Pleiotropic mutations affecting sporulation conditions and the syntheses of extracellular enzymes in Bacillus subtilis 168

Pleiotropic mutations of the chromosome of Bacillus subtilis 168 affecting simultaneously the levels of extracellular levansucrase and proteolytic activities are described. These mutations have been mapped at the sacU locus identified by PBS 1 mediated transduction. Several pleotropic hyperproducers and pleiotropic hypoproducers of these extracellular enzymatic activities, genotypically designated sacU^h and sacU⁻ respectively, have been isolated. sacU^h mutants are capable of sporulation in rich media or in mineral media containing amino acids in the presence of an excess of glucose in both cases; under these conditions the sporulation of the wild type strain 168 is inhibited. One pleiotropic mutation conferring hyperproduction of levansucrase and proteolytic activities was mapped at the sacQ locus distant from sacU.The sacU and sacQ mutants may be affected in a not yet identified regulation mechanism which controls simultaneously the production of several extracellular enzymatic activities and the sporulation conditions of B. subtilis 168.     

Chromosomal location of mutations affecting sucrose metabolism in Bacillus subtilis Marburg

Summary Mutations affecting sucrose metabolism have been mapped by PBS1 transduction on the Bacillus subtilis chromosome in seven loci sacA, sacB, sacQ, sacR, sacS, sacT and sacU. sacA and sacB are presumed to be the structural genes of a sucrase and a levansucrase respectively. sacR, sacS and sacT correspond to groups of mutations leading to constitutive synthesis of sucrase or both sucrase and levansucrase. In sacQ, sacS and sacU are located either mutations increasing the level of synthesis of levansucrase specifically (sacQ ^h , sacS ^h , sacU ^h ) or mutations abolishing specifically the synthesis of levansucrase (sacU ^– ). sacA, sacS and sacT map to the left of purA16. sacQ is located to the left of thr5, sacB and sacR between cysB3 and hisA1 and sacU between uvr1 and gtaB.     

Mapping of mutations affecting synthesis of exocellular enzymes in Bacillus subtilis

Summary The sacU ^h , amyB and pap mutations are identical with respect to their pleiotropic phenotype and their genetic location. Strains bearing these mutations overproduce several exocellular enzymes: -amylase, lavansucrase and proteases, they are poorly or not at all transformable and most of them are devoid of flagella. These mutations are tightly linked to the sacU ^- mutations by transformation and therefore lie between the hisA1 and gtaB290 markers. It is possible that the sacU ^h , amyB and pap mutations on one hand and the sacU ^- mutations on the other are two different classes of alterations of the same regulatory gene controlling the synthesis of some exocellular enzymes and several other cellular functions. Furthermore an amy ^- mutation, leading to the lack of -amylase activity, was mapped between the lin2 and aroI906 markers which are not linked to the sacU locus.     

Characterization of an lrp -like ( IrpC ) gene from Bacillus subtilis

In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4 kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilislrp-likegene is a bona fide Lrp protein – the first one to be detected in gram-positive bacteria. When expressed in E.␣coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B. subtilis Lrp-like protein plays a role in the growth phase transition. Received: 28 January 1997 / Accepted: 18 April 1997     

Stepwise Introduction of Regulatory Genes Stimulating Production of α-Amylase into Bacillus subtilis : Construction of an α-Amylase Extrahyper Producing Strain

The production of extracellular α-amylase in Bacillus subtilis is probably regulated by many genetic elements, such as amyR, tmrA7, pap, amyB and sacU. Additional genetic elements, C-108 and A-2 for production of the α-amylase were found in D-cycloserine and ampicillin resistant mutants (C108 and A2) of B. subtilis 6160, respectively. Strain C108 increased the production of α-amylase about 5 times and protease about 80 times compared to parental 6160 strain. Strain A2 showed a nearly 6-fold increased α-amylase production.

