A two-gene ABC-type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore

A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na at elevated pH, was deficient in energy-dependent Na extrusion. The capacity of the mutant JC901 for Na -dependent pH homeostasis was unaffected relative to the wild-type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3 -terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane-spanning regions at its C-terminus, and exhibits modest sequence similarity to regions of eukaryotic Na⁺/H⁺ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP-binding proteins that are components of transport systems of the ATP-binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natAB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p-chlorophenylhydrazone (CCCP), and, more modestly, by Na⁺, and K⁺, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na⁺-sensitive phe-notype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol-resistance does not result from NatAB-dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na⁺     

A Bacillus subtilis locus encoding several gene products affecting transport of cations

A 3.6-kb DNA fragment from Bacillus subtilis was found to complement the K⁺ uptake-deficient Escherichia coli strain TK2420. Transformation with a pKLO61 plasmid harboring this fragment conferred the capacity to grow on a minimal medium containing only 10 mM K⁺. Insertional mutagenesis and subcloning identified a single gene responsible for the complementation. This gene coded for an apparent homolog of E. coli TrkA. Sequence analysis of the cloned region also revealed three additional open reading frames. These included: a gene encoding a homolog to the czcD gene product of Alcaligenes eutrophus, a lysR-type regulatory gene which was found to enhance Na⁺ resistance in E. coli NM81 (ΔnhaA) in a separate complementation test, and an orfD with no significant similarity to sequences deposited in Genbank.     

pH homeostasis and ATP synthesis: studies of two processes that necessitate inward proton translocation in extremely alkaliphilic Bacillus species

Alkaliphilic Bacillus species that are isolated from nonmarine, moderate salt, and moderate temperature environments offer the opportunity to explore strategies that have developed for solving the energetic challenges of aerobic growth at pH values between 10 and 11. Such bacteria share many structural, metabolic, genomic, and regulatory features with nonextremophilic species such as Bacillus subtilis. Comparative studies can therefore illuminate the specific features of gene organization and special features of gene products that are homologs of those found in non-extremophiles, and potentially identify novel gene products of importance in alkaliphily. We have focused our studies on the facultative alkaliphile Bacillus firmus OF4, which is routinely grown on malate-containing medium at either pH 7.5 or 10.5. Current work is directed toward clarification of the characteristics and energetics of membrane-associated proteins that must catalyze inward proton movements. One group of such proteins are the Na⁺/H⁺ antiporters that enable cells to adapt to a sudden upward shift in pH and to maintain a cytoplasmic pH that is 2–2.3 units below the external pH in the most alkaline range of pH for growth. Another is the proton-translocating ATP synthase that catalyzes robust production of ATP under conditions in which the external proton concentration and the bulk chemiosmotic driving force are low. Three gene loci that are candidates for Na⁺/H⁺ antiporter encoding genes with roles in Na⁺- dependent pH homeostasis have been identified. All of them have homologs in B. subtilis, in which pH homeostasis can be carried out with either K⁺ or Na⁺. The physiological importance of one of the B. firmus OF4 loci, nhaC, has been studied by targeted gene disruption, and the same approach is being extended to the others. The atp genes that encode the alkaliphile's F₁F_O-ATP synthase are found to have interesting motifs in areas of putative importance for proton translocation. As an initial step in studies that will probe the importance and possible roles of these motifs, the entire atp operon from B. firmus OF4 has been cloned and functionally expressed in an Escherichia coli mutant that has a full deletion of its atp genes. The transformant does not exhibit growth on succinate, but shows reproducible, modest increases in the aerobic growth yields on glucose as well as membrane ATPase activity that exhibits characteristics of the alkaliphile enzyme. Received: January 22, 1998 / Accepted: February 16, 1998     

The Na+/H+ antiporter of alkaliphilic Bacillus sp.

The Na⁺/H⁺ antiporter, which appears to predominantly contribute to the alkaliphily of Bacillus halodurans C-125, was studied in an alkali-sensitive mutant of this strain and a transformant with restored alkaliphily. The alkali-sensitive mutant, strain 38154, which has lost the ability to grow above pH 9.5, was found to lack electro-genic Na⁺/H⁺ antiport activity driven by ΔΨ (membrane potential, interior negative), and it showed defective regulation of intracellular pH under alkaline conditions. On the other hand, a transformant carrying a 2.0-kb DNA fragment from the parental genome that complemented this defect was able to maintain an intracellular pH lower than that of the external milieu, and it was found to have recovered the Na⁺/H⁺ antiport activity driven by ΔΨ. Sequence analyses found that a 5.1-kb DNA region contained four open reading frames (ORF-1 to ORF-4). Direct sequencing of the corresponding region in mutant 38154 revealed a G-to-A substitution, which resulted in an amino acid substitution from Gly-393 to Arg in the putative ORF-1 product. It has been recently found that a region homologous to the DNA fragment responsible for the alkaliphily of strain C-125 exists in the genomes of Bacillus subtilis, Sinorhizobium (Rhizobium) meliloti, and Staphylococcus aureus. These homologues are present as a cluster of seven ORFs in each case. The shaA gene product of B. subtilis shows significant similarity to the ORF-1 product of strain C-125. Disruption of the shaA gene resulted in a decrease in Na⁺/H⁺ antiport activity, and growth of the shaA-disrupted strain was impaired when the external Na⁺ concentration was increased. We conclude that the shaA gene encodes a Na⁺/H⁺ antiporter, which plays an important role in extrusion of cytotoxic Na⁺. Received: May 29, 2000 / Accepted: July 18, 2000     

