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.

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.

Salipaludibacillus daqingensis sp. nov., a moderately halophilic bacterium isolated from an oilfield.

A novel alkaliphilic, Gram-stain-positive, moderately halophilic, rod-shaped, endospore-forming, motile, facultatively anaerobic bacterium (DQ-9T) was isolated from a sediment sample collected from Daqing oilfield in China, and characterized by a polyphasic taxonomic approach. Strain DQ-9T formed yellow pigment and grew occurred at salinities of 1-12 % (w/v) NaCl (optimum, 8 %) and at 10-40 °C (optimum, 30-35 °C), at pH 7.5-10.5 (optimum, pH 9.0-9.5). It was catalase-positive, but oxidase-negative. Based on the analysis of 16S rRNA gene sequences, DQ-9T was classified into the genus Salipaludibacillus and exhibited the highest similarities (98.37 %) to Salipaludibacillus neizhouensis JSM 071004T. Digital DNA-DNA hybridization and average nucleotide identity values between strain DQ-9T and the most closely related strain, S. neizhouensis DSM 19794T, were determined to be 72.0 and 21.6 %, respectively. The polar lipids were constituted by diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids (>5 %) comprised anteiso-C15 : 0, anteiso-C17 : 0, iso-C17 : 0, iso-C15 : 0 and C16 : 0. The cell-wall peptidoglycan contained meso-diaminopimelic acid, and menaquinone-7 was identified as the primary respiratory quinone. The DNA G+C content was 37.5 mol%. Through chemotaxonomic, physiological, and biochemical characterization, strain DQ-9T could be clearly distinguished from the closest Salipaludibacillus species. Based on provided data, strain DQ-9T is proposed to represent a novel species, Salipaludibacillus daqingensis sp. nov., within the genus Salipaludibacillus. The type strain is DQ-9T (=ACCC 60415T=KCTC 33936T).

Quantitative evaluation of endogenous reference genes for ddPCR under salt stress using a moderate halophile.

Droplet digital PCR (ddPCR) is being increasingly adopted for gene detection and quantification because of its higher sensitivity and specificity. According to previous observations and our laboratory data, it is essential to use endogenous reference genes (RGs) when investigating gene expression at the mRNA level under salt stress. This study aimed to select and validate suitable RGs for gene expression under salt stress using ddPCR. Six candidate RGs were selected based on the tandem mass tag (TMT)-labeled quantitative proteomics of Alkalicoccus halolimnae at four salinities. The expression stability of these candidate genes was evaluated using statistical algorithms (geNorm, NormFinder, BestKeeper and RefFinder). There was a small fluctuation in the cycle threshold (Ct) value and copy number of the pdp gene. Its expression stability was ranked in the vanguard of all algorithms and was the most suitable RG for quantification of expression by both qPCR and ddPCR of A. halolimnae under salt stress. Single RG pdp and RG combinations were used to normalize the expression of ectA, ectB, ectC and ectD under four salinities. The present study constitutes the first systematic analysis of endogenous RG selection for halophiles responding to salt stress. This work provides a valuable theory and an approach reference of internal control identification for ddPCR-based stress response models.

Alteribacter keqinensis sp. nov., a moderately halophilic bacterium isolated from a soda lake.

A Gram-stain-positive, aerobic, endospore-forming and rod-shaped bacterium (KQ-3T), which grew at 10-45 °C (optimum 35 °C), pH 8.0-10.5 (optimum pH 9.0) and in the presence of 0-16 % (w/v) NaCl (optimum 3.0 %), was isolated from a soda lake and identified as representing a novel species using a polyphasic taxonomic approach. Strain KQ-3T was catalase-positive, oxidase-negative and non-motile. Phylogenetic analysis based on 16S rRNA gene sequence affiliated KQ-3T to the genus Alteribacter and showed the highest similarities to Alteribacter natronophilus M30T (97.90 %), Alteribacter aurantiacus K1-5T (97.84 %) and Alteribacter populi FJAT-45347T (97.22 %). Digital DNA-DNA hybridization and average nucleotide identity analyses revealed that KQ-3T displayed 21.4 and 72.81% genomic DNA relatedness with the most closely related strain, A. natronophilus M30T, respectively. KQ-3T contained all of the conserved signature indels that are specific for members of the genus Alteribacter. The DNA G+C content was 45.03 mol%. The cell-wall peptidoglycan contained meso-diaminopimelic acid and the polar lipids consisted of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and one unidentified phospholipid. The predominant menaquinone was MK-7 (100%) and the major fatty acids (>10 %) comprised anteiso-C15 : 0, iso-C15 : 0 and iso-C16 : 0. Based on the data from the current polyphasic studies, KQ-3T represents a novel species of the genus Alteribacter, for which the name Alteribacter keqinensis sp. nov. is proposed. The type strain is KQ-3T (=ACCC 61799T=KCTC 33933T).

