Isolation and characterization of a Halomonas species for non-axenic growth-associated production of bio-polyesters from sustainable feedstocks.

Biodegradable plastics are urgently needed to replace petroleum-derived polymeric materials and prevent their accumulation in the environment. To this end, we isolated and characterized a halophilic and alkaliphilic bacterium from the Great Salt Lake in Utah. The isolate was identified as a Halomonas species and designated "CUBES01." Full-genome sequencing and genomic reconstruction revealed the unique genetic traits and metabolic capabilities of the strain, including the common polyhydroxyalkanoate (PHA) biosynthesis pathway. Fluorescence staining identified intracellular polyester granules that accumulated predominantly during the strain's exponential growth, a feature rarely found among natural PHA producers. CUBES01 was found to metabolize a range of renewable carbon feedstocks, including glucosamine and acetyl-glucosamine, as well as sucrose, glucose, fructose, and further glycerol, propionate, and acetate. Depending on the substrate, the strain accumulated up to ~60% of its biomass (dry wt/wt) in poly(3-hydroxybutyrate), while reaching a doubling time of 1.7 h at 30°C and an optimum osmolarity of 1 M sodium chloride and a pH of 8.8. The physiological preferences of the strain may not only enable long-term aseptic cultivation but also facilitate the release of intracellular products through osmolysis. The development of a minimal medium also allowed the estimation of maximum polyhydroxybutyrate production rates, which were projected to exceed 5 g/h. Finally, also, the genetic tractability of the strain was assessed in conjugation experiments: two orthogonal plasmid vectors were stable in the heterologous host, thereby opening the possibility of genetic engineering through the introduction of foreign genes. IMPORTANCE: The urgent need for renewable replacements for synthetic materials may be addressed through microbial biotechnology. To simplify the large-scale implementation of such bio-processes, robust cell factories that can utilize sustainable and widely available feedstocks are pivotal. To this end, non-axenic growth-associated production could reduce operational costs and enhance biomass productivity, thereby improving commercial competitiveness. Another major cost factor is downstream processing, especially in the case of intracellular products, such as bio-polyesters. Simplified cell-lysis strategies could also further improve economic viability.

The polyextremophile Natranaerobius thermophilus adopts a dual adaptive strategy to long-term salinity stress, simultaneously accumulating compatible solutes and K.

The bacterium Natranaerobius thermophilus is an extremely halophilic alkalithermophile that can thrive under conditions of high salinity (3.3-3.9 M Na+), alkaline pH (9.5), and elevated temperature (53°C). To understand the molecular mechanisms of salt adaptation in N. thermophilus, it is essential to investigate the protein, mRNA, and key metabolite levels on a molecular basis. Based on proteome profiling of N. thermophilus under 3.1, 3.7, and 4.3 M Na+ conditions compared to 2.5 M Na+ condition, we discovered that a hybrid strategy, combining the "compatible solute" and "salt-in" mechanisms, was utilized for osmotic adjustment dur ing the long-term salinity adaptation of N. thermophilus. The mRNA level of key proteins and the intracellular content of compatible solutes and K+ support this conclusion. Specifically, N. thermophilus employs the glycine betaine ABC transporters (Opu and ProU families), Na+/solute symporters (SSS family), and glutamate and proline synthesis pathways to adapt to high salinity. The intracellular content of compatible solutes, including glycine betaine, glutamate, and proline, increases with rising salinity levels in N. thermophilus. Additionally, the upregulation of Na+/ K+/ H+ transporters facilitates the maintenance of intracellular K+ concentration, ensuring cellular ion homeostasis under varying salinities. Furthermore, N. thermophilus exhibits cytoplasmic acidification in response to high Na+ concentrations. The median isoelectric points of the upregulated proteins decrease with increasing salinity. Amino acid metabolism, carbohydrate and energy metabolism, membrane transport, and bacterial chemotaxis activities contribute to the adaptability of N. thermophilus under high salt stress. This study provides new data that support further elucidating the complex adaptation mechanisms of N. thermophilus under multiple extremes.IMPORTANCEThis study represents the first report of simultaneous utilization of two salt adaptation mechanisms within the Clostridia class in response to long-term salinity stress.

