycle. So far, only a few bacterial strains have been described that can couple this reaction to cell growth. Continuous removal of the produced sulfide, for instance by oxidation and/or precipitation with metal ions such as iron, is essential to keep the reaction exergonic. Hitherto, the process has exclusively been reported for neutrophilic anaerobic bacteria. Here, we report for the first time disproportionation of elemental sulfur by three pure cultures of haloalkaliphilic bacteria isolated from soda lakes: the Deltaproteobacteria Desulfurivibrio alkaliphilus and Desulfurivibrio sp. AMeS2, and a member of the Clostridia, Dethiobacter alkaliphilus. All cultures grew in saline media at pH 10 by sulfur disproportionation in the absence of metals as sulfide scavengers. Our data indicate that polysulfides are the dominant sulfur species under highly alkaline conditions and that they might be disproportionated. Furthermore, we report the first organism (Dt. alkaliphilus) from the class Clostridia that is able to grow by sulfur disproportionation.

Cloning, over expression and functional attributes of serine proteases from Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12

Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50°C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis.

The Lipid A from the haloalkaliphilic bacterium Salinivibrio sharmensis strain BAG(T).

Lipid A is a major constituent of the lipopolysaccharides (or endotoxins), which are complex amphiphilic macromolecules anchored in the outer membrane of Gram-negative bacteria. The glycolipid lipid A is known to possess the minimal chemical structure for LPSs endotoxic activity, able to cause septic shock. Lipid A isolated from extremophiles is interesting, since very few cases of pathogenic bacteria have been found among these microorganisms. In some cases their lipid A has shown to have an antagonist activity, i.e., it is able to interact with the immune system of the host without triggering a proinflammatory response by blocking binding of substances that could elicit such a response. However, the relationship between the structure and the activity of these molecules is far from being completely clear. A deeper knowledge of the lipid A chemical structure can help the understanding of these mechanisms. In this manuscript, we present our work on the complete structural characterization of the lipid A obtained from the lipopolysaccharides (LPS) of the haloalkaliphilic bacterium Salinivibrio sharmensis. Lipid A was obtained from the purified LPS by mild acid hydrolysis. The lipid A, which contains different number of fatty acids residues, and its partially deacylated derivatives were completely characterized by means of electrospray ionization Fourier transform ion cyclotron (ESI FT-ICR) mass spectrometry and chemical analysis.

Cultivation-Dependant Assessment, Diversity, and Ecology of Haloalkaliphilic Bacteria in Arid Saline Systems of Southern Tunisia

Haloalkaliphiles are polyextremophiles adapted to grow at high salt concentrations and alkaline pH values. In this work, we isolated 122 haloalkaliphilic bacteria upon enrichments of 23 samples from 5 distinct saline systems of southern Tunisia, growing optimally in media with 10% salt and at pH 10. The collection was classified into 44 groups based on the amplification of the 16S–23S rRNA internal transcribed spacers (ITS-PCR). Phylogenetic analysis and sequencing of the 16S rRNA genes allowed the identification of 13 genera and 20 distinct species. Three gram-positive isolates showing between 95 and 96% of 16S rRNA sequence homology with Bacillus saliphilus could represent new species or genus. Beside the difference in bacterial diversity between the studied sites, several species ecological niches correlations were demonstrated such as Oceanobacillus in salt crust, Nesterenkonia in sand, and Salinicoccus in the rhizosphere of the desert plant Salicornia. The collection was further evaluated for the production of extracellular enzymes. Activity tests showed that gram-positive bacteria were mostly active, particularly for protease, lipase, DNase, and amylase production. Our overall results demonstrate the huge phenotypic and phylogenetic diversity of haloalkaliphiles in saline systems of southern Tunisia which represent a valuable source of new lineages and metabolites.

Structural characterization of the core oligosaccharide isolated from the lipopolysaccharide of the haloalkaliphilic bacterium Salinivibrio sharmensis strain BAGT

Salinivibrio genus is included in the family Vibrionaceae and up to now is constituted by only five members. All the species are moderately halophilic bacteria found in salted meats, brines, and several hypersaline environments. Halophilic microorganisms are good sources of biomolecules, such as proteases, that have a great industrial interest as demonstrated by recent studies.All these bacteria possess on their outer membrane amphiphilic molecules named lipopolysaccharides, which are of great interest because of their involvement in the mechanisms of interaction between the microbial life and environmental factors.A novel haloalkaliphilic, facultative anaerobic and Gram-negative Salinivibrio-like microorganism, named S. sharmensis strain BAGT, was recovered from a saline lake in Ras Mohammed Park (Egypt). The aim of this work is the isolation and structural characterization of the core oligosaccharidic fraction of the lipopolysaccharide from this bacterium. By means of HPAEC-PAD we were able to purify two glycoforms, fully depicted by ESI FT-ICR mass spectrometry, chemical analysis, and NMR spectroscopy. Like other haloalkaliphilic bacteria, the core region was found to be characterized by the presence of several negatively charged residues, such as uronic acids. All the data contributed to give the following structure ▪.