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     

Diverse genes of alkaliphilic Bacillus firmus OF4 that complement K+-uptake-deficient Escherichia coli include an ftsH homologue

Seven clones isolated from libraries of DNA from alkaliphilic Bacillus firmus OF4 restored the growth of a K⁺-uptake-deficient Escherichia coli mutant on only 10mM K⁺. None of the clones contained genes with apparent homology to known K⁺ transport systems in other organisms. Based on sequence homologies, the newly isolated alkaliphile loci included: ftsH; a dipeptide transport system; a gerC locus with hydrophobic open reading frames not found in the comparable locus of Bacillus subtilis; a sugar phosphotransferase enzyme; and a capBC homologue. The ftsH gene provided a new and striking example of a recognized property of extracellular and external regions of polytopic alkaliphile proteins: a significant paucity of basic amino acid residues relative to neutrophile counterparts. The alkaliphile ftsH gene was able to complement a mutant of E. coli with a temperature-sensitive ftsH gene product. Received: 5 August 1996 / Accepted: 14 October 1996     

The Na transport in gram-positive bacteria defect in the Mrp antiporter complex measured with Na nuclear magnetic resonance

²³Na nuclear magnetic resonance (NMR) has previously been used to monitor Na⁺ translocation across membranes in gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na⁺. In this work, the ²³Na NMR method was adapted for measurements of internal Na⁺ concentration in the gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na⁺ translocation activity of the Mrp (multiple resistance and pH) antiporter complex, a member of the cation proton antiporter-3 (CPA-3) family. The sodium-sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted could indeed be correlated to the inability of this strain to maintain a lower internal Na⁺ concentration than an external one.     

NhaK,a novel monovalent cation/H<Superscript>+</Superscript> antiporter of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Four Na⁺/H⁺ antiporters, Mrp, TetA(L), NhaC, and MleN have so far been described in Bacillus subtilis 168. We identified an additional Na⁺/H⁺ antiporter, YvgP, from B. subtilis that exhibits homology to the cation: proton antiporter-1 (CPA-1) family. The yvgP-dependent complementation observed in a Na⁺(Ca²⁺)/H⁺ antiporter-defective Escherichia coli mutant (KNabc) suggested that YvgP effluxed Na⁺ and Li⁺. In addition, effects of yvgP expression on a K⁺ uptake-defective mutant of E. coli indicated that YvgP also supported K⁺ efflux. In a fluorescence-based assay of everted membrane vesicles prepared from E. coli KNabc transformants, YvgP-dependent Na⁺ (K⁺, Li⁺, Rb⁺)/H⁺ antiport activity was demonstrated. Na⁺ (K⁺, Li⁺)/H⁺ activity was higher at pH 8.5 than at pH 7.5. Mg²⁺, Ca²⁺ and Mn²⁺ did not serve as substrates but they inhibited YvgP antiport activities. Studies of yvgP expression in B. subtilis, using a reporter gene fusion, showed a significant constitutive level of expression that was highest in stationary phase, increasing as stationary phase progressed. In addition, the expression level was significantly increased in the presence of added K⁺ and Na⁺.     

Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

Summary The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu⁺ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B·leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI^* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI^* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu ⁺. However, B. subtilis ilvB and ilvC auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. -Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.     

recE-Dependent Gene Amplification Induced by a Tunicamycin-resistant Mutation (tmr A7) in Bacillus subtilis

A recombinant Bacillus subtilis phage, ρ11-AA248, contains the tmr A7-amy R2-amy E⁺-tmr B⁺-aroI⁺ region of the B. subtilis N7 chromosome on a 22.4 kb DNA fragment. The amy E⁺-tmr B⁺ gene region in the phage genome of the B. subtilis 207-21 transductants by ρ11-AA248 was amplified to approximately 10 copies after cultivation in the presence of tunicamycin (10 μg/ml) and to two copies without tunicamycin. The amplification of the gene region caused hyper-production of extracellular α-amylase. In contrast, no amplification of the gene region was detected in the transductants of B. subtilis 207-25, a recE-deficient derivative of 207-21 strain.     

