Moderate Halophile Research Articles

Determination of Minimum Inhibitory Concentrations of Sophorolipids Against Selected Halotolerant and Moderately Halophilic Bacteria, and Extremely Halophilic Archaea Isolated from Salt Samples and Salted Skins

Determination of Minimum Inhibitory Concentrations of Sophorolipids Against Selected Halotolerant and Moderately Halophilic Bacteria, and Extremely Halophilic Archaea Isolated from Salt Samples and Salted Skins

Bioprospecting for moderately halophilic eubacteria for potential biotechnological applications from Sambhar Lake, Rajasthan, India

Sambhar Lake an athalassohaline habitat located at 27°58′N 75°55′E, Rajasthan, India is a major source of salt production in the country. From surface lake water, soil, and shore sediments, fifty-nine moderate halophiles were isolated which were subsequently grouped according to shape, colony characteristics, and staining into twenty-two isolates. Fourier transform infrared spectroscopy profiling identifiedthese isolates as eubacterial with characteristic C=O stretching of ester functional groups. Observations further indicatedsimilarity within some Halomonasisolates indicating potential phylogenetic lineages. The FASTA sequences obtained after sequencing with universal bacterial primers were processed for phylogenetic analysis. Predominantly Gram-positive genera like Alkalibacillus, Amphibacillus, Marinococcus, Piscibacillus, Planococcus, Salinicoccus, Staphylococcusand Virgibacilluswith only two Gram-negative strains of Halomonaswere identified. The genus Amphibacilluswas recognizedfor the first time in the study of Sambhar Lake. Despite being moderately halophilic, several isolates exhibited high salt tolerance with growth in 25% salt. All isolates were mesophilic with growth observed between 18-42°C which matches the temperature profile of the region. Analysis of hydrolytic potential identified eighteen isolates as protease producers, thirteen as lipase producers, and ten as cellulase-producing strains. Further evaluation showed the dominance of C10:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, C18:0, C18:1 FAMEs, among which presence of C16:0 and C18:1 fatty acid indicated probable antimicrobial potentials of these strains.

Aromatic compounds depurative and plant growth promotion rhizobacteria abilities of Allenrolfea vaginata (Amaranthaceae) rhizosphere microbial communities from a solar saltern hypersaline soil

IntroductionThis work investigates whether rhizosphere microorganisms that colonize halophyte plants thriving in saline habitats can tolerate salinity and provide beneficial effects to their hosts, protecting them from environmental stresses, such as aromatic compound (AC) pollution.MethodsTo address this question, we conducted a series of experiments. First, we evaluated the effects of phenol, tyrosine, 4-hydroxybenzoic acid, and 2,4-dichlorophenoxyacetic (2,4-D) acids on the soil rhizosphere microbial community associated with the halophyteAllenrolfea vaginata. We then determined the ability of bacterial isolates from these microbial communities to utilize these ACs as carbon sources. Finally, we assessed their ability to promote plant growth under saline conditions.ResultsOur study revealed that each AC had a different impact on the structure and alpha and beta diversity of the halophyte bacterial (but not archaeal) communities. Notably, 2,4-D and phenol, to a lesser degree, had the most substantial decreasing effects. The removal of ACs by the rhizosphere community varied from 15% (2,4-D) to 100% (the other three ACs), depending on the concentration.Halomonasisolates were the most abundant and diverse strains capable of degrading the ACs, with strains ofMarinobacter,Alkalihalobacillus,Thalassobacillus,Oceanobacillus, and the archaeaHaladaptatusalso exhibiting catabolic properties. Moreover, our study found that halophile strains Halomonas sp. LV-8T andMarinobactersp. LV-48T enhanced the growth and protection ofArabidopsis thalianaplants by 30% to 55% under salt-stress conditions.DiscussionThese results suggest that moderate halophile microbial communities may protect halophytes from salinity and potential adverse effects of aromatic compounds through depurative processes.