Leucobacter chinensis sp. nov., with plant growth-promoting potential isolated from field soil after seven-years continuous maize cropping.

A novel Gram-stain-positive, aerobic, non-motile and rod-shaped bacterium, designated strain NC76-1T, was isolated from soil from a field that had undergone seven years continuous maize cropping from Liuba town located in Zhangye city, Gansu province, PR China. Colonies of strain NC76-1T were white, opaque and circular with a convex shape. The isolate was found to be able to grow at 10-40 °C (optimum 30 °C), pH 6.0 to 12.0 (optimum 7.0-8.0) and with 0-5.0 % (w/v) NaCl (optimum 0%). On the basis of the results of 16S rRNA gene sequence analysis, the strain fell within the clade of the genus Leucobacter, showing the highest sequence similarities with Leucobacter iarius 40T (97.4%), Leucobacter aridicollis CIP 108388T (97.0%), Leucobacter chromiireducens subsp. solipictus TAN 31504T (96.7%) and Leucobacter denitrificans M1T8B10T (96.7%). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between NC76-1T and its closest relatives, L. iarius 40T, L. aridicollis CIP 108388T, L. chromiireducens subsp. solipictus TAN 31504T and L. denitrificans M1T8B10T were ≤73.5 % and 20.3%, respectively. The genomic DNA G+C content of NC76-1T was 61.5 mol%. It presented MK-11 as the predominant menaquinone. The major cellular fatty acids were anteiso-C15 : 0 (49.2 %) and iso-C16 : 0 (35.7%). The major polar lipids were found to be diphosphatidyglycerol, phosphatidylglycerol, phosphatidylethanolamine, aminoglycolipid, five glycolipid and one unidentified lipids. The cell wall amino acids were 2,4-diaminobutyric acid, alanine, glutamic acid, glycine and threonine. On the basis of the phylogenetic, phenotypic and chemotaxonomic characteristics, strain NC76-1T is concluded to represent a novel species within the genus Leucobacter, for which the name Leucobacter chinensis sp. nov. is proposed. The type strain is NC76-1T (GDMCC 1.2286T= JCM 34651T).

Bacillus lacisalsi sp. nov., a moderately haloalkaliphilic bacterium isolated from a saline-alkaline lake.

An alkaliphilic and moderately halophilic strain, designated YSP-3T, characterised by optimal growth at pH 9.0 and at 8.0% (w/v) NaCl, was isolated from Yangshapao Lake, Jilin Province, China. Cells of this strain is Gram-positive, straight rods and form a central or sub-terminal ellipsoidal endospore. Phylogenetic analysis based on 16S rRNA gene sequences indicated that it was grouped in the genus Bacillus with Bacillus aurantiacus K1-5T and Bacillus populi FJAT-45347T as the close relative (97.5 and 97.2% 16S rRNA gene sequence similarity, respectively). Genomic relatedness between strain YSP-3T and its close relative was evaluated using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity with the values of 70.3-85.1%, 19.7-20.1% and 71.5-71.6%, respectively. Comparative genomics analysis showed that strain YSP-3T has distinct amino acid bias and significantly differences from foreign invasion events during evolution relative to the reference strains. Cell-wall peptidoglycan contains meso-diaminopimelic acid. The predominant polar lipids are phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol. The predominant quinone is menaquinone-7. The major fatty acids of strain YSP-3T are anteiso-C15:0, iso-C15:0, iso-C16:0, anteiso-C17:0 and Iso-C14:0. DNA G + C content of strain YSP-3T is 48.3 mol%. Based on genomics analysis, physiological, biochemical and chemotaxonomic data, strain YSP-3T represent a novel species, for which the name Bacillus lacisalsi sp. nov. is proposed. The type strain is YSP-3T (  = ACCC 60365T = KCTC 33934T).