These genetic elements displayed a synergistic effect with other genetic factors in production of extracellular α-amylase when these elements were transferred by DNA mediated transformation. By stepwise introduction of these and other genetic elements into B. subtilis 6160 by transformation and mutation, strains with higher α-amylase producing activity were obtained. The finally obtained strain, T2N26, produced about 1,500-2,000 times more α-amylase than parental 6160 strain.     

Inducible Secretion of a Cellulase from Clostridium thermocellum in Bacillus subtilis

A host-vector system for inducible secretion during the logarithmic growth phase in Bacillus subtilis has been developed. The B. subtilis levansucrase gene promoter and the region encoding its signal sequence have been used. The endoglucanase A of Clostridium thermocellum was used as a model protein to test the efficiency of the system. Effective inducible secretion of the endoglucanase A was observed when either the levansucrase signal sequence or its own signal sequence was used. Expression of the endoglucanase A in different genetic backgrounds of B. subtilis showed that its regulation was similar to that of levansucrase, and high enzyme activity was recovered from the culture supernatant of a hyperproducing B. subtilis sacU(Hy) strain. The molecular weight of 46,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the secreted endoglucanase A is compatible with the calculated molecular weight of the mature polypeptide.     

Parasporin 1Ac2, a Novel Cytotoxic Crystal Protein Isolated from Bacillus thuringiensis B0462 Strain

Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100 % identical even in nucleotide sequence level with that of parasporin-1Aa (PS1Aa1) from B. thuringiensis A1190 strain. The other (PS1Ac2) showed significant homology (99 % identity) to that of PS1Ac1 from B. thuringiensis 87-29 strain. The 15 kDa (S¹¹³–R²⁵⁰) and 60 kDa (I²⁵¹–S⁷⁷⁷) fragments consisting of an active form of PS1Ac2 were expressed as His-tag fusion. Upon purification under denaturing condition and refolding, the recombinant polypeptides were applied to cancer cells to analyze their cytotoxicities. 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay revealed that either of 15 or 60 kDa polypeptide exhibited no cytotoxicity to HeLa cells, but they became cytotoxic upon mixed together. Our results suggested that PS1Ac2 was responsible for the cytotoxicity of B. thuringiensis B0462 strain, and that the formation of hetero-dimer of 15 and 60 kDa polypeptide was required for their cytotoxicity.     

Xanthine dehydrogenase (XDH) cross-reacting material in mutants of Drosophila melanogaster deficient in XDH activity

Rocket immunoelectrophoresis was used to estimate xanthine dehydrogenase cross-reacting material (XDH-CRM) in strains containing the cin ¹ and cin ⁹ mutant genes, which are deficient in XDH enzymatic activity. CRM levels were determined as percentages of CRM in the Oregon-R wild-type strain. The mutant strains contain 72 and 76% of Oregon-R CRM, respectively. CRM levels in strains containing the XDH-deficient mutant genes lxd and mal are 93 and 105%, respectively. The high levels of CRM in these four mutant strains indicate that the primary effects of the mutant genes are on the function of XDH protein rather than its accumulation.This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to L. W. B.     

Cloning and Characterisation of the aroA and aroD Genes of Shigella dysenteriae Type 1

The aroA and aroD genes from Shigella dysenteriae type 1, encoding 5-enolpyruvylshikimate 3-phosphate synthase and 3-dehydroquinase, respectively, were cloned by polymerase chain reaction (PCR). Their nucleotide sequences were determined and predicted to code for 46 kDa and 27.5 kDa proteins, respectively. Protein expressed from these genes using the minicell system, corresponded to the size of the predicted protein products. The cloned genes were shown to be functional by complementation of Escherichia coli aroA and aroD^? mutants. The predicted amino acid sequences of the cloned aroA (427 amino acids) and aroD (252 amino acids) genes of S. dysenteriae type 1 were found to be highly homologous to the corresponding genes in other bacterial species, indicating the high conservation of these housekeeping genes. The use of the cloned aroA and aroD genes in the development of a vaccine strain against S. dysenteriae is discussed.     