Breakdown of polyethylene therepthalate microplastics under saltwater conditions using engineered Vibrio natriegens

AbstractPoly(ethylene terephthalate) (PET) is a highly recyclable plastic that has been extensively used and manufactured. Like other plastics, PET resists natural degradation, thus accumulating in the environment. Several recycling strategies have been applied to PET, but these tend to result in downcycled products that eventually end up in landfills. This accumulation of landfilled PET waste contributes to the formation of microplastics, which pose a serious threat to marine life and ecosystems, and potentially to human health. To address this issue, our project leveraged synthetic biology to develop a whole‐cell biocatalyst capable of depolymerizing PET in seawater environments by using the fast‐growing, nonpathogenic, moderate halophile Vibrio natriegens. By leveraging a two‐enzyme system—comprising a chimera of IsPETase and IsMHETase from Ideonella sakaiensis—displayed on V. natriegens, we constructed whole‐cell catalysts that depolymerize PET and convert it into its monomers in salt‐containing media and at a temperature of 30°C.

Tandem mass tag-based quantitative proteomics reveals osmotic adaptation mechanisms in Alkalicoccus halolimnae BZ-SZ-XJ29T , a halophilic bacterium with a broad salinity range for optimal growth.

The moderate halophilic bacterium Alkalicoccus halolimnae BZ-SZ-XJ29T exhibits optimum growth over a wide range of NaCl concentrations (8.3-12.3%, w/v; 1.42-2.1 mol L-1 ). However, its adaptive mechanisms to cope with high salt-induced osmotic stress remain unclear. Using TMT-based quantitative proteomics, the cellular proteome was assessed under low (4% NaCl, 0.68 mol L-1 NaCl, control (CK) group), moderate (8% NaCl, 1.37 mol L-1 NaCl), high (12% NaCl, 2.05 mol L-1 NaCl), and extremely high (16% NaCl, 2.74 mol L-1 NaCl) salinity conditions. Digital droplet PCR confirmed the transcription of candidate genes related to salinity. A. halolimnae utilized distinct adaptation strategies to cope with different salinity conditions. Mechanisms such as accumulating different amounts and types of compatible solutes (i.e., ectoine, glycine betaine, glutamate, and glutamine) and the uptake of glycine betaine and glutamate were employed to cope with osmotic stress. Ectoine synthesis and accumulation were critical to the salt adaptation of A. halolimnae. The expression of EctA, EctB, and EctC, as well as the intracellular accumulation of ectoine, significantly and consistently increased with increasing salinity. Glycine betaine and glutamate concentrations remained constant under the four NaCl concentrations. The total content of glutamine and glutamate maintained a dynamic balance and, when exposed to different salinities, may play a role in low salinity-induced osmoadaptation. Moreover, cellular metabolism was severely affected at high salt concentrations, but the synthesis of amino acids, carbohydrate metabolism, and membrane transport related to haloadptation was preserved to maintain cytoplasmic concentration at high salinity. These findings provide insights into the osmoadaptation mechanisms of moderate halophiles and can serve as a theoretical underpinning for industrial production and application of compatible solutes.

A Novel Halo-Acid-Alkali-Tolerant and Surfactant Stable Amylase Secreted from Halophile Bacillus siamensis F2 and Its Application in Waste Valorization by Bioethanol Production and Food Industry.

The extracellular amylase production level by the moderate halophile Bacillus siamensis F2 was optimized, and the enzyme was biochemically characterized. The culture parameters for NaCl, carbon, nitrogen, pH, and temperature were optimized for high titers of amylase production. Growing B. siamensis F2 cultures in Great Salt Lake-2 medium with additions of (in g/L) NaCl (100), starch (30), yeast extract (2), KNO3 (2), and MgSO4 (1) at pH 8, 30 °C resulted in the maximum amylase production (4.2 U/ml). The amylase was active across a wide range of salinities (0 to 30% NaCl), pH (5.0-10.0), and temperatures (20-70 °C) and showed good stability with surfactants (sodium dodecyl sulfate (SDS) and Triton X-100); hence, it was identified as halo-acid-alkali-tolerant and surfactant stable. Temperature, pH, and salinity were optimal for amylase activity at 50 °C, pH 7, and 5% NaCl, respectively. It also generates amylase by utilizing agricultural wastes like sugarcane bagasse, sweet potato peel, and rice husk. Based on the performance of B. siamensis F2 using agricultural wastes and synthesizing amylase, the current study attempted to produce bioethanol by coculturing with baker's yeast using sugarcane bagasse and sweet potato peel as a substrate, which yielded 47 and 57 g/L of bioethanol, respectively. Besides bioethanol production, amylase secreted by F2 was also employed for juice clarification for better yield and clarity and for softening dough to produce better-quality buns. This novel amylase may have many potential applications in waste valorization, biorefinery sectors, and food industries.