Utilization of gene manipulation system for advancing the biotechnological potential of halophiles: A review.

Halophiles are salt-loving microorganisms known to have their natural resistance against media contamination even when cultivated in nonsterile and continuous bioprocess system, thus acting as promising cell factories for Next Generation of Industrial Biotechnology (NGIB). NGIB - a successor to the traditional industrial biotechnology, is a more sustainable and efficient bioprocess technology while saving energy and water in a more convenient way as well as reducing the investment cost and skilled workforce requirement. Numerous studies have achieved intriguing outcomes during synthesis of different metabolite using halophiles such as polyhydroxyalkanoates (PHA), ectoine, biosurfactants, and carotenoids. Present-day development in genetic maneuverings have shown optimistic effects on the industrial applications of halophiles. However, viable and competent genetic manipulation system and gene editing tools are critical to accelerate the process of halophile engineering. With the aid of such powerful gene manipulation systems, exclusive microbial chassis are being crafted with desirable features to breed another innovative area of research such as synthetic biology. This review provides an aerial perspective on how the expansion of adaptable gene manipulation toolkits in halophiles are contributing towards biotechnological advancement, and also focusses on their subsequent application for production improvement. This current methodical and comprehensive review will definitely help the scientific fraternity to bridge the gap between challenges and opportunities in halophile engineering.

Complete genome sequence of PETase type IIa-harboring Marinobacter nanhaiticus D15-8W, isolated from a South China Sea sediment.

Marinobacter nanhaiticus D15-8W is known for its ability to metabolize polycyclic aromatic hydrocarbons. Here, we report the complete circular genome sequence of this strain to be 5,336,660 bp (G + C content, 58.6%; 4,869 protein-coding sequences) with one plasmid (69,655 bp).

Lipingzhangella rawalii sp. nov., a novel halophile isolated from Sambhar Salt Lake, Rajasthan, India.

A haloalkaliphilic actinobacterial strain LS1_29T, isolated from an inland hypersaline Sambhar Salt Lake, situated in Rajasthan, India, was subjected to taxonomic characterisation using the polyphasic approach. Cells of the strain were Gram stain positive and aerobic, having reticulate and aerial hyphae. The major fatty acids detected were iso C16:0 (38.23%), anteiso C17:0 (20.52%), iso C18:0 10 methyl (8.09%), iso C18:0 (7.74%) and iso C17:0 (7.48%). The major polar lipids identified were diphosphatidyl glycerol, phosphatidylglycerol, phosphatidylcholine and phospholipids. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the strain belonged to the genus Lipingzhangella with the highest similarity of 94.09% to Lipingzhangella halophila EGI 80537 T and formed a separate clade within the genus Lipingzhangella. The DNA G + C content of genomic DNA of strain LS1_29T was 67.99 mol%. The strain showed the highest orthologous average nucleotide identity (OrthoANI) value of 72.80% and digital DNA-DNA hybridization (dDDH) value of 20.3% with Lipingzhangella halophila EGI 80537 T. Based on the phenotypic, chemotaxonomic and phylogenetic characteristics, strain LS1_29T represents a novel species within the genus Lipingzhangella, for which the name Lipingzhangella rawalii sp. nov. is proposed. The type strain of the type species is LS1_29T (= KCTC 49199 T = JCM 32979 T = MCC 3420 T).

'All About' Extremophiles.

Despite common perception, most of Earth is what is often referred to as an 'extreme environment.' Yet to the organisms that call these places home, it is simply that (home). They have adapted to thrive in these environments and, in the process, have evolved many unique adaptations at the molecular- and 'omic-level. Scientists' interest in these organisms has typically been in how they and their products can be harnessed for biotechnological applications and the environments where they are found, while the general public's veers more toward a fascination with their deviation from the 'norm'. However, these organisms have so much more to tell us about Life and the myriad ways there are to perform 'simple' biological processes.