Salipaludibacillus keqinensis sp. nov., a moderately halophilic bacterium isolated from a saline-alkaline lake.

A novel Gram-stain positive, short rod, forming sub-terminal endospores of ellipsoidal shape, halophilic, alkaliphilic and aerobic bacterium, designated strain KQ-12T, was isolated from a saline-alkaline lake in China, and characterised by a polyphasic taxonomic approach. The isolate grew at 4-40 °C (optimum, 25 °C), at pH 8.0-10.0 (pH 9.0) and in the presence of 0-16% (w/v) NaCl (8%). 16S rRNA gene sequence similarity of KQ-12T to species in the genera Salipaludibacillus ranged from 96.6 to 98.1%. Phylogenetic trees indicated that the strain should be assigned to the genus Salipaludibacillus. The polar lipids of KQ-12T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unidentified phospholipid and its major cellular fatty acids were anteiso-C15:0, anteiso-C17:0, iso-C15:0, and C16:0. The isoprenoid quinone was MK-7. These key chemotaxonomic properties also confirmed the affiliation of the strain to the genus Salipaludibacillus. However, some physiological, biochemical properties, low average nucleotide identity and low digital DNA-DNA hybridization relatedness values enabled the strain to be differentiated from closely related species of the genus Salipaludibacillus. Thus, KQ-12T can be classified as a novel species in the genus Salipaludibacillus, for which the name Salipaludibacillus keqinensis sp. nov. is proposed. The type strain is KQ-12T ( =  ACCC 60430T   =  KCTC 33935T).

Glycine Betaine Monooxygenase, an Unusual Rieske-Type Oxygenase System, Catalyzes the Oxidative N-Demethylation of Glycine Betaine in Chromohalobacter salexigens DSM 3043.

Although some bacteria, including Chromohalobacter salexigens DSM 3043, can use glycine betaine (GB) as a sole source of carbon and energy, little information is available about the genes and their encoded proteins involved in the initial step of the GB degradation pathway. In the present study, the results of conserved domain analysis, construction of in-frame deletion mutants, and an in vivo functional complementation assay suggested that the open reading frames Csal_1004 and Csal_1005, designated bmoA and bmoB, respectively, may act as the terminal oxygenase and the ferredoxin reductase genes in a novel Rieske-type oxygenase system to convert GB to dimethylglycine in C. salexigens DSM 3043. To further verify their function, BmoA and BmoB were heterologously overexpressed in Escherichia coli, and 13C nuclear magnetic resonance analysis revealed that dimethylglycine was accumulated in E. coli BL21(DE3) expressing BmoAB or BmoA. In addition, His-tagged BmoA and BmoB were individually purified to electrophoretic homogeneity and estimated to be a homotrimer and a monomer, respectively. In vitro biochemical analysis indicated that BmoB is an NADH-dependent flavin reductase with one noncovalently bound flavin adenine dinucleotide (FAD) as its prosthetic group. In the presence of BmoB, NADH, and flavin, BmoA could aerobically degrade GB to dimethylglycine with the concomitant production of formaldehyde. BmoA exhibited strict substrate specificity for GB, and its demethylation activity was stimulated by Fe2+ Phylogenetic analysis showed that BmoA belongs to group V of the Rieske nonheme iron oxygenase (RO) family, and all the members in this group were able to use quaternary ammonium compounds as substrates.IMPORTANCE GB is widely distributed in nature. In addition to being accumulated intracellularly as a compatible solute to deal with osmotic stress, it can be utilized by many bacteria as a source of carbon and energy. However, very limited knowledge is presently available about the molecular and biochemical mechanisms for the initial step of the aerobic GB degradation pathway in bacteria. Here, we report the molecular and biochemical characterization of a novel two-component Rieske-type monooxygenase system, GB monooxygenase (BMO), which is responsible for oxidative demethylation of GB to dimethylglycine in C. salexigens DSM 3043. The results gained in this study extend our knowledge on the catalytic reaction of microbial GB degradation to dimethylglycine.