Biodegradation of paraffin wax using Bacillus sp. SASH for quality improvement of crude oil properties

Biodegradation has become the most preferred and sustainable mean of reducing paraffin wax, removing paraffin deposition in pipelines and improving the flow of petroleum products. The current study focuses on the isolation of bacteria that has the capability to utilize paraffin as a sole carbon source and has been screened using 2,6-dichlorophenol indophenol (DCPIP) dye as redox indicator. The bacterium was identified as Bacillus sp. SASH according to its 16S rRNA gene sequence analysis. It is a moderate halophile that can grow in seawater at pH 7 and 37 °C. Bacillus sp. SASH was able to degrade various paraffinic products such as paraffin wax, heavy wax distillates, and liquid paraffin and it showed up to 84% paraffin wax degradation. Gas chromatography–mass spectrometry analysis revealed that higher carbon chains after retention time of 18 min were removed after treatment of the wax with the bacterium. Bacillus sp. SASH enhanced the flow of different petroleum products by reducing their viscosity and pour point values. It was also able to reduce the viscosity and improve the flow of crude oil by 60%.

Ecophysiological Properties and Hydrolytic Activity of Chemoorganotrophic Bacteria from Holosiivskyi National Nature Park

Any natural ecosystem contains a specific range of microorganisms. The anthropogenic impact can cause a change in the growth conditions of soil and rhizospheric microbiome and affect the number and the physiological properties of microorganisms. The aim of the study was to isolate the representative microorganisms from terrestrial ecosystems of Holosiivskyi National Nature Park (Ukraine) that are not exposed to extreme factors, to study their ecophysiological properties (resistance to UV radiation, dehydration, hypersalinity, temperature), and to study the extracellular glycoside and proteolytic activities. Methods. Aerobic chemoorganotrophic bacteria isolated at 30°C from soil and phytocenoses of Holosiivskyi National Nature Park were studied. Meat-peptone agar was used to cultivate bacteria. Bacterial UV irradiation was performed with a BUF-15 lamp (λ=254 nm) in the range of 30—1350 J/m2. The temperature range of growth and halotolerance of microorganisms was determined in the range of 1—42°C and 0.1—150 g NaCl/L, respectively. Bacterial isolates were cultivated in submerged conditions at 28°C for 4 days. Synthetic p-nitrophenyl substrates, soluble starch, and guar galactomannan were used to determine glycosidase activity. To study proteolytic activity, casein, elastin, and gelatin were used. Results. The study of 14 soil and plant samples revealed the number of bacteria detected from 9.3×104 to 4.8×105 CFU/g in winter, and 4.8×105 to 4.2×106 CFU/g in summer. The microorganisms were represented by 1—4 morphotypes. There were isolated 37 isolates of aerobic chemoorganotrophic microorganisms, and 69% of them were represented by gram-positive rods. Th e dominance of pigmented isolates was not detected. Most of the microorganisms studied were psychrotolerant and moderate halophiles. The isolates 3g3, 8g1, 8g2, 8g3 from chornozem and dark gray soil showed high resistance to UV radiation. Th e LD99.99 ranged from 800 to 1100 J/m2. The isolates from chornozem, birch moss, green moss with sand and soil, and green moss from oak (1g, 4g2, 9g1, 14g2) were moderately resistant. The LD99.99 was 280—650 J/m2. The UV resistance was shown to be independent of pigmentation. It correlated with dehydration. The phenomenon of resistance to such UV radiation and dehydration may indicate the presence of active reparation mechanisms of DNA damage. All isolates showed cellulose and hemicellulose degrading activities as well as caseinolytic activity. Isolate 9g1 showed high β-xylosidase activity. Conclusions. The high resistance to UV radiation and dehydration of non-adapted microorganisms as well as the wide range of exohydrolase activity indicate the wide adaptive capacity of microorganisms from natural ecosystems, which goes beyond the influence of surrounding factors. No data existed in the available literature defining hydrolytic activity and resistance of microorganisms of the temperate region of Ukraine to extreme factors. The obtained experimental data will allow for a better understanding of the resistance level of microorganisms of temperate regions to extreme factors. As a result of the work, new bacteria with high degrading activity were isolated. The studied isolates require further characterization and analysis for biotechnological applications.