Microbiology of Lonar Lake and other soda lakes.

Soda lakes are saline and alkaline ecosystems that are believed to have existed throughout the geological record of Earth. They are widely distributed across the globe, but are highly abundant in terrestrial biomes such as deserts and steppes and in geologically interesting regions such as the East African Rift valley. The unusual geochemistry of these lakes supports the growth of an impressive array of microorganisms that are of ecological and economic importance. Haloalkaliphilic Bacteria and Archaea belonging to all major trophic groups have been described from many soda lakes, including lakes with exceptionally high levels of heavy metals. Lonar Lake is a soda lake that is centered at an unusual meteorite impact structure in the Deccan basalts in India and its key physicochemical and microbiological characteristics are highlighted in this article. The occurrence of diverse functional groups of microbes, such as methanogens, methanotrophs, phototrophs, denitrifiers, sulfur oxidizers, sulfate reducers and syntrophs in soda lakes, suggests that these habitats harbor complex microbial food webs that (a) interconnect various biological cycles via redox coupling and (b) impact on the production and consumption of greenhouse gases. Soda lake microorganisms harbor several biotechnologically relevant enzymes and biomolecules (for example, cellulases, amylases, ectoine) and there is the need to augment bioprospecting efforts in soda lake environments with new integrated approaches. Importantly, some saline and alkaline lake ecosystems around the world need to be protected from anthropogenic pressures that threaten their long-term existence.

The sulfate-reducing bacterial community of sulfide-rich water of the Ust’-Kachka resort spring, Perm Krai, Russia

Microbiological investigation of the highly mineralized, sulfide-rich cold spring of the Ust’-Kachka resort was carried out. The total number and biomass of microbial cells were 50 × 103 cell/mL and 15 μg/L, respectively. While the total microbial number was low, the sulfate reduction rate determined by the radioisotope method was relatively high (0.575 mg S L−1 day−1). An enrichment culture was obtained on Widdel medium. According to the results of cloning the 16S rRNA gene fragments with subsequent restriction analysis, the dominant organisms were group 6 sulfate-reducing bacteria (Desulfovibrio-Desulfomicrobium) and the microorganism exhibiting 99% similarity to the anaerobic haloalkaliphilic bacterium Halanaerobium hydrogeniformans.

Comparative structural and functional analysis of two octaheme nitrite reductases from closely related Thioalkalivibrio species

Octaheme nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrioparadoxus was isolated and characterized. A comparative structural and functional analysis of two homologous octaheme nitrite reductases from closely related Thioalkalivibrio species was performed. It was shown that both enzymes have similar catalytic properties, owing to high structural similarity. Both enzymes are characterized by specific structural features distinguishing them from pentaheme cytochromec nitrite reductases, such as the Tyr-Cys bond in the active site, the hexameric structure resulting in the formation of a void space inside the hexamer, and the product channel that opens into the void interior space of the hexamer. It is suggested that these specific structural features are responsible for the higher nitrite reductase activity, the greater preference for nitrite than for sulfite as a substrate, and the wider pH range of the catalytic activity of octaheme nitrite reductases than of pentaheme homologs. Nucleotide sequence data are available in the GenBank database under the accession number HQ665012.1. Structural data are available in the RCSB Protein Data Bank database under the accession numbers 3SXQ and 3TTB STRUCTURED DIGITAL ABSTRACT: TvPaRandTvPaRbindbyx-ray crystallography(View interaction).

Whole-Genome Shotgun Sequencing of the Extremophile Alkalibacillus haloalkaliphilus C-5, of Indian Origin

Alkalibacillus haloalkaliphilus C-5 is a haloalkaliphilic bacterium that was isolated from a soil sample from the salty Sambhar Lake, Rajasthan, India. The organism is capable of alkaline protease production under conditions of pH 10 and 10% (wt/vol) salt. We sequenced and have reported the whole genome of Alkalibacillus haloalkaliphilus C-5, of Indian origin, for the first time.