Xinghamide A, a New Cyclic Nonapeptide Found in Streptomyces xinghaiensis.

Xinghamide A (1), a new nonapeptide, was discovered in Streptomyces xinghaiensis isolated from a halophyte, Suaeda maritima (L.) Dumort. Based on high-resolution mass and NMR spectroscopic data, the planar structure of 1 was established, and, in particular, the sequence of nine amino acids was determined with ROESY and HMBC NMR spectra. The absolute configurations of the α-carbon of each amino acid residue were determined with 1-fluoro-2,4-dinitrophenyl-l-and -d-leucine amide (Marfey's reagents) and 2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate, followed by LC-MS analysis. The anti-inflammatory activity of xinghamide A (1) was evaluated by inhibitory abilities against the nitric oxide (NO) secretion and cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.

Halorubrum hochsteinianum sp. nov., an ancient haloarchaeon from a natural experiment.

A single extremely halophilic strain was isolated from salt brine produced when a fresh water lake flooded a large salt mine located beneath the lake. The water that entered this mine contained less than 0.34 M NaCl, but over time, this sealed brine became saturated by Cenozoic age salt (121-125 million-year BCE). The isolated strain requires at least 1.7 M NaCl for survival and grows optimally in 3.1 M NaCl. Therefore, it could not have survived or been present in the waters that flooded this salt mine. The strain grows at a pH range from 6.5 to 9.0 and has a wide tolerance to temperatures from 25 ℃ to at least 60 ℃. The comparison of 16S rRNA and rpoB' genes revealed that strain 1-13-28T is related to Halorubrum tebenquichense DSM 14210T showing 98.6% and 98.1% similarities, respectively. Phylogenetic analyses based on 16S rRNA, rpoB' genes and 122 concatenated archaeal genes show that the strain 1-13-28T consistently forms a cluster with Halorubrum tebenquichense of the genus Halorubrum. Strain 1-13-28T contained sulfated mannosyl glucosyl diether, and the polar lipid profile was identical to those of most Halorubrum species. Based on the overall combination of physiological, phylogenetic, polar lipids and phylogenomic characteristics, strain 1-13-28T (= ATCC 700083T = CGMCC 1.62627T) represents a newly identified species within the genus Halorubrum for which the name Halorubrum hochsteinianum is proposed.

Enchelyothrix muria n. gen, n. sp., (Ciliophora, Litostomatea, Spathidiida), an extreme halotroph spathidiid from Maharloo Lake, Iran.

Although free-living ciliated protists (Ciliophora) commonly inhabit aquatic and terrestrial biotopes of mild to moderate salinity (PSS-practical salinity scale S = 10-150), very few have adapted to life at more extreme salinities (>150 to saturation). Such extreme halophiles or halotrophs are of interest from the standpoints of evolution, cell physiology, ecology, and even astrobiology. In this work, we present the morphology, 18S rRNA gene sequence, and phylogenetic analysis of a novel spathidiid ciliate (Ciliophora, Litosatomatea) that thrives in saturated brines of Maharloo Lake, Iran. Based on its unique combination of morphologic features, its molecular characterization, and its unusual ecology, it is assigned to a newly erected monotypic genus incertae sedis in order Spathidiida. The new species differs from other spathidiids by: a cell with a very short acutely pointed tail, the absence of a circumoral kinety with, instead, inclined, interrupted circumoral kinetofragments at the anterior end of somatic kineties and brush rows abutting the oral bulge, the absence of a contractile vacuole, a heteromorphic dorsal brush comprising five or six rows, and an extreme (at or near saturation) hypersaline habitat. We briefly discuss the persistent uncertainties regarding the phylogenetic relationships within the order Spathidiida.

Draft genome sequence of Hahella sp. CR1 and its ability in producing cellulases for saccharifying agricultural biomass.