Massilia violaceinigra sp. nov., a novel purple-pigmented bacterium isolated from glacier permafrost.

A Gram-stain-negative, motile and rod-shaped bacterium, designated strain B2T, which can synthesize purple pigments of violacein and dexyoviolacein, was isolated from Tianshan glacier in Xinjiang, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that it was grouped in the genus Massilia with Massilia glaciei B448-2T, Massilia eurypsychrophila B528-3T and Massilia psychrophila B1555-1T as its closest relatives (98.2, 97.9 and 97.0 % 16S rRNA gene sequence similarity, respectively). Genomic relatedness between strain B2T and its closest relatives was evaluated using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity, with values of 77.93-85.08 %, 22.4-23.4 % and 71.54-72.99 %, respectively. Q-8 was the major ubiquinone. The major fatty acids (>5 %) of strain B2T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C12 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The major polar lipids included phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content of strain B2T was 63.51 mol%. Based on genomic relatedness, physiological, biochemical and chemotaxonomic data, strain B2T (=CGMCC 1.6993T=DSM 19531T=KCTC 32446T) is considered to represent a novel species within the genus Massilia, for which the name Massilia violaceinigra sp. nov. is proposed.

Natronolimnobius aegyptiacus sp. nov., an extremely halophilic alkalithermophilic archaeon isolated from the athalassohaline Wadi An Natrun, Egypt.

An obligately aerobic extremely halophilic alkalithermophilic archaeon, strain JW/NM-HA 15T, was isolated from the sediments of Wadi An Natrun in Egypt. Phylogenetic analysis based on 16S rRNA and rpoB' gene sequences indicated that it belongs to the family Natrialbaceae of the order Natrialbales. The closest relatives were Natronolimnobius baerhuensis IHC-005T and Natronolimnobius innermongolicus N-1311T (95.3 and 94.5 % 16S rRNA gene sequence similarity, respectively). Genome relatedness between strain JW/NM-HA 15T and its neighbours was evaluated using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity with the values of 75.7-85.0, 18.1-20.0, and 70.2-71.0%, respectively. Cells were obligately aerobic, rod-shaped, non-motile, Gram-stain-negative and chemo-organotrophic. The strain grew in the presence of 2.57 M to saturating Na+ (optimum 3.25-4.60 M Na+), at pH55 °C 7.5-10.5 (optimum pH55 °C 9.0-9.5), and at 30-56 °C (optimum 52 °C). The major polar lipids consisted of phosphatidylglycerol, methylated phosphatidylglycerolphosphate and two phospholipids. The complete genome size of strain JW/NM-HA 15T is approximately 3.93 Mb, with a DNA G+C content of 64.1 mol%. On the basis of phylogenetic features, genomic relatedness, phenotypic and chemotaxonomic data, strain JW/NM-HA 15T was thus considered to represent a novel species within the genus Natronolimnobius, for which the name Natronolimnobius aegyptiacus sp. nov. is proposed. The type strain is JW/NM-HA 15T (=ATCC BAA-2088T =DSM 23470T).

Reclassification of Bacillus saliphilus as Alkalicoccus saliphilus gen. nov., comb. nov., and description of Alkalicoccus halolimnae sp. nov., a moderately halophilic bacterium isolated from a salt lake.

