Analysis of the genome of an alkaliphilic Bacillus strain from an industrial point of view

Bacillus species and other microbes with pH optima for growth higher than pH 9 are defined as alkaliphiles. A large number of alkaliphilic Bacillus strains producing useful enzymes, have been isolated from various environments. Some of these enzymes, such as proteases and cellulases from alkaliphilic Bacillus strains, have been commercialized and have brought great advantages to industry and domestic life. To support further development of the enzyme industry, we initiated analysis of the genome of Bacillus halodurans C-125, which is 4.25 Mb in size, and constructed a physical and genetic map for comparison with the Bacillus subtilis chromosome. Systematic sequencing of the whole genome of Bacillus halodurans C-125 has been automated since the beginning of May 1998, and sequencing of 98% of the whole genome has been done so far. Through genome analysis, it became apparent that the genome organization of alkaliphilic Bacillus halodurans C-125 is totally different from that of B. subtilis orthologues. Received: July 11, 1999 / Accepted: December 27, 1999     

Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis

The 4 202 353 bp genome of the alkaliphilic bacterium Bacillus halodurans C-125 contains 4066 predicted protein coding sequences (CDSs), 2141 (52.7%) of which have functional assignments, 1182 (29%) of which are conserved CDSs with unknown function and 743 (18.3%) of which have no match to any protein database. Among the total CDSs, 8.8% match sequences of proteins found only in Bacillus subtilis and 66.7% are widely conserved in comparison with the proteins of various organisms, including B.subtilis. The B.halodurans genome contains 112 transposase genes, indicating that transposases have played an important evolutionary role in horizontal gene transfer and also in internal genetic rearrangement in the genome. Strain C-125 lacks some of the necessary genes for competence, such as comS, srfA and rapC, supporting the fact that competence has not been demonstrated experimentally in C-125. There is no paralog of tupA, encoding teichuronopeptide, which contributes to alkaliphily, in the C-125 genome and an ortholog of tupA cannot be found in the B.subtilis genome. Out of 11 σ factors which belong to the extracytoplasmic function family, 10 are unique to B.halodurans, suggesting that they may have a role in the special mechanism of adaptation to an alkaline environment.     

Cloning and characterization of dihydrofolate reductase from a facultative alkaliphilic and halotolerant bacillus strain

Elucidation of the molecular basis of the stability of enzymes from extremophilic organisms is of fundamental importance for various industrial applications. Due to the wealth of structural data from various species, dihydrofolate reductase (DHFR, EC 1.5.1.3) provides an excellent model for systematic investigations. In this report, DHFR from alkaliphilic Bacillus halodurans C-125 was cloned and expressed in E. coli. Functional analyses revealed that BhDHFR exhibits the most alkali-stable phenotype of DHFRs characterized so far. Optimal enzyme activity was observed in a slightly basic pH region ranging from 7.25 to 8.75. Alkali-stability is associated with a remarkable resistance to elevated temperatures (half-life of 60 min at 52.5°C) and to high concentrations of urea (up to 3 M). Although the secondary structure shows distinct similarities to those of mesophilic DHFR molecules, BhDHFR exhibits molecular features contributing to its alkaliphilic properties. Interestingly, the unique phenotype is diminished by C-terminal addition of a His-tag sequence. Therefore, His-tag-derivatized BhDHFR offers the opportunity to obtain deeper insights into the specific mechanisms of alkaliphilic adaption by comparison of the three dimensional structure of both BhDHFR molecules.     

Digestion of Algal Biomass for Electricity Generation in Microbial Fuel Cells

We developed a semi-automated genome analysis system called GAMBLER in order to support the current whole-genome sequencing project focusing on alkaliphilic Bacillus halodurans C-125. GAMBLER was designed to reduce the human intervention required and to reduce the complications in annotating thousands of ORFs in the microbial genome. GAMBLER automates three major routines: analyzing assembly results provided by genome assembler software, assigning ORFs, and homology searching. GAMBLER is equipped with an interface for convenience of annotation. All processes and options are manipulatable through a WWW browser that enables scientists to share their genome analysis results without choosing computer platforms.     