Spontaneous granulation of moderately halophilic sludge inoculated with saltern sediments from single granule into multi-granule aggregation

There is very limited research on the application of moderate halophiles for biotreatment of hypersaline wastewater widely generated from some industries. This study demonstrated the development of moderate halophiles inoculated from saltern sediments into aerobic granule sludge (AGS) to treat hypersaline wastewater with a salinity of 100 g/L. The granulation of moderate halophiles can occur without applying the settling velocity selective pressure. The saltern sediment initially aggregated into single small granules and finally developed into 1200 ± 50 μm multiparticle granules. The halophiles affiliated in Halomonas was dominant in the granular bacterial community, with a relative abundance of 94.52%. Halomonas ventosae secreted sulfated polysaccharides. The sulfated polysaccharides content accounted for 63.95 ± 2.10% in the polysaccharides (PS), having an adhesive role in connecting single granules. Multiparticle granules showed the clear stratified structure, with α-D-glucopyranose polysaccharides in the inner bounders and β-D-glucopyranose polysaccharides in the outer. The moderately granular sludge showed the stable chemical oxygen demand (COD) removal efficiency of >90% and the aerobic total inorganic nitrogen (TIN) removal efficiency (equal to ammonia removal) of 70 ± 5.00%. This paper contributes new insight into the formation of moderately halophilic granular sludge and accelerates the application of moderately halophilic granular sludge to treat hypersaline wastewater.

Red algae industrial residues as a sustainable carbon platform for the co-production of poly-3-hydroxybutyrate and gluconic acid by Halomonas boliviensis.

Polyhydroxyalkanoate (PHA) production using halophilic bacteria has been revisited because less severe operational conditions with respect to sterility can be applied, also alleviating production costs. Halomonas boliviensis was selected because it is a moderate halophile able to grow and attain high poly-3-hydroxybutyrate (P3HB) contents under 5–45 g/L NaCl concentrations, conditions that discourage microbial contamination. Industrial residues of the red alga Gelidium corneum after agar extraction were used as sugar platform to reduce costs associated with the carbon source. These residues still comprise a high carbohydrate content (30–40% w/w) of mainly cellulose, and their hydrolysates can be used as substrates for the bioproduction of value-added products. Preliminary assays using glucose were carried out to determine the best conditions for growth and P3HB production by H. boliviensis in bioreactor fed-batch cultivations. Two strategies were addressed, namely nitrogen or phosphorus limitation, to promote polymer accumulation. Similar P3HB cell contents of 50% (gpolymer/gCDW) and yields Y P3HB/glucose of 0.11–0.15 g polymer/g glucose were attained under both conditions. However, higher specific productivities were reached under P-limitation, and thus, this strategy was adopted in the subsequent study. Two organic acids, resulting from glucose metabolism, were identified to be gluconic and 2-oxoglutaric acid. Reducing the oxygen concentration in the cultivation medium to 5% sat was found to minimize organic acid production and enhance the yield of polymer on sugar to 0.20 gP3HB/gglucose. Finally, fed-batch cultivations using G. corneum hydrolysates as the only C-source achieved an overall volumetric productivity of 0.47 g/(L.h), 40% polymer accumulation, and negligible gluconic acid production.

Molecular Characterization and Biocompatibility of Exopolysaccharide Produced by Moderately Halophilic Bacterium Virgibacillus dokdonensis from the Saltern of Kumta Coast.

