Draft genome sequence of Dietzia sp. strain CH92 isolated from oil reservoir.

We report the draft genome sequence of Dietzia sp. strain CH92, isolated from a high temperature oil well in Baolige oilfield, China. The estimated genome is 3.73 Mb, with 3,479 protein-coding sequences.

Draft Genome Sequence of a Tepidicella baoligensis Strain Isolated from an Oil Reservoir.

We report the draft genome sequence of Tepidicella baoligensis strain B18-50T, isolated from a high-temperature oil well in Baolige Oilfield, China. The estimated genome is 2.87 Mb, with 2,653 protein-coding sequences.

Tepidiphilus baoligensis sp. nov., a Novel Bacterium of the Family Hydrogenophilaceae Isolated from an Oil Reservoir.

A Gram-negative, aerobic, motile, non-spore-forming and rod-shaped bacterium, designated strain B18-69 T, was isolated from oil-well production liquid in Baolige oilfield, China. The strain was able to grow at pH 6-9.5 (optimum at pH 7), in 0-4% (w/v) NaCl (optimum at 0.5-1%, w/v) and at 35-60 °C (optimum at 55 °C). Major cellular fatty acids were C16:0, C19:0 cyclo ω8c, C17:0 cyclo and C18:1 ω7c. The predominant respiratory quinone was ubiquinone 8. Major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and phosphatidylcholine (PC). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain B18-69 T was most closely related to Tepidiphilus margaritifer DSM 15129 T (98.8% similarity). The draft genome of strain B18-69 T was composed of 2,250,419 bp, and the G+C content was 64.6 mol%. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain B18-69 T and T. margaritifer DSM 15129 T were 90.9% and 68.9%, respectively. Genotypic and phenotypic features indicate that strain B18-69 T represents a novel species of the genus Tepidiphilus, for which the name Tepidiphilus baoligensis sp. nov. is proposed. The type strain is B18-69 T (= CGMCC 1.13573 T = KCTC 62782 T).

Terphenyl derivatives and terpenoids from a wheat-born mold Aspergillus candidus.

A new p-terphenyl derivative aspergicandidusin A (1), a new cleistanthane diterpenoid 6-deoxyaspergiloid C (13), and 12 known compounds (2-12, and 14) were isolated from the mold Aspergillus candidus. The structures of the new compounds were elucidated by spectral analysis of NMR and MS data. The absolute configuration of C-1 in 13 was determined via the circular dichroism data of the [Rh2(OCOCF3)4] complex. Compounds 2-8 and 11 showed moderate inhibitory activity against K562 cell lines with the IC50 value in the range from 17.9 to 46.3 µM. Compound 13 exhibited moderate cytotoxicity against HepG2 cells with the IC50 value of 47.7 µM. Compounds 11 and 12 exhibited moderate activity against the growth of S. aureus with MIC value of 6.25 µM, respectively.

Tepidicella baoligensis sp. nov., A Novel Member of Betaproteobacterium Isolated from an Oil Reservoir.

A Gram-negative, non-pigmented, aerobic bacterium, designated strain B18-50T was isolated from oil-well production water in Baolige oilfield, China. The strain was able to grow at pH 6.5-10.5 (optimum at pH 7.5-8.5), in 0-3% (w/v) NaCl (optimum at 0-0.5%, w/v) and at 20-60 °C (optimum at 45 °C). Cells of the isolate were motile with a single polar flagellum and non-spore-forming rods. Organic acids and amino acids were used as carbon and energy sources, but sugars and polyols were not assimilated. The major cellular fatty acids were C16:0, C16:1ω6c/ω7c, and C18:1ω7c. Ubiquinone 8 was the predominant respiratory quinone. The major polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The genomic DNA G+C content of the isolate was 62.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain B18-50T was most closely related to Tepidicella xavieri DSM 19605T (97.5% similarity). Comparative analysis of genotypic and phenotypic features indicate that strain B18-50T represents a novel species of the genus Tepidicella, for which the name Tepidicella baoligensis sp. nov. is proposed. The type strain is B18-50T (= CGMCC 1.13575T = KCTC 62779T).