Genetic analysis of the chromosome of alkaliphilic Bacillus halodurans C-125

Seventeen Sse8387I linking clones isolated from the chromosome of Bacillus halodurans C-125 for the purpose of constructing a physical map were sequenced and analyzed by comparison with the BSORF database and the nonredundant protein databank. The orientations of Sse8387I or AscI linking clones serving to join adjacent fragments were determined by southern blot analysis using specific DNA probes. One-third of the open reading frames (ORFs) identified in the Sse8387I linking clones showed no significant similarity to any protein so far reported. The ORFs showing significant similarities to those of Bacillus subtilis were mapped in the chromosome of strain C-125, and the locations of the putative genes on the map were not well conserved between B. halodurans C-125 and B. subtilis. Received: March 26, 1999 / Accepted: April 27, 1999     

Isolation and characterization of new facultative alkaliphilic Bacillus flexus strains from maize processing waste water (nejayote)

Aims: This work describes the isolation and characterization of two new alkaliphilic micro‐organisms present in nejayote. Methods and Results: Samples of fresh industrial nejayote were plated on nejayote medium and incubated for 4 days at 37°C. Isolates were identified based on morphological and physiological characteristics, as well as 16S rDNA sequence analysis. Two gram‐positive strains, NJY2 and NJY4, able to hydrolyse starch, xylan, and gelatin were isolated from nejayote. Comparative sequence analysis of 16S rDNA and phylogenetic studies indicate that the micro‐organisms studied were closely related to members of the Bacillus flexus species. The strains were identified as facultative alkaliphilic salt tolerant bacteria. Isolate NJY2 produced cell associated phenolic acid esterases, able to release ferulic acid from nixtamalised corn bran and ethyl and methyl esters. Conclusions: The isolated strains of B. flexus NJY2 and NJY4 showed important physiological properties to produce high‐value molecules from agroindustrial by‐products. Significance and Impact of the Study: This is the first report about the isolation of alkaliphilic micro‐organisms from nejayote and the first report of phenolic acid esterases synthesised by alkaliphiles. The new alkaliphilic micro‐organisms have potential application in the treatment and transformation of tortilla industry residues.     

Characterization of a thermostable xylanase from an alkaliphilic Bacillus sp.

A xylanase gene (xyn10) from alkaliphilic Bacillus sp. N16-5 was cloned and expressed in Pichia pastoris. The deduced amino acid sequence has 85% identity with xylanase xyn10A from B. halodurans and contains two potential N-glycosylation sites. The glycosylated Xyn10 with MW 48 kDa can hydrolyze birchwood and oatspelt xylan. The enzyme had optimum activity at pH 7 and 70°C, with the specific activity of 92.5U/mg. The Xyn10 retained over 90% residual activity at 60°C for 30 min but lost all activity at 80°C over 15 min. Most tested ions showed no or slight inhibition effects on enzyme activity.     

Antibacterial activity of cyclodextrins against <Emphasis Type="Italic">Bacillus</Emphasis> strains

Growth of alkaliphilic Bacillus halodurans C-125 both on agar plates and in liquid culture was inhibited by methyl-β-cyclodextrin (CD). Furthermore, resting cells of the strain were lysed by contact with methyl-β-CD higher than 10 mM. α-CD also showed lysis activity against Bacillus and related strains. The activity was not observed with Gram-negative and Gram-positive bacteria except for Bacillus strains. Fluorescence staining and scanning electron microscopy of cells revealed that methyl-β-CD disrupted cell membranes, and consequently, the cells were lysed. This is a novel physiological property of CDs.     

Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database.     

Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris

A gene encoding a lipolytic enzyme amplified from the alkaliphilic bacterium Bacillus halodurans LBB2 was cloned into the pPICZαB vector and integrated into the genome of the protease deficient yeast strain Pichia pastoris SMD1168H. This previously undescribed enzyme was produced in active form, and cloning in frame with the Saccharomyces cerevisiae secretion signal (α-factor) enabled extracellular accumulation of correctly processed enzyme, with an apparent molecular mass of 30 kDa. In shake-flask cultivations, very low production levels were obtained, but these were significantly improved by use of a “batch-induced” cultivation technique which allowed a maximum enzyme activity of 14,000 U/l using p-nitrophenyl butyrate (C-4) as a substrate and a final extracellular lipolytic enzyme concentration of approximately 0.2 g/l. Partial characterization of the produced enzyme (at pH 9) revealed a preference for the short-chain ester (C-4) and significant but lower activity towards medium (C5-C6) and long (C16 and C18) fatty acid chain-length esters. In addition, the enzyme exhibited true lipase activity (7,300 U/l) using olive oil as substrate and significant levels of phospholipase activity (6,400 U/l) by use of a phosphatidylcholine substrate, but no lysophospholipase activity was detected using a lysophosphatidylcholine substrate.     

High-Level Heterologous Expression of Bacillus halodurans Putative Xylanase Xyn11A (BH0899) in Kluyveromyces lactis

The putative xyn11A structural gene (BH0899) encoding a family-11 xylanase from alkaliphilic Bacillus halodurans strain C-125 was heterologously expressed in the yeast Kluyveromyces lactis CBS 1065 and secreted to a level of 156 μg/ml under selective culture conditions in shake flasks. The Xyn11A production level in shake flask cultures of K. lactis CBS 1065 was higher than that reported for other xylanase genes placed under the control of the regulated LAC4 promoter on a plasmid containing an entire sequence of pKD1 from Kluyveromyces drosophilarium. Recombinant Xyn11A was highly active over pH range from 3 to 10, with maximal activity around pH 7. The enzyme showed a specific activity of 628 U/mg-protein on birchwood xylan as substrate, but no cellulase or β-xylosidase activity.     