Hahella is a genus that has not been well-studied, with only two identified species. The potential of this genus to produce cellulases is yet to be fully explored. The present study isolated Hahella sp. CR1 from mangrove soil in Tanjung Piai National Park, Malaysia, and performed whole genome sequencing (WGS) using NovaSeq 6000. The final assembled genome consists of 62 contigs, 7,106,771 bp, a GC ratio of 53.5%, and encoded for 6,397 genes. The CR1 strain exhibited the highest similarity with Hahella sp. HN01 compared to other available genomes, where the ANI, dDDH, AAI, and POCP were 97.04%, 75.2%, 97.95%, and 91.0%, respectively. In addition, the CAZymes analysis identified 88 GTs, 54 GHs, 11 CEs, 7 AAs, 2 PLs, and 48 CBMs in the genome of strain CR1. Among these proteins, 11 are related to cellulose degradation. The cellulases produced from strain CR1 were characterized and demonstrated optimal activity at 60 ℃, pH 7.0, and 15% (w/v) sodium chloride. The enzyme was activated by K+, Fe2+, Mg2+, Co2+, and Tween 40. Furthermore, cellulases from strain CR1 improved the saccharification efficiency of a commercial cellulase blend on the tested agricultural wastes, including empty fruit bunch, coconut husk, and sugarcane bagasse. This study provides new insights into the cellulases produced by strain CR1 and their potential to be used in lignocellulosic biomass pre-treatment.

Microbial Influence on the Mobility of +3 Actinides from a Salt-Based Nuclear Waste Repository.

Biologically enhanced transport of radionuclides is one of several processes that can affect the performance of a nuclear waste repository. In this work, several microbial isolates from the Waste Isolation Pilot Plant (WIPP) were tested for their influence on the concentration of neodymium, as an analog for +3 actinides, in simple sodium chloride solutions and in anoxic WIPP brines. Batch sorption experiments were carried out over a period of 4-5 weeks. In many cases, the effect on neodymium in solution was immediate and extensive and assumed to be due to surface complexation. However, over time, the continued loss of Nd from the solution was more likely due to biologically induced precipitation and/or mineralization and possible entrapment in extracellular polymeric substances. The results showed no correlation between organism type and the extent of its influence on neodymium in solution. However, a correlation was observed between different test matrices (simple NaCl versus high-magnesium brine versus high-NaCl brine). Further experiments were conducted to test these matrix effects, and the results showed a significant effect of magnesium concentration on the ability of microorganisms to remove Nd from solution. Possible mechanisms include cation competition and the alteration of cell surface structures. This suggests that the aqueous chemistry of the WIPP environs could play a larger role in the final disposition of +3 actinides than the microbiology.

Development of Martian saline seep models and their implications for planetary protection.

While life on Mars has not been found, Earth-based microorganisms may contaminate the Red Planet during rover expeditions and human exploration. Due to the survival advantages conferred by the biofilm morphology to microorganisms, such as resistance to UV and osmotic stress, biofilms are particularly concerning from a planetary protection perspective. Modeling and data from the NASA Phoenix mission indicate that temporary liquid water might exist on Mars in the form of high salinity brines. These brines could provide colonization opportunities for terrestrial microorganisms brought by spacecraft or humans. To begin testing for potential establishment of microbes, results are presented from a simplified laboratory model of a Martian saline seep inoculated with sediment from Hailstone Basin, a terrestrial saline seep in Montana (USA). The seep was modeled as a sand-packed drip flow reactor at room temperature fed media with either 1 M MgSO4 or 1 M NaCl. Biofilms were established within the first sampling point of each experiment. Endpoint 16S rRNA gene community analysis showed significant selection of halophilic microorganisms by the media. Additionally, we detected 16S rRNA gene sequences highly similar to microorganisms previously detected in two spacecraft assembly cleanrooms. These experimental models provide an important foundation for identifying microbes that could hitch-hike on spacecraft and may be able to colonize Martian saline seeps. Future model optimization will be vital to informing cleanroom sterilization procedures.

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.