Exploration and characterization of agriculturally and industrially important haloalkaliphilic bacteria from environmental samples of hypersaline Sambhar lake, India

Screening of bacteria from Sambhar lake, an extreme hypersaline environment of India, led to the isolation of 93 haloalkaliphilic bacteria growing optimally in media with 2-25% salt and 6-12 pH. Based on 16S rRNA gene sequences, 93 isolates were further categorized into 32 groups, with each group representing a different taxa belonging to 3 phyla (Firmicutes, Proteobacteria and Actinobacteria). Majority of the isolates (53.12%) showed similarity with phylum Firmicutes which was followed by Proteobacteria (40.63%) and Actinobacteria (6.25%). The isolates belonging to 32 representative groups were further evaluated for the production of extracellular enzymes viz. amylase, cellulase, protease and xylanase, plant growth promoting attributes and BIOLOG™ substrate usage. Among all the isolates, xylanase producing isolates were in maximum (68%) as compared to protease (56%), cellulase (40%), and amylase (37%) producing strains. Similarly, among plant growth promoting activities, ammonia producing isolates were highest (56%) when compared to those producing ACC deaminase (53%), IAA (50%), hydrogen cyanide (28%), siderophore (21%) and solubilizing P (34%). Isolates showing enzymatic and PGP activities could be further utilized for promoting plant growth in saline affected area.

Desulfohalophilus alkaliarsenatis gen. nov., sp. nov., an extremely halophilic sulfate- and arsenate-respiring bacterium from Searles Lake, California

A haloalkaliphilic sulfate-respiring bacterium, strain SLSR-1, was isolated from a lactate-fed stable enrichment culture originally obtained from the extreme environment of Searles Lake, California. The isolate proved capable of growth via sulfate-reduction over a broad range of salinities (125–330 g/L), although growth was slowest at salt-saturation. Strain SLSR-1 was also capable of growth via dissimilatory arsenate-reduction and displayed an even broader range of salinity tolerance (50–330 g/L) when grown under these conditions. Strain SLSR-1 could also grow via dissimilatory nitrate reduction to ammonia. Growth experiments in the presence of high borate concentrations indicated a greater sensitivity of sulfate-reduction than arsenate-respiration to this naturally abundant anion in Searles Lake. Strain SLSR-1 contained genes involved in both sulfate-reduction (dsrAB) and arsenate respiration (arrA). Amplicons of 16S rRNA gene sequences obtained from DNA extracted from Searles Lake sediment revealed the presence of close relatives of strain SLSR-1 as part of the flora of this ecosystem despite the fact that sulfate-reduction activity could not be detected in situ. We conclude that strain SLSR-1 can only achieve growth via arsenate-reduction under the current chemical conditions prevalent at Searles Lake. Strain SLSR-1 is a deltaproteobacterium in the family Desulfohalobiacea of anaerobic, haloalkaliphilic bacteria, for which we propose the name Desulfohalophilus alkaliarsenatis gen. nov., sp. nov.

Pathways of Sulfide Oxidation by Haloalkaliphilic Bacteria in Limited-Oxygen Gas Lift Bioreactors

Physicochemical processes, such as the Lo-cat and Amine-Claus process, are commonly used to remove hydrogen sulfide from hydrocarbon gas streams such as landfill gas, natural gas, and synthesis gas. Biodesulfurization offers environmental advantages, but still requires optimization and more insight in the reaction pathways and kinetics. We carried out experiments with gas lift bioreactors inoculated with haloalkaliphilic sulfide-oxidizing bacteria. At oxygen-limiting levels, that is, below an O(2)/H(2)S mole ratio of 1, sulfide was oxidized to elemental sulfur and sulfate. We propose that the bacteria reduce NAD(+) without direct transfer of electrons to oxygen and that this is most likely the main route for oxidizing sulfide to elemental sulfur which is subsequently oxidized to sulfate in oxygen-limited bioreactors. We call this pathway the limited oxygen route (LOR). Biomass growth under these conditions is significantly lower than at higher oxygen levels. These findings emphasize the importance of accurate process control. This work also identifies a need for studies exploring similar pathways in other sulfide oxidizers such as Thiobacillus bacteria.

Catalysis and stability of an alkaline protease from a haloalkaliphilic bacterium under non-aqueous conditions as a function of pH, salt and temperature

A haloalkaliphilic bacterium, isolated from Coastal Gujarat (India) was identified as Oceanobacillus sp. (GQ162111) based on 16S rRNA gene sequence. The organism grew and secreted extra cellular protease in presence of various organic solvents. At 30% (v/v) concentration of hexane, heptane, isooctane, dodecane and decane, significant growth and protease production was evident. The alkaline protease was purified in a single step on phenyl sepharose 6 FF with 28% yield. The molecular mass as judged by SDS-PAGE was 30 kDa. The temperature optimum of protease was 50°C and the enzyme retained 70% activity in 10% (v/v) isooctane. Effect of salt and pH was investigated in combination to assess the effect of isooctane. In organic solvents, the enzyme was considerably active at pH 8-11, with optimum activity at pH 10. Salt at 2 M was optimum for activity and enzyme maintained significant stability up to 18 h even at 3 M salt concentration. Patters of growth, protease production, catalysis and stability of the enzyme are presented. The study resumes significance as limited information is available on the interaction of haloalkaliphilic bacteria and their enzymes with organic solvents.