Cloning and Characterization of Xylanase A from the Strain <Emphasis Type="Italic">Bacillus</Emphasis> sp. BP-7: Comparison with Alkaline pI-Low Molecular Weight Xylanases of Family 11

The xynA gene encoding a xylanase from the recently isolated Bacillus sp. strain BP-7 has been cloned and expressed in Escherichia coli. Recombinant xylanase A showed high activity on xylans from hardwoods and cereals, and exhibited maximum activity at pH 6 and 60°C. The enzyme remained stable after incubation at 50°C and pH 7 for 3 h, and it was strongly inhibited by Mn²⁺, Fe³⁺, Pb²⁺, and Hg²⁺. Analysis of xylanase A in zymograms showed an apparent molecular size of 24 kDa and a pI of above 9. The amino acid sequence of xylanase A, as deduced from xynA gene, shows homology to alkaline pI-low molecular weight xylanases of family 11 such as XynA from Bacillus subtilis. Analysis of codon usage in xynA from Bacillus sp. BP-7 shows that the G+C content at the first and second codon positions is notably different from the mean values found for glycosyl hydrolase genes from Bacillus subtilis.     

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.     

Industrial production of α-amylase by genetically engineered Bacillus

A genetically engineered Bacillus subtilis strain (ALKO 84) has been introduced for industrial production of α-amylase. This strain carries the α-amylase gene from a traditionally developed production strain B. amyloliquefaciens (ALKO 89) on the multicopy plasmid pUB110.⁸At laboratory scale the recombinant strain ALKO 84 produced in industrial medium about twice as much α-amylase as the traditional strain ALKO 89. The process for production of the enzyme was scaled-up to 60m³. At this scale B. subtilis ALKO 84 retained its relative superiority to B. amyloliquefaciens ALKO 89, producing about 85% of the activity obtained at laboratory scale. Stability of the recombinant plasmid was found acceptable during the large-scale cultivations with over 90% of cells retaining plasmid-encoded characteristics throughout.     

Organization and characterization of three genes involved in d-xylose catabolism in Lactobacillus pentosus

Summary A cluster of three genes involved in d-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis d-xylose isomerase (68% and 77%, respectively), and to E. coli d-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment d-xylose. NMR analysis confirmed that ¹³C-xylose was converted into ¹³C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of d-xylose isomerases of different bacteria suggests that L. pentosus d-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli d-xylose isomerase and not to a second similarity group comprising d-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5-xylR (1167 bp, repressor) — xylA (1350 bp, D-xylose isomerase) — xylB (1506 bp, d-xylulose kinase) — 3 is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB.     

Streptomyces blattellae,a novel actinomycete isolated from the in vivo of a Blattella germanica