Phylogenetic assessment of the halophilic Australian gastropod Coxiella and South African Tomichia resolves taxonomic uncertainties, uncovers new species and supports a Gondwanan link.

Genetic and morphological data have suggested a Gondwanan connection between the three non-marine aquatic gastropod genera Coxiella Smith, 1894, Tomichia Benson, 1851 and Idiopyrgus Pilsbry, 1911. These genera have recently been included in the family Tomichiidae Wenz, 1938, however, further assessment of the validity of this family is warranted. Coxiella is an obligate halophile that occurs in Australian salt lakes while Tomichia occurs in saline and freshwater environments in southern Africa and Idiopyrgus is a freshwater taxon from South America. Despite their novel evolutionary and ecological characteristics, Coxiella, Tomichia and Idiopyrgus are poorly studied, and the lack of a contemporary taxonomic framework restricts our ability to assess the risk of declining habitat quality to these gastropods. We used data from mitochondrial (COI and 16S) and nuclear (28S and 18S) genes in 20 species from all three genera to undertake the most comprehensive phylogenetic test of the Tomichiidae to date. Bayesian and Maximum Likelihood phylogenetic analyses of a concatenated dataset (2974 bp) of all four genes strongly supported a monophyletic Tomichiidae. The COI analysis (n = 307) identified 14 reciprocally monophyletic lineages in Coxiella that comprised eight of the nine currently described species and at least six putative new species. Four distinct genetic clades of species with somewhat distinctive morphologies were found, each of which may constitute a distinct genera. In addition, four species of Tomichia were identified, including three described and one putatively new species. Current species descriptions of Coxiella do not account for the range of morphological variation observed within most described species, and although morphology is reasonably effective at delineating between clades, it is of limited use when trying to separate closely related Coxiella species. The improved understanding of the taxonomy and diversity of Tomichia and especially Coxiella will underpin future studies and conservation planning for these taxa.

Microbial community on industrial salty bovine hides: From the slaughterhouse to the salting.

The leather-making industry is an age-old industry and desiccation with salt has been one of the most used methodologies for obtaining valuable skins. However, halophiles may proliferate and affect the integrity of the hide-collagen structure, as well as leading to undesirable red colorations or less-frequent purple stains. To understand the basis of these industrial hide contaminations, the microbial community from raw hide samples, salt-cured samples and four different industrial salts, was analyzed by 16S rRNA gene metabarcoding together with standard cultivation methods. Comparison of raw hides and correctly cured hides revealed a core microbiome that was absent from contaminated hides. In addition, archaea were missing from well-cured hides, whereas Psychrobacter and Acinetobacter were highly represented (23 % and 17.4 %, respectively). In damaged hides, only a few operational taxonomic units (OTUs), from among the hundreds detected, were able to proliferate and, remarkably, a single Halomonas OTU represented 57.66 % of the reads. Halobacteria, mainly Halovenus, Halorubrum and Halovivax, increased by up to 36.24-39.5 % in the red- and purple-affected hides. The major contaminants were isolated and hide infections, together with collagenase activity, were evaluated. The results showed that hides enriched with the non-pigmented isolate Halomonas utahensis COIN160 damaged the collagen fibers similarly to Halorubrum, and together they were considered to be one of the major causes. Putative degrading inhibitors were also identified from among the Alkalibacillus isolates. It was concluded that hide contaminations were driven by clonal outbreaks of a few specific microbes, which may have been non-pigmented collagen degraders. Acinetobacter and Alkalibacillus, members of the core microbiome of raw and well-cured salted hides, are suggested as hide contaminant inhibitors that need further analysis.

Paracoccus onchidii sp. nov., a moderately halophilic bacterium isolated from a marine invertebrate from the South China Sea.