Coralloluteibacterium thermophilus sp. nov., A Gammaproteobacterium Isolated from an Oil Reservoir.

A Gram-negative, yellow-pigmented, aerobic bacterium, designated strain B51-30T, was isolated from oil-well production liquid in Baolige oilfield, China. The strain was able to grow at pH 6-10 (optimum at pH 7.5), in 0-6% (w/v) NaCl (optimum at 1%, w/v) at 15-55 °C (optimum at 45 °C). Cells of the isolate were non-motile and non-spore-forming rods. The major cellular fatty acids were iso-C15:0, iso-C11:0, iso-C11:0 3OH, iso-C17:1 ω9c, and iso-C17:0. Ubiquinone 8 was the predominant respiratory quinone. The major polar lipids consisted of phosphatidylethanolamine and diphosphatidylglycerol. The genomic DNA G+C content of the isolate was 70.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain B51-30T was most closely related to Coralloluteibacterium stylophorae KCTC 52167T (98.7% similarity). The two strains showed DNA-DNA relatedness values of 58.5%. Genotypic and phenotypic features indicate that strain B51-30T represents a novel species of the genus Coralloluteibacterium, for which the name Coralloluteibacterium thermophilus sp. nov. is proposed. The type strain is B51-30T (= CGMCC 1.13574T = KCTC 62780T).

Decalin-Containing Tetramic Acids and 4-Hydroxy-2-pyridones with Antimicrobial and Cytotoxic Activity from the Fungus Coniochaeta cephalothecoides Collected in Tibetan Plateau (Medog).

New tetramic acid derivatives, (±)-conipyridoins A-D (1-4), conipyridoins E (5) and F (6), and new 4-hydroxy-2-pyridone alkaloids (±)-didymellamide E (7), (+)-didymellamide B (8), (+)-N-hydroxyapiosporamide (9), and didymellamides F-H (10-12) were isolated and identified from the solid culture of the fungus Coniochaeta cephalothecoides. Chiral resolution of 1, 2, 3, 4, and 7 gave five pairs of enantiomers: 1a/1b, 2a/2b, 3a/3b, 4a/4b, and 7a/7b, respectively. Stereochemistry of 1a and 1b, and 2a and 2b was established and confirmed by the single-crystal X-ray diffraction and electronic circular dichroism (ECD) methods. Absolute configuration in 3a, 3b, 4a, 4b, 7a, and 7b was assigned by ECD calculations. Compounds 1-6 possess an unprecedented chemical skeleton featuring a decalin ring and a tetramic acid moiety. Compound 11 significantly inhibited the growth of Candida albicans and Aspergillus fumigatus with minimum inhibitory concentration (MIC) of 3.13 and 1.56 µM, respectively, and was further confirmed to be a new chitin synthesis inhibitor. Compound 5 exhibited the strongest activity against the growth of both Staphylococcus aureus and MRSA with MIC value of 0.97 µM. In the light of a co-occurrence of 3-acyl tetramic acids and biogenetically related pyridine alkaloids, the biosynthetic pathway for 1-12 was postulated.

Two unexpected promiscuous activities of the iron-sulfur protein IspH in production of isoprene and isoamylene.