A Gram-stain-positive, cocci-shaped, non-spore-forming and moderately halophilic bacterium, designed BZ-SZ-XJ29T, was isolated from a salt lake of China. On the basis of 16S rRNA gene sequence similarity, the closest phylogenetic relatives were Bacillus saliphilus 6AGT (97.3 % 16S rRNA gene sequence similarity) and five other species of the genus Bacillus(95.4-96.3 %). However, strain BZ-SZ-XJ29T shared only 89.5 % 16S rRNA gene sequence similarity with Bacillus subtilis subsp. subtilis DSM 10T, indicating that this isolate might not be a member of the genus Bacillus. The genomic DNA G+C content was 40.0 mol% (Tm). The DNA-DNA relatedness value with B. saliphilus 6AGT was 45±2 %. Strain BZ-SZ-XJ29T formed yellow pigment and grew in the presence of 0.74-4.15 M Na+ [optimum 1.42-2.10 M Na+], at pH 6.0-10.5 (optimum pH 7.5), and at 5-41 °C (optimum 33 °C). The predominant (>10 %) fatty acids were anteiso-C15 : 0 and anteiso-C15 : 0. The dominant polar lipids consisted of diphosphatidylglycerol and the respiratory quinone was menaquinone-7 (MK-7). The peptidoglycan type of the cell wall was A1γ, based on meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of the combined phylogenetic data, phenotypic features and chemotaxonomic properties, it is proposed that B. saliphilus and strain BZ-SZ-XJ29T should be assigned to a single novel genus as two separate species. Bacillus. saliphilus is reclassified in a new genus, Alkalicoccus gen. nov., as Alkalicoccus saliphilus comb. nov., and is the type species of the new genus; the type strain of the type species is 6AGT (=DSM 15402T=ATCC BAA-957T). Strain BZ-SZ-XJ29T (=DSM 29191T=JCM 30193T=CGMCC 1.12936T) is placed in the genus Alkalicoccus as a novel species, Alkalicoccus halolimnae sp. nov.

Establishment of a markerless gene deletion system in Chromohalobacter salexigens DSM 3043.

Chromohalobacter salexigens DSM 3043 can grow over a wide range of salinity, which makes it as an excellent model organism for understanding the mechanism of prokaryotic osmoregulation. Functional analysis of C. salexigens genes is an essential way to reveal their roles in cellular osmoregulation. However, the lack of an effective markerless gene deletion system has prevented construction of multiple gene deletion mutants for the members in the genus. Here, we report the development of a markerless gene deletion system in C. salexigens using allelic exchange method. In this system, the in vitro mutant allele of target gene was inserted into a pK18mobsacB-based integrative vector pMDC21, which contained a chloramphenicol resistance cassette as the positive selection marker and a sacB gene from Bacillus subtilis as the counterselectable marker. To validate this system, two single-gene deletion mutants and a double-gene deletion mutant were constructed. In addition, our results showed that growth of the merodiploids and sucrose screening at 25 °C were more effective to decrease the occurrence of spontaneous sucrose resistance colonies than at higher temperature (30 or 37 °C), and growth of the merodiploids in mineral salt medium instead of the complex medium was critical to increase the recovery rate of deletion mutants.

Bacillusurumqiensis sp. nov., a moderately haloalkaliphilic bacterium isolated from a salt lake.