During a screening for novel and useful actinobacteria in desert animal, a new actinomycete was isolated and designated strain TRM63209^T. The strain was isolated from in vivo of a Blattella germanica in Tarim University in Alar City, Xinjiang, north-west China. The strain was found to exhibit an inhibitory effect on biofilm formation by Candida albicans ATCC 18,804. The strain was observed to form abundant aerial mycelium, occasionally twisted and which differentiated into spiral spore chains. Spores of TRM63209^T were observed to be oval-shaped, with a smooth surface. Strain TRM63209^T was found to grow optimally at 28 °C, pH 8 and in the presence of 1% (w/v) NaCl. The whole-cell sugars of strain TRM63209^T were rhamnose ribose, xylose, mannose, galactose and glucose, and the principal polarlipids were found to be diphosphatidylglycerol, phos-phatidylethanolamine, phosphatidylcholine, phosphatidylinositol mannoside, phosphatidylinositol and an unknown phospholipid(L). The diagnostic cell wall amino acid was identified as LL-diaminopimelic acid. The predominant menaquinone was found to be MK-9(H6) (14.64%), MK-9(H2) (19.65%), MK-9(H8) (22.34%), MK-10(H2) (25.37%). The major cellular fatty acids were identified as iso-C16:0, 16:0, anteiso-C15:0, anteiso-C17:0, iso-C15:0 and Sum in Feature 3. Analysis of the 16S rRNA sequence showed that strain TRM63209^T exhibits high sequence similarity to Streptomyces bungoensis strain DSM 41781^T 98.20%. A multi-locus sequence analysis of five house-keeping genes (atpD, gyrB, rpoB, recA and trpB) and phylogenomic analysis also illustrated that strain TRM63209^T should be assigned to the genus Streptomyces. The DNA G?+?C content of the strain was determined to be 70.2 mol%. Average nucleotide identity (ANI) between strain TRM63209^T and S. bungoensis DSM 41781^T, Streptomyces phyllanthi PA1-07^T, Streptomyces longwoodensis DSM 41677^T and Streptomyces caeruleatus NRRL B-24802^T were 82.76%, 82.54%, 82.65%, 84.02%, respectively. Digtal DNA-DNA (dDDH) hybridization were 26.30%, 25.10%, 26.20%, 29.50%, respectively. Therefore, it is concluded that strain TRM63209^T represents a novel species of the genus Streptomyces, for which the name Streptomyces blattelae is proposed. The type strain is TRM63209^T (CCTCC AA 2018093^T?=?LMG 31,403?=?TRM63209^T).

     

The tagGH operon of Bacillus subtilis 168 encodes a two-component ABC transporter involved in the metabolism of two wall teichoic acids

We report the nucleotide sequence and the characterization of the Bacillus subtilis tagGH operon. The latter is controlled by a σ^A-dependent promoter and situated in the 308° chromosomal region which contains genes involved in teichoic acid biosynthesis. TagG is a hydrophobic 32.2 kDa protein which resembles integral membrane proteins belonging to polymerexport systems of Gram-negative bacteria. Gene tagH encodes a 59.9 kDa protein whose N-moiety contains the ATP-binding motif and shares extensive homology with a number of ATP-binding proteins, particularly with those associated with the transport of capsular polysaccharides and O-antigens. That the tagGH operon is essential for cell growth was established by the failure to inactivate tagG and the 5′ -moiety of tagH by insertional mutagenesis. During limited tagGH expression, cells exhibited a cocoid morphology while their walls contained reduced amounts of phosphate as well as galactosamine. These observations, revealing impaired metabolism of both wall teichoic acids of B. subtilis 168, i.e. poly(glycerol phosphate), and poly(glucose galactosamine phosphate), combined with sequence homologies, suggest that TagG and TagH are involved in the translocation through the cytoplasmic membrane of the latter teichoic acids or their precursors.     

Members of the pogo superfamily of DNA-mediated transposons in the human genome

Bacillus subtilis, likeEscherichia coli, possesses several sets of genes involved in the utilization of-glucosides. InE. coli, all these genes are cryptic, including the genes forming thebgl operon, thus leading to a Bgl^– phenotype. We screened forB. subtilis chromosomal DNA fragments capable of reverting the Bgl⁺ phenotype associated with anE. coli hns mutant to the Bgl^– wild-type phenotype. OneB. subtilis chromosomal fragment having this property was selected. It contained a putative Ribonucleic AntiTerminator binding site (RAT sequence) upstream from thebglP gene. Deletion studies as well as subcloning experiments allowed us to prove that the putativeB. subtilis bglP RAT sequence was responsible for the repression of theE. coli bgl operon. We propose that this repression results from the titration of the BglG antiterminator protein ofE. coli bgl operon by our putativeB. subtilis bglP RAT sequence. Thus, we report evidence for a new cross interaction between heterologous RAT-antiterminator protein pairs.     