BACKGROUND: Bacillus species, possessing the methylerythritol phosphate (MEP) pathway for the synthesis of isoprenoid feedstock, are the highest producers of isoprene among bacteria; however, the enzyme responsible for isoprene synthesis has not been identified. The iron-sulfur protein IspH is the final enzyme of the MEP pathway and catalyses the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to form isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). In this study, we demonstrated two unexpected promiscuous activities of IspH from alkaliphilic Bacillus sp. N16-5, which can produce high levels of isoprene. RESULTS: Bacillus sp. N16-5 IspH could catalyse the formation of isoprene from HMBPP and the conversion of DMAPP into a mixture of 2-methyl-2-butene and 3-methyl-1-butene. Both reactions require an electron transfer system, such as that used for HMBPP dehydration. Isoprene and isoamylene synthesis in Bacillus sp. N16-5 was investigated and the reaction system was reconstituted in vitro, including IspH, ferredoxin and ferredoxin-NADP(+)-reductase proteins and NADPH. The roles of specific IspH protein residues were also investigated by site-directed mutagenesis experiments; two variants (H131N and E133Q) were found to have lost the HMBPP reductase activity but could still catalyse the formation of isoprene. Overexpression of IspH H131N in Bacillus sp. N16-5 resulted in a twofold enhancement of isoprene production, and the yield of isoprene from the strain expressing E133Q was increased 300% compared with the wild-type strain. CONCLUSIONS: IspH from Bacillus sp. N16-5 is a promiscuous enzyme that can catalyse formation of isoprene and isoamylene. This enzyme, especially the H131N and E133Q variants, could be used for the production of isoprene from HMBPP.

[Identification of Hydrocarbon-Oxidizing Dietzia Bacteria from Petroleum Reservoirs Based on Phenotypic Properties and Analysis of the 16S rRNA and gyrB Genes].

The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol%, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002(T), respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classiffication of strain 32d as a D. maris strain and of strain 263, as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

Global Microarray Analysis of Alkaliphilic Halotolerant Bacterium Bacillus sp. N16-5 Salt Stress Adaptation.

The alkaliphilic halotolerant bacterium Bacillus sp. N16-5 is often exposed to salt stress in its natural habitats. In this study, we used one-colour microarrays to investigate adaptive responses of Bacillus sp. N16-5 transcriptome to long-term growth at different salinity levels (0%, 2%, 8%, and 15% NaCl) and to a sudden salt increase from 0% to 8% NaCl. The common strategies used by bacteria to survive and grow at high salt conditions, such as K+ uptake, Na+ efflux, and the accumulation of organic compatible solutes (glycine betaine and ectoine), were observed in Bacillus sp. N16-5. The genes of SigB regulon involved in general stress responses and chaperone-encoding genes were also induced by high salt concentration. Moreover, the genes regulating swarming ability and the composition of the cytoplasmic membrane and cell wall were also differentially expressed. The genes involved in iron uptake were down-regulated, whereas the iron homeostasis regulator Fur was up-regulated, suggesting that Fur may play a role in the salt adaption of Bacillus sp. N16-5. In summary, we present a comprehensive gene expression profiling of alkaliphilic Bacillus sp. N16-5 cells exposed to high salt stress, which would help elucidate the mechanisms underlying alkaliphilic Bacillus spp. survival in and adaptation to salt stress.

Identification and characterization of a novel salt-tolerant esterase from a Tibetan glacier metagenomic library.

A salt-tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p-nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three-dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat-resistant features.

Cloning, expression, and characterization of a novel alkali-tolerant xylanase from alkaliphilic Bacillus sp. SN5.

A xylanase gene (xyn11A) was cloned from the genomic library of alkalophilic Bacillus sp. SN5. It encoded a polypeptide of 366 amino acids, consisting of a family 11 glycoside hydrolase, a short linker region, and a family 36 carbohydrate-binding module (CBM). The intact xylanase Xyn11A and the CBM-linker-truncated Xyn11A-LC were expressed in Escherichia coli BL21 (DE3). Both purified recombinant proteins exhibited the highest activity at 55 °C. The optimal pH for Xyn11A activity was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5-8.5) with optimal activity at pH 5.5 and 7.5-8.0. They had high alkali tolerance, retaining over 80% residual activity after preincubation at pH 8.5-11.0 at 37 °C for 1 H. Xyn11A-LC showed better thermal stability, lower affinity, and lower catalytic activity to insoluble xylan than Xyn11A, whereas its specific activity for soluble beechwood xylan (4,511.9 U/mg) was greater than that of Xyn11A (3,136.4 U/mg). These results implied that the CBM of Xyn11A could change the enzymatic properties and play a role in degrading insoluble xylan. Xyn11A-LC is a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which qualifies it as a potential candidate for industrial applications, especially in the paper industry.