A Gram-stain-positive, rod-shaped, aerobic and moderately haloalkaliphilic bacterium, designated BZ-SZ-XJ18T, was isolated from the mixed water and sediment of a saline-alkaline lake located in the Xinjiang Uyghur Autonomous Region of China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BZ-SZ-XJ18T was a member of the genus Bacillus. The closest phylogenetic relatives were Bacillus saliphilus 6AGT (96.7 % 16S rRNA gene sequence similarity), 'Bacillusdaqingensis' X10-1 (96.6 %), Bacillus luteus JC167T (96.5 %), Bacillus daliensis DLS13T (96.2 %), Bacillus chagannorensis CG-15T (95.2 %) and Bacillus polygoni YN-1T (95.0 %). DNA-DNA relatedness between strain BZ-SZ-XJ18T and the reference type strains of the related species of the genus Bacillus was lower than 27 %. The isolate formed yellow pigment and grew in the presence of 0.22-4.32 M Na+ (equivalent to 1.3-25.3 %, w/v, NaCl) (optimum 1.08 M Na+, equivalent to 6.3 %, w/v, NaCl), at pH 6.5-10.0 (optimum pH 8.5-9.5) and at 8-41 ºC (optimum 37 ºC). The major cellular fatty acids were anteiso-C15:0 (43.0 %), C16:0 (18.1 %), iso-C15:0 (11.3 %), anteiso-C17:0 (8.0 %) and iso-C16:0 (7.0 %). The major polar lipids consisted of diphosphatidylglycerol and phosphatidylglycerol. The main respiratory quinone was menaquinone-7 (MK-7), and the peptidoglycan type of the cell wall was A1γ based on meso-diaminopimelic acid as the diagnostic diamino acid. The genomic DNA G+C content was 42.3 mol% (HPLC) or 41.4 mol% (Tm). On the basis of phenotypic, chemotaxonomic and phylogenetic features, strain BZ-SZ-XJ18T is proposed to represent a novel species, Bacillusurumqiensis within the genus Bacillus. The type strain is BZ-SZ-XJ18T (=DSM 29145T=JCM 30195T).

Halomonas urumqiensis sp. nov., a moderately halophilic bacterium isolated from a saline-alkaline lake.

A moderately halophilic, aerobic bacterium, strain BZ-SZ-XJ27T, belonging to the genus Halomonas, was isolated from a saline-alkaline lake in the Xinjiang Uyghur Autonomous Region of China. Phylogenetic analysis based on 16S rRNA gene sequences and a multilocus sequence analysis using the 16S rRNA, gyrB and rpoD genes demonstrated that strain BZ-SZ-XJ27T represents a member of the genus Halomonas. On the basis of 16S rRNA gene sequence similarity, the closest relatives were Halomonas campaniensis 5AGT, H. fontilapidosi 5CRT, H. korlensis XK1T and H. sinaiensis ALO SharmT, with similarities of 96.2-97.2 %. DNA-DNA hybridization with H. korlensis CGMCC 1.6981T (the nearest phylogenetic neighbour) and H. campaniensis DSM 15293T (the highest 16S rRNA gene sequence similarity) showed relatedness values of 53 and 38 %, respectively, demonstrating the separateness of the three taxa. The bacterium stained Gram-negative and the cells were motile and rod-shaped. The strain formed creamy-white colonies and grew under optimal conditions of 1.42 M Na+ (range 0.22-4.32 M Na+), pH 8.0-8.5 (range pH 6.0-10.0) and 39 °C (range 4-43 °C). The dominant fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c; 36.6 %), C16 : 0 (25.9 %) and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c; 21.2 %). The dominant polar lipids were two unknown phospholipids, phosphatidylethanolamine and phosphatidylglycerol, and the main respiratory quinones were ubiquinone 9 (Q-9; 89 %) and ubiquinone 8 (Q-8; 10 %). The genomic DNA G+C content was 61.7 ± 0.8 mol% (Tm). On the basis of phenotypic, chemotaxonomic and phylogenetic features, strain BZ-SZ-XJ27T is proposed to represent a novel species, Halomonas urumqiensis sp. nov., within the genus Halomonas of the family Halomonadaceae. The type strain is BZ-SZ-XJ27T ( = JCM 30202T = CGMCC 1.12917T).

Identification of important charged residues for alkali cation exchange or pH regulation of NhaH, a Na(+)/H(+) antiporter of Halobacillus dabanensis.