The use of natural polysaccharides as biomaterials is gaining importance in tissue engineering due to their inherent biocompatibility. In this direction, the present study aims to explore the structure and biocompatibility of the EPS produced by Virgibacillus dokdonensis VITP14. This marine bacterium produces 17.3 g/L of EPS at 96 h of fermentation. The EPS was purified using ion exchange and gel permeation chromatographic methods. The porous web-like structure and elemental composition (C, O, Na, Mg, P, S) of the EPS were inferred from SEM and EDX analysis. AFM analysis revealed spike-like lumps with a surface roughness of 84.85 nm. The zeta potential value of −10 mV indicates the anionic nature of the EPS. Initial molecular characterization showed that the EPS is a heteropolysaccharide composed of glucose (25.8%), ribose (18.6%), fructose (31.5%), and xylose (24%), which are the monosaccharide units in the HPLC analysis. The FTIR spectrum indicates the presence of functional groups/bonds typical of EPSs (O-H, C-H, C-O-H, C-O, S=O, and P=O). The polymer has an average molecular weight of 555 kDa. Further, NMR analysis revealed the monomer composition, the existence of two α- and six β-glycosidic linkages, and the branched repeating unit as → 1)[α-D-Xylp-(1 → 2)-α-D-Glcp-(1 → 6)-β-D-Glcp-(1 → 5)]-β-D-Frup-(2 → 2)[β-D-Xylp-(1 → 4)]-β-D-Xylp-(1 → 6)-β-D-Fruf-(2 → 4)-β-D-Ribp-(1 →. The EPS is thermally stable till 251.4 °C. X-ray diffraction analysis confirmed the semicrystalline (54.2%) nature of the EPS. Further, the EPS exhibits significant water solubility (76.5%), water-holding capacity (266.8%), emulsifying index (66.8%), hemocompatibility (erythrocyte protection > 87%), and cytocompatibility (cell viability > 80% on RAW264.7 and keratinocyte HaCaT cells) at higher concentrations and prolongs coagulation time in APTT and PT tests. Our research unveils the significant biocompatibility of VITP14 EPS for synthesizing a variety of biomaterials.

Sediminibacillus dalangtanensis sp. nov., a moderate halophile isolated from hypersaline sediments of the Qaidam Basin in Northwest China.

A Gram-stain-positive, moderately halophilic, aerobic, endospore-forming, rod-shaped bacterium, designated strain DP4-553-ST, was isolated from hypersaline sediment collected from the Dalangtan Playa in the Qaidam Basin, Northwest PR China. Growth occurred within 0-21.6% (w/v) NaCl (optimum 7.2%) at pH 5.5-9.0 (optimum pH 7.0) and at 4-45 °C (optimum 37 °C). Phylogeny based on 16S rRNA gene sequences indicated that strain DP4-553-ST belonged to the genus Sediminibacillus, with high 16S rRNA gene sequence similarity to Sediminibacillus halophilus EN8dT (99.5 %), Sediminibacillus terrae JSM 102062T (99.4 %), Virgibacillus senegalensis SK-1T (99.3 %) and Sediminibacillus albus NHBX5T (98.3 %). The G+C content of the chromosomal DNA was 43.6 mol %. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values between strain DP4-553-ST and the four close type strains were 71.2-93.3, 74.0-90.5 and 20.0-41.4 %, respectively. The whole genomic analysis showed that strain DP4-553-ST constituted a different taxon separated from the recognized Sediminibacillus species. The major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0, C16 : 0 and iso-C15 : 0. The type strain contained cell-wall peptidoglycan based on diaminopimelic acid and possessed menaquinone-7 as the major respiratory isoprenoid quinone. The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, four unidentified glycolipids, phosphatidylcholine, aminophospholipid, aminolipid and seven unidentified phospholipids. The combined data from phenotypic and genotypic studies demonstrated that strain DP4-553-ST represents a novel species of the genus Sediminibacillus, for which the name Sediminibacillus dalangtanensis sp. nov. is proposed, the type strain is DP4-553-ST (=MCCC 1K03838T= KCTC 43250T).

Role of carnitine in adaptation of Chromohalobacter salexigens DSM 3043 and its mutants to osmotic and temperature stress in defined medium.