[A novel gene (Aa-accA ) encoding acetyl-CoA carboxyltransferase alpha-subunit of Alkalimonas amylolytica N10 enhances salt and alkali tolerance of Escherichia coli and tobacco BY-2 cells].

OBJECTIVE: Acetyl-CoA carboxylase (ACC) catalyzes the first step of fatty acid synthesis. In most bacteria, ACC is composed of four subunits encoded by accA, accB, accC, and accD. Of them, accA encodes acetyl-CoA carboxyltransferase alpha-subunit. Our prior work on proteomics of Alkalimonas amylolytica N10 showed that the expression of the Aa-accA has a remarkable response to salt and alkali stress. This research aimed to find out the Aa-accA gene contributing to salt and alkali tolerance. METHODS: The Aa-accA was amplified by PCR from A. amylolytica N10 and expressed in E. coli K12 host. The effects of Aa-accA expression on the growth of transgenic strains were examined under different NaCl concentration and pH conditions. Transgenic tobacco BY-2 cells harboring Aa-accA were also generated via Agrobacterium-mediated transformation. The viability of BY-2 cells was determined with FDA staining method after salt and alkali shock. RESULTS: The Aa-accA gene product has 318 amino acids and is homologous to the carboxyl transferase domain of acyl-CoA carboxylases. It showed 76% identity with AccA (acetyl-CoA carboxylase carboxyltransferase subunit alpha) from E. coli. Compared to the wild-type strains, transgenic E. coli K12 strain containing Aa-accA showed remarkable growth superiority when grown in increased NaCl concentrations and pH levels. The final cell density of the transgenic strains was 2.6 and 3.5 times higher than that of the control type when they were cultivated in LB medium containing 6% (W/V) NaCl and at pH 9, respectively. Complementary expression of Aa-accA in an accA-depletion E. coli can recover the tolerance of K12 delta accA to salt and alkali stresses to some extent. Similar to the transgenic E. coli, transgenic tobacco BY-2 cells showed higher percentages of viability compared to the wild BY-2 cells under the salt or alkali stress condition. CONCLUSION: We found that Aa-accA from A. amylolytica N10 overexpression enhances the tolerance of both transgenic E. coli and tobacco BY-2 cells to NaCl and alkali stresses.

Cloning, expression and characterization of a novel salt-tolerant xylanase from Bacillus sp. SN5.

A xylanase gene (xyn10A) was cloned from Bacillus sp. SN5 and expressed in Escherichia coli. It encoded a 348-residue polypeptide of ~45 kDa. The deduced amino acid sequence had 68 % identity with the endo-1,4-beta-xylanase from Paenibacillus lactis 154 that belonged to family 10 of the glycoside hydrolases. Purified recombinant Xyn10A had maximum activity at 40 °C and pH 7.0, with the specific activity of 105 U/mg and a Km of 0.6 mg/ml for beechwood xylan. Xyn10A retained more than 80 % activity between 25 and 45 °C and 29 % activity at 5 °C. It exhibited the highest activity (134 %) in 0.5 M NaCl and still retained 90 % activity in 2.5 M NaCl. It retained about 87 % activity after incubation in 2 M NaCl for 24 h. The cold-active and halo-tolerant properties of Xyn10A make it promising for application in the food industry, especially in the processing of saline food and sea food.

A thermostable esterase from Thermoanaerobacter tengcongensis opening up a new family of bacterial lipolytic enzymes.