NhaH is a novel Na(+)/H(+) antiporter identified from the moderate halophile Halobacillus dabanensis. In this study, six conserved charged residues located in the putative transmembrane segments (TMS) including TMSV, TMSVI, TMSVIII and TMSXI of NhaH as well as two His residues in Loop III were replaced by site-directed mutagenesis for the identification of their potential roles in the antiport activity and pH regulation. Substitutions D137A, D166A and R325A caused a complete loss of Na(+)(Li(+))/H(+) antiport activity, revealing that D137, D166 and R325 are indispensable for the antiport activity. Substitution D137E led to a significant increase of the apparent Km values for Na(+) and Li(+) without affecting the changes of pH profile, confirming that D137 plays vital roles in alkali cation binding/translocation. Substitution D166E resulted in not only a significant increase of the apparent Km values for Na(+) and Li(+) but also an alkaline shift of pH profile, suggesting that D166 is involved in alkali cation binding/translocation as well as H(+) binding or pH regulation. Substitutions E161N, D224A and D224E caused a significant increase of Km for Na(+) and Li(+), indicating that E161 and D224 partly contribute to alkali cation binding/translocation. Substitution E229K caused an over 50% elevation of the apparent Km for Li(+), without affecting that for Na(+), suggesting that E229 may be mainly responsible for Li(+) binding/translocation. Substitutions H87A and H88A resulted in an acidic shift of pH profile without an effect on Km for Na(+) and Li(+), indicating that H87 and H88 are involved in H(+) binding or pH regulation.

How could haloalkaliphilic microorganisms contribute to biotechnology?

Haloalkaliphiles are microorganisms requiring Na(+) concentrations of at least 0.5 mol·L(-1) and an alkaline pH of 9 for optimal growth. Their unique features enable them to make significant contributions to a wide array of biotechnological applications. Organic compatible solutes produced by haloalkaliphiles, such as ectoine and glycine betaine, are correlated with osmoadaptation and may serve as stabilizers of intracellular proteins, salt antagonists, osmoprotectants, and dermatological moisturizers. Haloalkaliphiles are an important source of secondary metabolites like rhodopsin, polyhydroxyalkanoates, and exopolysaccharides that play essential roles in biogeocycling organic compounds. These microorganisms also can secrete unique exoenzymes, including proteases, amylases, and cellulases, that are highly active and stable in extreme haloalkaline conditions and can be used for the production of laundry detergent. Furthermore, the unique metabolic pathways of haloalkaliphiles can be applied in the biodegradation and (or) biotransformation of a broad range of toxic industrial pollutants and heavy metals, in wastewater treatment, and in the biofuel industry.

Long-term fertilization coupled with rhizobium inoculation promotes soybean yield and alters soil bacterial community composition.

Microbial diversity is an important indicator of soil fertility and plays an indispensable role in farmland ecosystem sustainability. The short-term effects of fertilization and rhizobium inoculation on soil microbial diversity and community structure have been explored extensively; however, few studies have evaluated their long-term effects. Here, we applied quantitative polymerase chain reaction (qPCR) and amplicon sequencing to characterize the effect of 10-year fertilizer and rhizobium inoculation on bacterial communities in soybean bulk and rhizosphere soils at the flowering-podding and maturity stages. Four treatments were examined: non-fertilization control (CK), phosphorus and potassium fertilization (PK), nitrogen and PK fertilization (PK + N), and PK fertilization and Bradyrhizobium japonicum 5821 (PK + R). Long-term co-application of rhizobium and PK promoted soybean nodule dry weight by 33.94% compared with PK + N, and increased soybean yield by average of 32.25%, 5.90%, and 5.00% compared with CK, PK, and PK + N, respectively. The pH of PK + R was significantly higher than that of PK and PK + N at the flowering-podding stage. The bacterial abundance at the flowering-podding stage was positively correlated with soybean yield, but not at the maturity stage. The significant different class Gemmatimonadetes, and the genera Gemmatimonas, and Ellin6067 in soil at the flowering-podding stage were negatively correlated with soybean yield. However, the bacterial community at class and genus levels at maturity had no significant effect on soybean yield. The key bacterial communities that determine soybean yield were concentrated in the flowering-podding stage, not at maturity stage. Rhizosphere effect, growth period, and treatment synergies resulted in significant differences in soil bacterial community composition. Soil organic matter (OM), total nitrogen (TN), pH, and available phosphorus (AP) were the main variables affecting bacterial community structure. Overall, long-term co-application of rhizobium and fertilizer not only increased soybean yield, but also altered soil bacterial community structure through niche reconstruction and microbial interaction. Rhizobium inoculation plays key role in reducing nitrogen fertilizer application and promoting sustainable agriculture practices.