Studies on the respiratory system in alkaliphilic Bacillus; a proposed new respiratory mechanism

Respiratory electron transfer systems in two alkaliphilic Bacillus species, YN-1 and YN-2000, were investigated. In the cyanide-sensitive pathway of the obligate alkaliphilic Bacillus YN-1, the terminal enzyme was a caa ₃-type cytochrome c oxidase constituting up to just 10% of the total oxygen-reducing activity, while 90% of the respiratory activity was due to cyanide-insensitive, nonproteinaceous material with a molecular weight of 662. These results were consistent with the cyanide-tolerant growth of the bacterium. The molecular and catalytic properties of the nonproteinaceous material were not identical with those of menaquinones extracted from the bacterium. Furthermore, the nonproteinaceous material was also found in the facultative alkaliphilic Bacillus YN-2000, when that bacterium was cultivated in alkaline conditions. A new respiratory oxygen-reducing mechanism comprising a nonproteinaceous component and a catalase is proposed for these alkaliphilic Bacillus species. Received: October 31, 1997 / Accepted: December 17, 1997     

Alkaline extracellular reduction: isolation and characterization of an alkaliphilic and halotolerant bacterium, Bacillus pseudofirmus MC02

Aims: To isolate an alkaliphilic bacterium and to investigate its ability of extracellular reduction. Methods and Results: An alkaliphilic and halotolerant humus‐reducing anaerobe, Bacillus pseudofirmus MC02, was successfully isolated from a pH 10·0 microbial fuel cell. To examine its ability of extracellular reduction, AQDS (anthraquinone‐2, 6‐disulfonae), humic acids (HA) and Fe(III) oxides were chosen as representative electron acceptors. All the experiments were conducted in a pH 9·5 carbonate buffer. The results are as follows: (i) Sucrose, lactate, glucose and glycerol were the favourable electron donors for AQDS reduction by the strain MC02; (ii) The strain had the ability of reducing HA in the presence of sucrose; (iii) It could effectively reduce Fe(III) oxides coupled with sucrose fermentation when AQDS was added as electron shuttle and its Fe(III) reducing capacity ranked as: lepidocrocite (γ‐FeOOH) > goethite (α‐FeOOH) > haematite(α‐Fe₂O₃); (iv) The strain could decolourize azo dye Orange I. Conclusions: Bacillus pseudofirmus MC02 was capable of extracellular reduction in AQDS, HA and Fe(III) oxides, and it can be used for decolourizing azo dye (Orange I) in alkaline conditions. Significance and Impact of the Study: This is the first report of an alkaliphlic strain of B. pseudofirmus capable of extracellular reduction in AQDS, HA, Fe(III) oxides and decolourization of Orange I. This study could provide valuable information on alkaline biotransformation in the printing and dyeing wastewater and saline‐alkali soil.     

Applicability of thermo-alkali-stable and cellulase-free xylanase from a novel thermo-halo-alkaliphilic Bacillus halodurans in producing xylooligosaccharides

An alkaliphilic, moderately thermophilic and halophilic bacterial isolate capable of producing a high titer of extracellular thermo-alkali-stable, cellulase-free endoxylanase was isolated from the paper mill effluents. It was identified as Bacillus halodurans. The purified xylanase was active from pH 7 to 12 and 30 to 100°C with optimal activity at pH 9.0 and 80°C. It had T_1/2 values of 40 and 15 min at 70 and 80°C, respectively. Activity was stimulated by dithiothreitol but strongly inhibited by N-bromosuccinimide. Its action on birchwood xylan and agro-residues liberated xylooligosaccharides of 2–7 degree of polymerization, and thus, the mode of action is similar to endoxylanases of the family 10 glucoside hydrolases.     

Characterization of a new rhamnogalacturonan acetyl esterase from Bacillus halodurans C-125 with a new putative carbohydrate binding domain

BH1115 is a gene from Bacillus halodurans strain C-125 that hypothetically encodes a rhamnogalacturonan acetyl esterase (RGAE) of the CE-12 family. As confirmation, this gene was cloned, and the product was expressed in Escherichia coli strain Rosetta (DE3) cells and purified. The enzyme obtained was monomeric, with a molecular mass of 45 kDa, and exhibited alkaliphilic properties. A study of the inhibition of the activity by some modulators confirmed that the catalytic triad for the esterase activity was Ser-His-Asp. This enzyme also presents broad substrate specificity and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, β-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, RGAE from B. halodurans achieves a synergistic effect with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common α/β hydrolase fold. The homology between the folds of RGAE from Aspergillus aculeatus and the hypothetical YxiM precursor from Bacillus subtilis, which both belong to the SGNH family, illustrates the divergence of such proteins from a common ancestor. Furthermore, the enzyme possesses a putative substrate binding region at the N terminus of the protein which has never been described to date for any RGAE.     