An unidentified α/ß hydrolase gene lipA3 from thermostable eubacterium species Thermoanaerobacter tengcongensis MB4 was cloned and heterologously expressed by Escherichia coli BL21(DE3)pLysS. The purified recombinant enzyme EstA3 turned out to be a monomeric thermostable esterase with optimal activity at 70°C and pH 9.5. The enzyme showed lipolytic activity towards a wide range of ester substrates including p-nitrophenyl esters and triacylglycerides, with the highest activity being observed for p-nitrophenyl caproate at 150 U/mg and for Triacetin at 126U/mg, respectively. Phylogenetic analysis revealed that EstA3 did not show homology to any identified bacterial lipolytic hydrolases. Sequence alignment showed that there was a common pentapeptide CHSMG with a cysteine replacing the first glycine in most esterase and lipase conserved motif GXSXG. The catalytic triad of EstA3 is Ser92, Asp269 and His292, which was confirmed by site directed mutagenesis. Based on the enzymatic properties and sequence alignment we concluded that the esterase EstA3 represented a novel bacterial lipolytic enzyme group and in chronological order this group was assigned as Family XIV.

[Purification and characterization of mannanase from an alkaliphilic mannanase producing bacterium HMTS15].

OBJECTIVE: To isolate and identify an alkaliphilic mannanase producing bacterium, purify and characterize mannanase thereof. METHODS: Mannanase-producing alkaliphilic bacterium HMTS15 was isolated by alkaline agar with konjak from water sample of Hamatai Lake in Inner Mongolia, China. The morphological, biochemical and physiological characteristics and 16S rRNA gene were analyzed to identify the taxonomic position of strain HMTS15. Mannanase produced by strain HMTS15 was purified by four steps including (NH4)2SO4 precipitation, cellulose DEAE-sepharose, twice Superdex 200. The enzyme properties including optimal temperature, optimal pH, thermal stability, pH stability, NaCl tolerance, metal ion tolerance, EDTA and SDS tolerance were tested. RESULTS: Strain HMTS15 was Gram-positive rod. Its growth pH ranged from 7.0 to 11.0 and growth temperature ranged from 10 degrees C to 45 degrees C. The G + C content of the DNA was 40 mol%. Phylogenetic analyses based on 16S rRNA gene sequence comparisons indicated that strain HMTS15 was a member of Bacillus. The extracellular mannanase from strain HMTS15 was purified as a single band with molecular weight of about 45 kD on SDS-PAGE. The optimal catalytic activity was showed at 75 degrees C and pH 10. The mananase was stable up to 60 degrees C and retained about 60% residual activity at 65 degrees C for 30 min. The ions Fe2+, Mn2+, Co2+, Zn2+, Ag+, Hg2+ and EDTA inhibited the acitivity of the mannanase. CONCLUSION: Polyphasic taxonomy revealed that strain HTMS15 was a new member of Bacillus agaradhaerens. The alkaline mannanase produced by strain HMTS15 hold the valuable property in stability at high temperature and broad range of pH.

Cloning, expression, and characterization of a highly active alkaline pectate lyase from alkaliphilic Bacillus sp. N16-5.

An alkaline pectate lyase, Bsp165PelA, was purified to homogeneity from the culture broth of alkaliphilic Bacillus sp. N16-5. The enzyme showed a specific activity as high as 1000 U/mg and had optimum activity at pH 11.5 and 50 degrees . It was composed of a single polypeptide chain with a molecular of 42 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 6.0. It could efficiently depolymerize polygalacturonate and pectin. Characterization of product formation revealed unsaturated digalacturonate and trigalacturonate as the main product. The pectate lyase gene (pelA) contained an open reading frame (ORF) of 1089 bp, encoding a 36-amino acids signal peptide and a mature protein of 326 amino acids with a calculated molecular mass of 35.943 Da. The deduced amino acid sequence from the pelA ORF exhibited significant homology to those of known pectate lyases in polysaccharide lyase family 1. Some conserved active-site amino acids were found in the deduced amino acid sequence of Bsp165PelA. Ca2+ was not required for activity on pectic substrates.

[Isolation, identification and enzyme profiling of an alkaliphilic protease producing bacterium].