Reidentification of facultatively alkaliphilic Bacillus firmus OF4 as Bacillus pseudofirmus OF4

With a view toward verifying the original classification of alkaliphilic Bacillus firmus OF4, physiological and biochemical characteristics were more extensively catalogued than in original studies, and this catalog was supplemented with 16S rDNA sequence homology and more extensive DNA–DNA hybridization analyses. Phylogenetic analysis of this alkaliphile based on the comparison of multiple 16S rDNA sequences from Bacillus species indicated that this strain is most closely related to Bacillus pseudofirmus. Consistently, in the DNA–DNA hybridization analysis of the alkaliphile and Bacillus reference strains, the highest level of DNA–DNA relatedness (96%) was found between the alkaliphile and the B. pseudofirmus type strain (DSM 8715^T). The findings support the conclusion that this alkaliphile strain is more closely related to B. pseudofirmus than to B. firmus, and we propose the future use of the designation B. pseudofirmus OF4. Received: April 20, 1999 / Accepted: August 31, 1999     

Identification of cry-Type Genes on 20-kb DNA Associated with Cry1 Crystal Proteins from Bacillus thuringiensis

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.     

High-level expression of an endoxylanase gene from Bacillus sp. in Bacillus subtilis DB104 for the production of xylobiose from xylan

To produce xylobiose from xylan, high-level expression of an endoxylanase gene from Bacillus sp. was carried out in Bacillus subtilis DB104. A 1.62-kb SmaI DNA fragment, coding for an endoxylanase of Bacillus sp., was ligated into the Escherichia coli/B. subtilis shuttle vector pJH27Δ88, producing pJHKJ4, which was subsequently transformed into B. subtilis DB104. A maximum endoxylanase activity of 105 U/ml was obtained from the supernatant of B. subtilis DB104 harboring pJHKJ4. The endoxylanase was purified to homogeneity by ion-exchange chromatography and the production profile of xylooligosaccharides from xylan by the endoxylanase was examined by HPLC with a carbohydrate analysis column. Xylobiose was the major product from xylan at 40 °C and its proportion in the xylan hydrolyzates increased with the reaction time; at 12 h, over 60% of the reaction products was xylobiose. These results suggest that xylobiose, which has a stimulatory effect on the selective growth of the intestinal bacterium Bifidobacterium, can be mass-produced effectively by the endoxylanase of Bacillus sp. cloned in B. subtilis. Received: 2 January 1998 / Received revision: 4 March 1998 / Accepted: 4 March 1998     

Multifarious potential applications of keratinase of Bacillus subtilis K-5

Abstract

Bacillus subtilis K-5, an isolate from compost, utilized a wide range of keratinous wastes viz. diverse feather types, nails, hair, scales, etc. for growth and produced a thermostable alkaline protease (keratinase) with broad proteolytic activity. Optimization of cultural and environmental variables using a Plackett–Burman design and response surface methodology resulted in enhanced keratinase production (89%). Keratinase was partially purified (15-fold) by ammonium sulfate precipitation and carboxymethyl cellulose chromatography. The optimum pH and temperature for keratinase activity were 9.0 and 60°C, however, considerable activity and stability was observed over broad pH (5–10) and temperature range (50–90°C). B. subtilis K-5 keratinase exhibited excellent stability toward detergents (cetyl trimethylammonium bromide, Tween 80, and sodium dodecyl sulfate) and organic solvents (benzene, acetonitrile, phenylmethylsulfonyl fluoride); however, metal ions like Mn²⁺, Cu²⁺, Na⁺, Hg²⁺, K⁺, Ca²⁺, and Zn²⁺ inhibited the activity. B. subtilis K-5 protease showed remarkable potential for diverse applications like blood stain removal, gelatin hydrolysis from waste X-ray films and dehairing of animal hide.     

Cloning and expression inEscherichia coli of the sucrase gene fromBacillus subtilis

Summary A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank ofE. coli harboring recombinant cosmids representative of theB. subtilis genome. It was shown that thesacA gene is located in a 2 kbEcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2 kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for inB. subtilis andE. coli. Complementation of asacA mutation was observed in Rec⁺ and Rec^- strains ofB. subtilis. Expression of sucrase was also demonstrated inE.coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic inE. coli since the presence of the recombinant plasmids did not allow the transport of [U¹⁴C] sucrose and the growth of the cells.It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport ofB. subtilis.     

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15–20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a λ phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis. Received: April 17, 1998 / Accepted: June 23, 1998     

Restriction of plasmid-mediated transformation in Bacillus subtilis 168.

Summary When plasmids carrying leucine genes of Bacillus subtilis 168 were isolated from a restriction and modification deficient (r^-m^-) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec ⁺ strain (transformation by integration of the donor DNA into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r⁺ or r^- phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r^-m^- strains should be used for construction of recombinant DNA molecules in B. subtilis 168.