L-Carnitine is widespread in nature, but little information is available on its metabolism and physiological functions in moderate halophiles. In this study, we found that Chromohalobacter salexigens DSM 3043 could utilize carnitine not only as a nutrient, but also as an osmolyte. When grown at 37 °C under salt-stress conditions, the strain utilized carnitine as an osmoprotectant by enzymatically converting it into GB. When grown at low and high temperature, both carnitine and its metabolic intermediate GB were simultaneously accumulated intracellularly, serving as cryoprotectants and thermoprotectants. The genes (csal_3172, csal_3173, and csal_3174) which were predicted to participate in L-carnitine degradation to GB were deleted to construct the corresponding mutants. The effects of salinity and temperature on the growth rates and cytoplasmic solute pools of the C. salexigens wild-type and mutant strains were investigated. 13C-NMR analysis revealed that GB was still detected in the Δcsal_3172Δcsal_3173Δcsal_3174 mutant grown in a defined medium with added DL-carnitine, but not with L-carnitine, indicating that an unidentified D-carnitine degradation pathway exists in C. salexigens. Taken together, the data presented in this study expand our knowledge on carnitine metabolism and its physiological functions in C. salexigens exposed to single or multiple environmental abiotic stress.

Characterization of Halophilic Bacteria Isolated from Khewra Salt Mines

Halophilic bacteria can populate every niche of earth. These halophiles have a great potential of exopolysaccharides production that is of considerable importance in various industries. To screen EPS producing halophiles, saline soil samples were collected from Khewra salt mines, Jehlum, Pakistan. Twenty-two morphologically different bacterial strains were isolated by serial dilution method. All strains were considered as moderate halophilic bacteria as they could grow at 3-15% of NaCl concentration whereas only three strains could grow at 15% of NaCl, which belonged to the genus Bacillus and Pseudomonas. For screening of EPS production, P-medium was used. While for the estimation of slime production, congo-red agar was used that exhibited positive results by appearance of black colored colonies by many strains. Moreover, EPS production was analyzed quantitatively and qualitatively. Isolated Staphylococcus and Bacillus species produced high amount of EPS (20g/L). “Moderate halophiles” play an important role in therapeutics, bioremediation, food and medicine, petroleum and tanning industries by producing EPS. Recently, growth of many agriculture crops has been improved by using beneficial halophiles in saline soils. Consequently, with the help of these beneficial halophiles we can give benefit to mankind

Utilization and accumulation of compatible solutes in Halomonas pacifica: a species of moderately halophilic bacteria isolated from a saline lake in South Libya.

When grown in high salt concentrations, halophilic bacteria often accumulate compatible solutes, which have major applications in biotechnology because they stabilize cells and proteins. Four Gram-negative bacterial strains, belonging to the family Halomonadaceae, were isolated from Qaberoun and Um-Alma lakes in South Libya using high-salinity medium. The strains were identified using 16S rRNA gene sequencing as belonging to Halomonas pacifica (strain ABQ1), Halomonas venusta (ABQ2), Halomonas elongata (ABU1) and Halomonas salifodinae (ABU2). H. pacifica ABQ1 is a moderate halophile (salinity range 0.05 to 2.5 M NaCl), with a broad tolerance to pH (7 to 9) and temperature (25–37 °C). Addition of the compatible solutes glycine betaine (betaine) and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) to the medium had a positive effect on growth of H. pacifica at 2 M NaCl. In rich LB medium, betaine was the major compatible solute accumulated, with ectoine only being accumulated at salinities in excess of 1 M NaCl. In minimal M9 medium, betaine was not produced, but increasing amounts of ectoine were synthesized with increasing salinity, and hydroxyectoine [(4S,5S)−5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] was also synthesized when the cells were grown in very high salt. We have thus identified H. pacifica as a producer of ectoine and hydroxyectoine, with more being produced at higher salinities. As industrial demand for these compatible solutes continues to increase, this system has biotechnological potential.

Adaptation to Varying Salinity in Halomonas elongata: Much More Than Ectoine Accumulation.