A novel dextransucrase is produced by <Emphasis Type="Italic">Leuconostoc</Emphasis><Emphasis Type="Italic">citreum</Emphasis> strain B/110-1-2: an isolate used for the industrial production of dextran and dextran derivatives

The industrial Leuconostoc strain B/110-1-2 producing dextran and dextran derivatives was taxonomically identified by 16S rRNA as L. citreum. Its dextransucrase enzymes were characterized according to their cellular location and reaction specificity. In the presence of sucrose, the strain B/110-1-2 produced two cell-associated dextransucrases (31.54% of the total glucosyltransferase activity) with molecular weights of 160 and 240 kDa and a soluble dextransucrase (68.46%) at 160–180 kDa. Two open reading frames (ORF) coding for L. citreum strain B/110-1-2 dextransucrases were identified. One of them shared a 52% identity with the alternansucrase ASR of L. citreum NRRL B-1355 and with a putative annotated alternansucrase sequence found in the genome of L. citreum KM20. The structural analysis (HPAEC-PAD, HPSEC, and ¹³C-NMR) of the polymer and oligodextrans produced by the B/110-1-2 dextransucrases suggest this novel glucansucrase has specificity similar to a dextransucrase but not to an alternansucrase, producing a soluble linear dextran with glucose molecules linked mainly in α-1,6 and α-1,3 with α-1,4 branches. These results enhance the understanding of this industrially significant strain and will aid in distinguishing between physiologically similar Leuconostoc spp. strains.     

Improving Toxicity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain Contains the <Emphasis Type="Italic">cry8Ca</Emphasis> Gene Specific to <Emphasis Type="Italic">Anomala corpulenta</Emphasis> Larvae

The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein, with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain of B. thuringiensis (HD-73⁻), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC₅₀) of the two mutants were 0.2334 × 10⁸ and 0.2591 × 10⁸ colony-forming units g⁻¹, considerably lower than the LC₅₀ of the wild-type strain HBF-1 (0.9583 × 10⁸ CFU g⁻¹) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 10⁸ CFU g⁻¹).     

Production of human papillomavirus type 33 L1 major capsid protein and virus-like particles from Bacillus subtilis to develop a prophylactic vaccine against cervical cancer

We developed a bacterial expression system to produce human papillomavirus (HPV) type 33 L1 major capsid protein and virus-like particles from a recombinant Bacillus subtilis strain. For the first time, we have isolated self-assembled virus-like particles (VLPs) of HPV type 33 from B. subtilis, a strain generally recognized as safe (GRAS). The gene encoding the major capsid protein L1 of HPV type 33 was amplified from viral DNA isolated from a Korean patient and expressed in B. subtilis; a xylose-induction system was used to control gene activity. HPV33 L1 protein was partially purified by 40% (w/v) sucrose cushion centrifugation and strong cation exchange column chromatography. Eluted samples exhibited immunosignaling in fractions of 0.5-1.0 M NaCl. The HPV33 L1 protein was shown to be approximately 56 kDa in size by SDS-PAGE and Western blotting; recovery and purity were quantified by indirect immuno-ELISA assay. The final yield and purity were approximately 20.4% and 10.3%, respectively. Transmission electron microscopic analysis of fractions immunoactive by ELISA revealed that the L1 protein formed self-assembled VLPs with a diameter of approximately 20-40 nm. Humoral and cellular immune responses provoked by the B. subtilis/HPV33 L1 strain were approximately 100- and 3-fold higher than those of the empty B. subtilis strain as a negative control, respectively. Development of a VLP production and delivery system using B. subtilis will be helpful, in that the vaccine may be convenient production as an antigen delivery system. VLPs thus produced will be safer for human use than those purified from Gram-negative strains such as Escherichia coli. Also, use of B. subtilis as a host may aid in the development of either live or whole cell vaccines administered by antigen delivery system.     

Characterization of an lrp-like (IrpC ) gene from Bacillus subtilis

In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4?kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilislrp-likegene is a bona fide Lrp protein – the first one to be detected in gram-positive bacteria. When expressed in E.?coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B. subtilis Lrp-like protein plays a role in the growth phase transition.