Isolation and characterization of two serine proteases from metagenomic libraries of the Gobi and Death Valley deserts

Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an l-rhamnose isomerase (l-RhI). The identified l -RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k _cat of 8,971 min⁻¹ and a k _cat/K _m of 17 min⁻¹ mM⁻¹ for l-rhamnose. Although l-RhIs have been characterized from several other sources, B. halodurans l-RhI is distinguished from other l-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans l-RhI a good choice for industrial applications. This work describes one of the most thermostable l-RhI characterized thus far.     

A cloned Bacillus halodurans multicopper oxidase exhibiting alkaline laccase activity

The gene product of open reading frame bh2082 from Bacillus halodurans C-125 was identified as a multicopper oxidase with potential laccase activity. A homologue of this gene, lbh1, was obtained from a B. halodurans isolate from our culture collection. The encoded gene product was expressed in Escherichia coli and showed laccase-like activity, oxidising 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid), 2,6-dimethoxyphenol and syringaldazine (SGZ). The pH optimum of Lbh1 with SGZ is 7.5–8 (at 45°C) and the laccase activity is stimulated rather than inhibited by chloride. These unusual properties make Lbh1 an interesting biocatalyst in applications for which classical laccases are unsuited, such as biobleaching of kraft pulp for paper production.     

TEMPERATURE EFFECTS ON MICROALGAL PHOTOSYNTHESIS‐LIGHT RESPONSES MEASURED BY O2 PRODUCTION,PULSE‐AMPLITUDE‐MODULATED FLUORESCENCE,AND 14C ASSIMILATION1

Short‐term temperature effects on photosynthesis were investigated by measuring O₂ production, PSII‐fluorescence kinetics, and ¹⁴C‐incorporation rates in monocultures of the marine phytoplankton species Prorocentrum minimum (Pavill.) J. Schiller (Dinophyceae), Prymnesium parvum f. patelliferum (J. C. Green, D. J. Hibberd et Pienaar) A. Larsen (Coccolithophyceae), and Phaeodactylum tricornutum Bohlin (Bacillariophyceae), grown at 15°C and 80 μmol photons · m^?2 · s^?1. Photosynthesis versus irradiance curves were measured at seven temperatures (0°C–30°C) by all three approaches. The maximum photosynthetic rate (P^C_max) was strongly stimulated by temperature, reached an optimum for Pro. minimum only (20°C–25°C), and showed a similar relative temperature response for the three applied methods, with Q₁₀ ranging from 1.7 to 3.5. The maximum light utilization coefficient (α^C) was insensitive or decreased slightly with increasing temperature. Absolute rates of O₂ production were calculated from pulse‐amplitude‐modulated (PAM) fluorometry measurements in combination with biooptical determination of absorbed quanta in PSII. The relationship between PAM‐based O₂ production and measured O₂ production and ¹⁴C assimilation showed a species‐specific correlation, with 1.2–3.3 times higher absolute values of P^C_max and α^C when calculated from PAM data for Pry. parvum and Ph. tricornutum but equivalent for Pro. minimum. The offset seemed to be temperature insensitive and could be explained by a lower quantum yield for O₂ production than the theoretical maximum (due to Mehler‐type reactions). Conclusively, the PAM technique can be used to study temperature responses of photosynthesis in microalgae when paying attention to the absorption properties in PSII.     

Physical map of alkaliphilic Bacillus firmus OF4 and detection of a large endogenous plasmid

Extremely alkaliphilic Bacillus firmus OF4 is among the best characterized of this group of alkaliphiles. Together with alkaliphilic Bacillus C-125 and numerous non-alkaliphilic Bacillus species whose chromosomes and gene organizations are currently being studied in detail, work on B. firmus OF4 offers the opportunity to discern whether there are features of chromosome and gene organization that are associated with alkaliphily. A physical map of the B. firmus OF4 is consistent with a circular chromosome of approximately 4 Mb, with an extrachromosomal element of 110 kb also detected. The previously identified cadmium-resistance locus and transposition functions in B. firmus OF4 were localized to the extrachromosomal element, whose genes exhibit a slightly different pattern of codon usage from chromosomal genes. No clustering of genes thus far identified with roles in alkaliphily has been found. Direct repeat sequences (DRS) were previously reported upstream of a gene encoding a Na⁺/H⁺ antiporter that has a role in pH homeostasis. In the current analyses, these sequences were found to be present in multiple copies on the chromosome, most of which are present in one 920-kb fragment. Such sequences might play a role in DNA rearrangements that allow amplification of important genes in this region. Received: March 3, 1998 / Accepted May 12, 1998