The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at salt concentrations well above 10 % NaCl (1.7 M NaCl). A well-known osmoregulatory mechanism is the accumulation of the compatible solute ectoine within the cell in response to osmotic stress. While ectoine accumulation is central to osmoregulation and promotes resistance to high salinity in halophilic bacteria, ectoine has this effect only to a much lesser extent in non-halophiles. We carried out transcriptome analysis of H. elongata grown on two different carbon sources (acetate or glucose), and low (0.17 M NaCl), medium (1 M), and high salinity (2 M) to identify additional mechanisms for adaptation to high saline environments. To avoid a methodological bias, the transcripts were evaluated by applying two methods, DESeq2 and Transcripts Per Million (TPM). The differentially transcribed genes in response to the available carbon sources and salt stress were then compared to the transcriptome profile of Chromohalobacter salexigens, a closely related moderate halophilic bacterium. Transcriptome profiling supports the notion that glucose is degraded via the cytoplasmic Entner-Doudoroff pathway, whereas the Embden-Meyerhoff-Parnas pathway is employed for gluconeogenesis. The machinery of oxidative phosphorylation in H. elongata and C. salexigens differs greatly from that of non-halophilic organisms, and electron flow can occur from quinone to oxygen along four alternative routes. Two of these pathways via cytochrome bo' and cytochrome bd quinol oxidases seem to be upregulated in salt stressed cells. Among the most highly regulated genes in H. elongata and C. salexigens are those encoding chemotaxis and motility proteins, with genes for chemotaxis and flagellar assembly severely downregulated at low salt concentrations. We also compared transcripts at low and high-salt stress (low growth rate) with transcripts at optimal salt concentration and found that the majority of regulated genes were down-regulated in stressed cells, including many genes involved in carbohydrate metabolism, while ribosome synthesis was up-regulated, which is in contrast to what is known from non-halophiles at slow growth. Finally, comparing the acidity of the cytoplasmic proteomes of non-halophiles, extreme halophiles and moderate halophiles suggests adaptation to an increased cytoplasmic ion concentration of H. elongata. Taken together, these results lead us to propose a model for salt tolerance in H. elongata where ion accumulation plays a greater role in salt tolerance than previously assumed.

A novel acidic and SDS tolerant halophilic lipase from moderate halophile Nesterenkonia sp. strain F: molecular cloning, structure analysis and biochemical characterization

A novel acidic and SDS tolerant halophilic lipase from moderate halophile Nesterenkonia sp. strain F: molecular cloning, structure analysis and biochemical characterization

ISOLATION OF BACTERIA FROM THE SITES OF FEED AND NESTING ACTIVITY OF LARUS DOMINICANUS (GALINDEZISLAND, THE MARITIMEANTARCTIC) AND THEIR CHARACTERISTICS

The aim of the study was to investigate the number of different groups of microorganisms in soil samples from sites of feed and nesting activity of Larus dominicanus (Galindez Island, the maritime Antarctic) and to characterize the physiological and biochemical properties of isolated microorganisms. Methods. In the work standard microbiological and biochemical research methods were used (cultural, microscopy methods, determination of enzymatic activity). Genomic DNA was isolated by soft lysis. The 16 S rRNA gene was amplified using universal primers 27F and 1492R. Identification of isolates was performed based on the determination of the 16 S rRNA gene sequence, physiological and biochemical properties. Results. The number of microorganisms of different groups in soil samples from sites of food and nesting activity of Larus dominicanus (Galindez Island, the maritime Antarctic) was established. 74 isolates of bacteria were isolated. Isolate 2U-K-37, that was isolated from upper layer of soil, and isolate 2B-K-54, that was isolated from a depth of 2–5 cm, were characterized by amylase, lipase, phospholipase, protease activities, the ability to form exopolysaccharides.They were identified by sequencing of the 16 S rRNA gene, physiological and biochemical properties as Pedobacter sp. 2U-K-37 and Pseudarthrobacter sp. 2B-K-54. In the soil samples from sites of feed and nesting activity of Larus dominicanus microorganisms that metabolize nitrogen of organic compounds were the most numerous. Oligotrophic microorganisms and microorganisms that metabolize nitrogen of inorganic compounds were less numerous. The number of microorganisms of groups in the samples from the soil surface and lower layers differed statistically significant. Isolated and identified obligate psychrophilic strain Pedobacter sp. 2U-K-37 and psychrotolerant strain Pseudarthrobacter sp. 2B-K-54 are moderate halophiles that are able to hydrolyze starch, gelatin, tween-20 and synthesize exopolysaccharides.