A novel moderately halophilic bacterial strain, designated Z330T, was isolated from the egg of a marine invertebrate of the genus Onchidium collected in the South China Sea. The 16S rRNA gene sequence of strain Z330T exhibited the highest similarity value to that of the type strain Paracoccus fistulariae KCTC 22803T (97.6%), Paracoccus seriniphilus NBRC 100798T (97.6%) and Paracoccus aestuarii DSM 19484T (97.6%). Phylogenomic and 16S rRNA phylogenetic analysis showed that strain Z330T was most closely related to P. seriniphilus NBRC 100798T and P. fistulariae KCTC 22803T. Strain Z330T grew optimally at 28-30 °C, pH 7.0-8.0 with the presence of 5.0-7.0% (w/v) NaCl. In addition, growth of strain Z330T occurred at 0.5-16% NaCl, indicated strain Z330T was a moderately halophilic and halotolerant bacterium of genus Paracoccus. The predominant respiratory quinone in strain Z330T was identified as ubiquinone-10. The major polar lipids of strain Z330T were phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, glycolipid and six unidentified polar lipids. The major fatty acids of strain Z330T was summed feature 8 (C18:1 ω6c and/or C18:1 ω7c). The draft genome sequence of strain Z330T includes 4,084,570 bp in total (N50 = 174,985 bp) with a medium read coverage of 463.6 × and 83 scaffolds. The DNA G + C content of strain Z330T was 60.5%. In silico DNA-DNA hybridization with the four type strains showed 20.5, 22.3, 20.1 and 20.1% relatedness to Paracoccus fistulariae KCTC 22803T, Paracoccus seriniphilus NBRC 100798T, Paracoccus aestuarii DSM 19484T and Paracoccus denitrificans 1A10901T, respectively. And the average nucleotide identity (ANIb) values between strain Z330T and these four type strains were 76.2, 80.0, 75.8 and 73.8%, respectively, lower than the 95-96% threshold value for dividing prokaryotic species. On the basis of the phenotypic, phylogenetic, phylogenomic and chemotaxonomic properties, a novel species of the genus Paracoccus, Paracoccus onchidii sp. nov. is proposed with the type strain Z330T (= KCTC 92727T = MCCC 1K08325T).

Prokaryotic Community Structure, Abundances, and Potential Ecological Functions in a Mars Analog Salt Lake.

Barkol Lake, situated northeast of the Tianshan Mountains, Xinjiang, is a hypersaline lake with abundant sulfate and chloride minerals, which can be a potential analog for microbial saline paleolakes on Mars. The lake water, sediments, and surrounding soils of Barkol Lake were sampled for geochemical analysis and 16S rRNA gene sequencing to investigate the prokaryotic community structure, abundances, interactions, and ecological functions. Results show that (1) prokaryotic community structure differs significantly between biotopes (water, sediment, and soil), with the highest abundances of archaea occurring in water samples and highest prokaryotic diversities in soil samples; (2) archaeal communities are dominated by Halobacterota, Nanoarchaeota, Thermoplasmatota, and Crenarchaeota, while bacterial communities are mainly Proteobacteria, Bacteroidetes, Actinobacteria, Desulfobacterota, Chloroflexi, Gemmatimonadetes, Firmicutes, and Cyanobacteria; (3) the prokaryotic community network for soil is far more complicated and stable than those for water and sediment; (4) soil prokaryotic communities could be significantly affected by environmental factors such as salinity, pH, total sulfur, and Ca2+; (5) archaeal communities may play an important role in the nitrogen cycle, while bacterial communities may mainly participate in the sulfur cycle. This study extends the data set of prokaryotic communities for hypersaline environments, which will provide perspectives into identification of the counterparts and help to understand potential microbial interactions and biogeochemical cycles occurring on Mars.

Genomic Analysis of Haloarchaea from Diverse Environments, including Permian Halite, Reveals Diversity of Ultraviolet Radiation Survival and DNA Photolyase Gene Variants.