ArxA, a new clade of arsenite oxidase within the DMSO reductase family of molybdenum oxidoreductases

Arsenotrophy, growth coupled to autotrophic arsenite oxidation or arsenate respiratory reduction, occurs only in the prokaryotic domain of life. The enzymes responsible for arsenotrophy belong to distinct clades within the DMSO reductase family of molybdenum-containing oxidoreductases: specifically arsenate respiratory reductase, ArrA, and arsenite oxidase, AioA (formerly referred to as AroA and AoxB). A new arsenite oxidase clade, ArxA, represented by the haloalkaliphilic bacterium Alkalilimnicola ehrlichii strain MLHE-1 was also identified in the photosynthetic purple sulfur bacterium Ectothiorhodospira sp. strain PHS-1. A draft genome sequence of PHS-1 was completed and an arx operon similar to MLHE-1 was identified. Gene expression studies showed that arxA was strongly induced with arsenite. Microbial ecology investigation led to the identification of additional arxA-like sequences in Mono Lake and Hot Creek sediments, both arsenic-rich environments in California. Phylogenetic analyses placed these sequences as distinct members of the ArxA clade of arsenite oxidases. ArxA-like sequences were also identified in metagenome sequences of several alkaline microbial mat environments of Yellowstone National Park hot springs. These results suggest that ArxA-type arsenite oxidases appear to be widely distributed in the environment presenting an opportunity for further investigations of the contribution of Arx-dependent arsenotrophy to the arsenic biogeochemical cycle.

Organic Solvent Tolerance of an α-Amylase from Haloalkaliphilic Bacteria as a Function of pH, Temperature, and Salt Concentrations

A haloalkaliphilic bacterium was isolated from salt-enriched soil of Mithapur, Gujarat (India) and identified as Bacillus agaradhaerens Mi-10-6₂ based on 16S rRNA sequence analysis (NCBI gene bank accession, GQ121032). The bacterium was studied for its α-amylase characteristic in the presence of organic solvents. The enzyme was quite active and it retained considerable activity in 30% (v/v) organic solvents, dodecane, decane, heptane, n-hexane, methanol, and propanol. At lower concentrations of solvents, the catalysis was quite comparable to control. Enzyme catalysis at wide range of alkanes and alcohol was an interesting finding of the study. Mi-10-6₂ amylase retained activity over a broader alkaline pH range, with the optimal pH at 10-11. Two molars of salt was optimum for catalysis in the presence of most of the tested solvents, though the enzyme retained significant activity even at 4 M salt. With dodecane, the optimum temperature shifted from 50 °C to 60 °C, while the enzyme was active up to 80 °C. Over all, the present study focused on the effect of organic solvents on an extracellular α-amylase from haloalkaliphilic bacteria under varying conditions of pH, temperature, and salt.

Extraction, purification and characterization of a protease from Micrococcus sp. VKMM 037

The haloalkaliphilic bacterium Micrococcus sp. VKMM 037, isolated from an effluent of the caustic soda industry, was found to produce a protease. Maximal proteolytic activity was observed in cell culture grown at 40 °C using 2% (w/v) glycerol, 2% (w/v) beef extract and 2% (w/v) peptone as nutrients in medium also containing 0.85 M NaCl with a pH of 10.0. An efficient purification procedure combining ammonium sulphate precipitation and Q‐Sepharose ion‐exchange chromatography was developed. The purified 41 kDa protease was stable in a temperature range between 20 °C and 60 °C. The protease remained active over a wide range of pH values (4.0–12.0) and NaCl concentrations (0–3.42 M) with an optimum at pH 10.0 and 0.85 M NaCl, respectively. Furthermore, the enzyme remained stable or was only marginally inhibited in the presence of various organic solvents, surfactants and reducing agents. The purified protease of Micrococcus sp. VKMM 037 efficiently removed blood stains within 40 minutes of treatment. Given the biochemical characteristics determined, this novel protease could be exploited as an additive in the detergent industry and also for the synthesis of biomolecules and the degradation of protein.