Cloning and characterization of the yjeA gene, encoding a novel deoxyribonuclease, from Bacillus subtilis

The yjeA gene, encoding a secreted protein, YjeA, of Bacillus subtilis, was cloned and characterized. A derivative of YjeA, the recombinant YjeA-H, which contained a C-terminal His(6)-tag, was purified from Escherichia coli for functional studies. YjeA-H was shown to be an endonuclease, which hydrolyses both single-stranded and double-stranded DNA, but not RNA. Covalently closed circular pBR322 DNA incubated with YjeA-H was shown by gel electrophoresis to be first nicked to an open circular form, and then to a linearized structure on a background of DNA smear, and finally to small species of linear molecules that accumulated gradually. When (32)P-labelled pBR322 DNA was used as substrate, YjeA-H was shown to progressively nick both DNA strands in a random fashion, creating intermediates of various structures, as well as DNA smears comprising linear molecules of different sizes. The final products were found to consist essentially of degraded species of DNA. The detection of a putative signal peptide at the N-terminus of YjeA, together with the purification of YjeA-H from the culture supernatants of E. coli yjeA-H clones, and the identification of YjeA in the culture medium of Bacillus subtilis, supports the conclusion that YjeA is a secretory protein of Bacillus subtilis.     

Cloning of a novel gene yrbB, encoding a protein located in the spore integument of Bacillus subtilis

A DNA fragment (2.7 kbp) containing three deduced open reading frames, orf1, orf2 and orf3 (partial sequence), was isolated from the genomic library of Bacillus subtilis using an antiserum raised against spore integument, and was sequenced. orf2 was 519 nucleotides long and encoded a protein of 172 amino acids with a predicted molecular size of 19552, corresponding to the protein which reacted with the antiserum. Immunoelectron microscopic observation indicated that YrbB, the product of orf2 , was located within the spore integument, mainly in the cortex layer with a part in the inner region of the coat layer.     

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli.     

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli.     

Cloning of the glycerol kinase gene of Bacillus subtilis

A 3.5 kb fragment of Bacillus subtilis DNA which contains wild type alleles of mutations in glpK (glycerol kinase) and glpD (glycerol-3-phosphate [G3P] dehydrogenase) was cloned in plasmid pHV32 in Escherichia coli. The cloned fragment expresses glycerol kinase in B. subtilis mutants carrying the mutations glpK11 and recE4 after induction with glycerol or G3P whereas it does not express G3P dehydrogenase. The cloned fragment thus contains the complete glpK but probably only part of glpD.     

Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes.     

Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis.

We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr (A. Sloma, C. Rudolph, G. Rufo, Jr., B. Sullivan, K. Theriault, D. Ally, and J. Pero, J. Bacteriol. 172:1024-1029, 1990). During purification, this novel protease (Vpr) was found bound in a complex in the void volume after gel filtration chromatography. The amino-terminal sequence of the purified protein was determined, and an oligonucleotide probe was constructed on the basis of the amino acid sequence. This probe was used to clone the structural gene (vpr) for this protease. The gene encodes a primary product of 806 amino acids. The amino acid sequence of the mature protein was preceded by a signal sequence of approximately 28 amino acids and a prosequence of approximately 132 amino acids. The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500, suggesting that Vpr undergoes C-terminal processing or proteolysis. The vpr gene maps in the ctrA-sacA-epr region of the chromosome and is not required for growth or sporulation.     

Cloning and characterization of human CAGLP gene encoding a novel EF-hand protein.

The EF-hand proteins, containing conserved Ca2+ binding motifs, play important roles in many biological processes. Through data mining, a novel human gene, CAGLP (calglandulin-like protein) was predicted and subsequently isolated from human skeleton muscle. The open reading frame of CAGLP is 543 bp in length, coding a putative Ca2+ binding protein with four EF-hand motifs. The deduced amino acid sequence of CAGLP displays high similarity with Bothrops insularis snake protein calglandulin (80%). The results of PCR amplification using cDNA from 17 human tissues indicated that human CAGLP is expressed in prostate, thymus, heart, skeleton muscle, bone marrow and ovary. Functional CAGLP::EGFP (enhanced green fluorescent protein) fusion protein revealed that CAGLP accumulated through-out Hela cells. Western blot using anti-EGFP antibodies indicated that the CAGLP protein has a molecular weight of about 19 kD. A phylogenetic tree showed that CAGLP and calglandulin may be orthologous proteins representing a distinct group in the EF-hand proteins.     