Net Charges of the Ribosomal Proteins of the S10 and spc Clusters of Halophiles Are Inversely Related to the Degree of Halotolerance.

ABSTRACTNet positive charge(s) on ribosomal proteins (r-proteins) have been reported to influence the assembly and folding of ribosomes. A high percentage of r-proteins from extremely halophilic archaea are known to be acidic or even negatively charged. Those proteins that remain positively charged are typically far less positively charged. Here, the analysis is extended to non-archaeal halophilic bacteria, eukaryotes, and halotolerant archaea. The net charges (pH 7.4) of the r-proteins that comprise the S10-spc operon/cluster from individual microbial and eukaryotic genomes were estimated and intercompared. It was observed that, as a general rule, the net charges of individual proteins remained mostly basic as the salt tolerance of the bacterial strains increased from 5 to 15%. The most striking exceptions were the extremely halophilic bacterial strains, Salinibacter ruber SD01, Acetohalobium arabaticum DSM 5501 and Selenihalanaerobacter shriftii ATCC BAA-73, which are reported to require a minimum of 18% to 21% salt for their growth. All three strains have higher numbers of acidic S10-spc cluster r-proteins than what is seen in the moderate halophiles or the halotolerant strains. Of the individual proteins, only uL2 never became acidic. uS14 and uL16 also seldom became acidic. The net negative charges on several of the S10-spc cluster r-proteins are a feature generally shared by all extremely halophilic archaea and bacteria. The S10-spc cluster r-proteins of halophilic fungi and algae (eukaryotes) were exceptions: these were positively charged despite the halophilicity of the organisms.IMPORTANCE The net charges (at pH 7.4) of the ribosomal proteins (r-proteins) that comprise the S10-spc cluster show an inverse relationship with the halophilicity/halotolerance levels in both bacteria and archaea. In non-halophilic bacteria, the S10-spc cluster r-proteins are generally basic (positively charged), while the rest of the proteomes in these strains are generally acidic. On the other hand, the whole proteomes of the extremely halophilic strains are overall negatively charged, including the S10-spc cluster r-proteins. Given that the distribution of charged residues in the ribosome exit tunnel influences cotranslational folding, the contrasting charges observed in the S10-spc cluster r-proteins have potential implications for the rate of passage of these proteins through the ribosomal exit tunnel. Furthermore, the universal protein uL2, which lies in the oldest part of the ribosome, is always positively charged irrespective of the strain/organism it belongs to. This has implications for its role in the prebiotic context.

Osmolyte Signatures for the Protection of Aspergillus sydowii Cells under Halophilic Conditions and Osmotic Shock.

Aspergillus sydowii is a moderate halophile fungus extensively studied for its biotechnological potential and halophile responses, which has also been reported as a coral reef pathogen. In a recent publication, the transcriptomic analysis of this fungus, when growing on wheat straw, showed that genes related to cell wall modification and cation transporters were upregulated under hypersaline conditions but not under 0.5 M NaCl, the optimal salinity for growth in this strain. This led us to study osmolyte accumulation as a mechanism to withstand moderate salinity. In this work, we show that A. sydowii accumulates trehalose, arabitol, mannitol, and glycerol with different temporal dynamics, which depend on whether the fungus is exposed to hypo- or hyperosmotic stress. The transcripts coding for enzymes responsible for polyalcohol synthesis were regulated in a stress-dependent manner. Interestingly, A. sydowii contains three homologs (Hog1, Hog2 and MpkC) of the Hog1 MAPK, the master regulator of hyperosmotic stress response in S. cerevisiae and other fungi. We show a differential regulation of these MAPKs under different salinity conditions, including sustained basal Hog1/Hog2 phosphorylation levels in the absence of NaCl or in the presence of 2.0 M NaCl, in contrast to what is observed in S. cerevisiae. These findings indicate that halophilic fungi such as A. sydowii utilize different osmoadaptation mechanisms to hypersaline conditions.