OBJECTIVE: To isolate and characterize alkaliphilic bacteria with protease activity. METHODS: Protease-producing alkaliphiles were isolated by skim milk agar method. The morphological, biochemical and physiological characteristics, 16S rRNA gene sequence and DNA-DNA hybridization were determined to identify the taxonomic position of strain ZL223. Meanwhile, the enzyme characterization of the protease including optimal temperature and pH range, stability and oxidation resistance were tested by using casein hydrolysis test. RESULTS: A new strain named ZL223 was isolated from Bange Lake in Tibet, China. Cells were Gram-positive, spore-forming, motile rods. Growth pH range was from 7.0 to 10.0, optimum at pH 9.0. Growth temperature range was from 15 degrees C to 45 degrees C, optimum at 37 degrees C. The G + C content of DNA was 40.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Bacillus and most closely related to Bacillus pseudofirmus OF4 with 98.6% sequence similarity. DNA-DNA hybridization analysis confirmed the close relationship of strain ZL223 with Bacillus pseudofirmu OF4 (86% relatedness). Strain ZL223 produced extracellular alkaline protease and its maximal enzyme activity was observed at 40 degrees C and pH 12.0. CONCLUSION: Base on polyphasic taxonomy, strain ZL223 was a new member of Bacillus pseudofirmus. It produced alkaline protease which was worth further research.

Cloning, expression, and characterization of a thermostable glucoamylase from Thermoanaerobacter tengcongensis MB4.

A thermostable glucoamylase (TtcGA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli. The full-length gene (2112 bp) encodes a 703-amino acid polypeptide including a predicted signal peptide of 21 residues. The recombinant mature protein was partially purified to 30-fold homogeneity by heat treatment and gel filtration chromatography. The mature protein is a monomer with the molecular weight of 77 kD. The recombinant enzyme showed maximum activity at 75 degrees C and pH 5.0. It is the most thermostable bacterial glucoamylase described to date with nearly no activity loss after incubation at 75 degrees C for 6 h. TtcGA can hydrolyze both alpha-1, 4- and alpha-1, 6-glycosidic linkages in various alpha-glucans. It showed preference for maltooligosaccharides over polysaccharides with specific activity of 80 U/mg towards maltose. Kinetic studies revealed that TtcGA had the highest activity on maltooligosaccharide with four monosaccharide units. The cations Ca2+, Mn2+, Co2+, Mg2+, and reducing agent DTT showed no obvious effects on the action of TtcGA. In contrast, the enzyme was inactivated by Zn2+, Pb2+, Cu2+, and EDTA.

Exploring membrane and cytoplasm proteomic responses of Alkalimonas amylolytica N10 to different external pHs with combination strategy of de novo peptide sequencing.

Identification of differentially proteomic responses to external pHs would pave an access for understanding of survival mechanisms of bacteria living at extreme pH environment. We cultured Alkalimonas amylolytica N10 (N10), a novel alkaliphilic bacterium found in Lake Chahannor, in media with three different pHs and extracted the correspondent membrane and cytoplasm proteins for proteomic analysis through 2-DE. The differential 2-DE spots corresponding to the altered pHs were delivered to MALDI TOF/TOF MS for protein identification. Since the genomic data of strain N10 was unavailable, we encountered a problem at low rate of protein identification with 18.1%. We employed, therefore, a combined strategy of de novo sequencing to analyze MS/MS signals generated from MALDI TOF/TOF MS. A significantly improved rate of protein identification was thus achieved at over than 70.0%. Furthermore, we extensively investigated the expression of these pH-dependent N10 genes using Western blot and real-time PCR. The conclusions drawn from immunoblot and mRNA measurements were mostly in agreement with the proteomic observations. We conducted the bioinformatic analysis to all the pH-dependent N10 proteins and found that some membrane proteins participated in iron transport were differentially expressed as external pH elevated and most of differential proteins with increased or bell-shape mode of pH-dependence were involved in bioenergetic process and metabolism of carbohydrates, fatty acid, amino acids, and nucleotides. Our data thus provide a functional profile of the pH-responsive proteins in alkaliphiles, leading to elucidation of alkaliphilic-adaptive mechanism.