Ultraviolet (UV) radiation responses of extremophilic and archaeal microorganisms are of interest from evolutionary, physiological, and astrobiological perspectives. Previous studies determined that the halophilic archaeon, Halobacterium sp. NRC-1, which survives in multiple extremes, is highly tolerant of UV radiation. Here, Halobacterium sp. NRC-1 UV tolerance was compared to taxonomically diverse Haloarchaea isolated from high-elevation salt flats, surface warm and cold hypersaline lakes, and subsurface Permian halite deposits. Haloterrigena/Natrinema spp. from subsurface halite deposits were the least tolerant after exposure to photoreactivating light. This finding was attributed to deviation of amino acid residues in key positions in the DNA photolyase enzyme or to the complete absence of the photolyase gene. Several Halobacterium, Halorubrum and Salarchaeum species from surface environments exposed to high solar irradiance were found to be the most UV tolerant, and Halorubrum lacusprofundi from lake sediment was of intermediate character. These results indicate that high UV tolerance is not a uniform character trait of Haloarchaea and is likely reflective of UV exposure experienced in their environment. This is the first report correlating natural UV tolerance to photolyase gene functionality among Haloarchaea and provides insights into their survival in ancient halite deposits and potentially on the surface of Mars.

HACCP plan for microbiological hazards associated with fermented crab, Episesarma mederi H. Milne Edwards 1853.

AIMS: To develop a model HACCP plan related to the microbiological hazards for the traditional fermented crab. METHODS AND RESULTS: The microbiological and chemical characteristics of commercial products were surveyed. Microbiological hazard analysis was performed for raw materials and during processing. Critical control points (CCPs) were determined using a decision tree, with CCP1 as saturated salt preparation and CCP2 as fermentation. The critical limit (CL) of CCP1 was at 100°C for 20 min applied to brining and of CCP2 was at 25% NaCl for the brine applied to fermented crab. Isolated microbial hazards and type strains were used for the validation of the CLs. Monitoring and verification of the proposed HACCP plan were carried out, and an effective HACCP plan was established. CONCLUSIONS: The HACCP plan promoted the safe consumption of fermented crab with the provided CCPs at the saturated salt preparation and fermentation steps. The effective CLs to ensure microbiological hazards as safe at the CCPs provide the best support for an effective plan. The hazards were reduced significantly after the HACCP plan had been applied.

Draft genome sequence of Joostella atrarenae M1-2T with cellulolytic and hemicellulolytic ability.

The halophilic genus Joostella is one of the least-studied genera in the family of Flavobacteriaceae. So far, only two species were taxonomically identified with limited genomic analysis in the aspect of application has been reported. Joostella atrarenae M1-2T was previously isolated from a seashore sample and it is the second discovered species of the genus Joostella. In this project, the genome of J. atrarenae M1-2T was sequenced using NovaSeq 6000. The final assembled genome is comprised of 71 contigs, a total of 3,983,942 bp, a GC ratio of 33.2%, and encoded for 3,416 genes. The 16S rRNA gene sequence of J. atrarenae M1-2T shows 97.3% similarity against J. marina DSM 19592T. Genome-genome comparison between the two strains by ANI, dDDH, AAI, and POCP shows values of 80.8%, 23.3%, 83.4%, and 74.1% respectively. Pan-genome analysis shows that strain M1-2T and J. marina DSM 19592T shared a total of 248 core genes. Taken together, strain M-2T and J. marina DSM 19592T belong to the same genus but are two different species. CAZymes analysis revealed that strain M1-2T harbors 109 GHs, 40 GTs, 5 PLs, 9 CEs, and 6 AAs. Among these CAZymes, while 5 genes are related to cellulose degradation, 12 and 24 genes are found to encode for xylanolytic enzymes and other hemicellulases that involve majorly in the side chain removal of the lignocellulose structure, respectively. Furthermore, both the intracellular and extracellular crude extracts of strain M1-2T exhibited enzymatic activities against CMC, xylan, pNPG, and pNPX substrates, which corresponding to endoglucanase, xylanase, ß-glucosidase, and ß-xylosidase, respectively. Collectively, description of genome coupled with the enzyme assay results demonstrated that J. atrarenae M1-2T has a role in lignocellulosic biomass degradation, and the strain could be useful for lignocellulosic biorefining.