Cloning, genetic organization, and characterization of a structural gene encoding bacillopeptidase F from Bacillus subtilis

Bacillopeptidase F is an extracellular serine protease that is expressed at the beginning of the stationary phase. To study its structure, regulation of expression, and physiological roles, we have cloned and characterized the structural gene (bpf) encoding this protease from Bacillus subtilis. DNA sequence analysis suggests this protease is synthesized as a preproenzyme (Mr = 92,000). Through processing at both the NH2 and COOH termini, it is gradually converted into various forms with molecular mass ranging from 80 to 48 kDa. Shortening the 3' end of bpf demonstrates that at least 290 amino acid residues from the COOH-terminus of bacillopeptidase F are not required for either catalytic activity or secretion. Bacillopeptidase F exhibits sequence similarity with several serine proteases. Its gene is found immediately downstream from the fts operon which was mapped at 135 degrees on the B. subtilis genetic linkage map. Inactivation of the chromosomal copy of bpf shows no effect on cell growth and sporulation. A triple protease-deficient strain (WB300 with the structural genes for bacillopeptidase F and two other major proteases inactivated) was constructed to serve as a better expression host for the production and secretion of foreign proteins.     

Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis.

We have cloned from Bacillus subtilis a novel protease gene (nprB) encoding a neutral protease by using a shotgun cloning approach. The gene product was determined to have a molecular mass of 60 kDa. It has a typical signal peptide-like sequence at the N-terminal region. The expression of nprB can be stimulated by using a B. subtilis strain, WB30, carrying a sacU(h)h mutation. Expression of this protease gene results in production of a 37-kDa protease in the culture medium. The first five amino acid residues from the N terminus of the mature protease were determined to be Ala-Ala-Gly-Thr-Gly. This indicates that the protease is synthesized in a preproenzyme form. The purified protease has a pH optimum of around 6.6, and its activity can be inhibited by EDTA, 1,10-phenanthroline (a zinc-specific chelator), and dithiothreitol. It retained 65% of its activity after treatment at 65 degrees C for 20 min. Sequence comparison indicates that the mature form of this protease has 66% homology with the two thermostable neutral proteases from B. thermoproteolyticus and B. stearothermophilus. It also shares 65, 61, and 56% homology with the thermolabile neutral proteases from B. cereus, B. amyloliquefaciens, and B. subtilis, respectively. The zinc-binding site and the catalytic residues are all conserved among these proteases. Sequence homology extends into the "propeptide" region. The nprB gene was mapped between metC and glyB and was not required for growth or sporulation.     

Cloning and characterization of a novel gene encoding keratin-like protein from nematode Nippostrongylus brasiliensis.

Nippostrongylus brasiliensis (Nb) is one of the most important parasites in studying Th2 immune response of the host, but little is known about its antigenic structures of the excretory-secretory or structural proteins of the parasite. Here we report cloning and characterization of a novel antigenic gene from cDNA library of Nb adult worm by immunoscreening. The positive clone, KLP-Nb, had an open reading frame of 612 bp that encodes a 203-amino-acid protein and was homologous to 'similar to keratins in a glycine-rich region' of Caenorhabditis elegans. Its expression was confirmed by Northern blotting and IgG enzyme-linked immunosorbent assay. This protein seems to be one of the components of cuticle that covers the nematode body.     

Cloning and characterization of a novel human gene encoding a zinc finger protein with 25 fingers

This study reports cloning and characterization of a human cDNA encoding a novel human zinc finger protein, ZFD25. ZFD25 cDNA is 6118 bp long and has an open reading frame of 2352 bp that encodes a 783 amino acid protein with 25 C2H2-type zinc fingers. The ZFD25 cDNA also contains a region with high sequence similarity to the Krüppel-associated box A and B domain in the 5'-untranslated region, suggesting that ZFD25 belongs to the Krüppel-associated box zinc finger protein family. The ZFD25 gene was localized to chromosome 7q11.2. Northern blot analysis showed that ZFD25 was expressed in a wide range of human organs. In cultured endothelial cells, the mRNA level was decreased upon serum starvation.     

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.     

Cloning of the sacS gene encoding a positive regulator of the sucrose regulon in Bacillus subtilis

The sacS gene controls the expression of 2 saccharolytic enzymes in Bacillus subtilis (sucrase and levansucrase).This paper describes a recombinant plasmid containing a mutant allele, sacS^c. The plasmid was isolated from a B. subtilis DNA bank established in Escherichia coli. Moreover, it was shown that the sacS^c allele, placed on a high-copy plasmid, is dominant over the wild-type chromosomal sacS⁺ allele. This result strongly suggests that the sacS gene encodes a positive regulatory protein.