Nucleotide sequence of the region encompassing the int gene of a cryptic prophage and the dna Y gene flanked by a curved DNA sequence of Escherichia coli K12

Summary The nucleotide sequence of a 2.5 kb region encompassing a curved DNA segment (BENT-9) randomly cloned from the total Escherichia coli chromosome was determined. This region was found to contain the dnaY gene encoding a transfer RNA. The curved DNA structure was demonstrated to be located just upstream of the dnaY promoter. The results of sequencing further revealed that the int gene of a cryptic prophage, qsr, which has been shown to be present in the E. coli genome, is located next to the dnaY gene.     

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.     

Cloning and sequencing of the hemA gene of Rhodobacter capsulatus and isolation of a δ-aminolevulinic acid-dependent mutant strain

Summary The Rhodobacter capsulatus hemA gene, coding for the enzyme -aminolevulinic acid synthase (ALAS), was isolated from a genome bank by hybridization with a hemT probe from Rhodobacter sphaeroides. Subcloning of the initial 3.9 kb HindIII fragment allowed the isolation of a 2.5 kb HindIII-BglII fragment which was able to complement the -aminolevulinic acid-requiring (ALA-requiring) Escherichia coli mutant SHSP19. DNA sequencing revealed an open reading frame coding for a protein with 401 amino acids which displayed similarity to the amino acid sequences of other known ALASs. However, no resemblance was seen to the HemA protein of E. coli K12. Based on the sequence data, an ALA-requiring mutant strain of R. capsulatus was constructed by site-directed insertion mutagenesis. Introduction of a plasmid, containing the hemA gene of R. capsulatus on the 3.9 kb HindIII fragment, restored ALA-independent growth of the mutant indicating that there is only one gene for ALA biosynthesis in R. capsulatus. Transfer of the R factor pRPS404 and hybridization analysis revealed that the ALAS gene is not located within the major photosynthetic gene cluster.Part of this research was presented at the Symposium on Molecular Biology of Membrane-Bound Complexes in Phototrophic Bacteria, Freiburg, FRG, 2–5 August 1989     

Sequence variation at theSec-1 locus of rye,Secale cereale (Poaceae)

This paper describes the structure of a 9.2-kb repeat unit of DNA, which represents one-secalin gene and spacer sequence located at theSec-1 locus on the short arm of chromosome 1 of rye. The gene units at theSec-1 locus comprise 1.1 kb representing the gene and 8.1 kb of spacer sequence separating the genes. A sequence comparison of nine genes and their promoter regions from theSec-1 locus, reveals that there is greater variation within the coding sequence than there is within the promoter regions. The gene sequence variation is discussed in terms of the size variation seen for the-secalin proteins in rye species. The results include a comparison of promoter sequences from members of the Triticeae to examine the degree of conservation between other seed storage protein genes.     

Molecular cloning and nucleotide sequence of the mutT mutator of Escherichia coli that causes A:T to C:G transversion

Summary The Escherichia coli mutator gene mutT, which causes A:TC:G transversion, was cloned in pBR 322. mutT ⁺ plasmids carry a 0.9 kb PvuII DNA fragment derived from the E. coli chromosome. Specific labelling of plasmid-encoded proteins by the maxicell method revealed that mutT codes for a polypeptide of about 15,000 daltons. The protein was overproduced when the mutT gene was placed under the control of the lac regulatory region on a multicopy runaway plasmid. The nucleotide sequence of the mutT gene was determined by the dideoxy method.Abbreviations Ap ampicillin - IPTG isopropyl--d-thiogalactopyranoside - kb kilobase pair(s) - kDa kilodalton(s) - SDS sodium dodecyl sulphate - Tc tetracycline     

Cloning of an Arabidopsis thaliana cDNA encoding cystathionine β-lyase by functional complementation in Escherichia coli

Cystathionine -lyase, the second enzyme involved in the methionine biosynthetic pathway in plants, catalyses the synthesis of homocysteine from cystathionine. A cDNA encoding cystathionine -lyase was cloned from an Arabidopsis thaliana expression library by complementation of an Escherichia coli mutant deficient in this enzyme. As deduced from the full-length nucleotide sequence (1.7 kb), the polypeptide contains 464 amino acids and presents a predicted M _r of 50372. A. thaliana cystathionine -lyase exhibits 22% sequence identity with the E. coli corresponding enzyme and contains a 70 amino acid N-terminal additional sequence compared with the bacterial protein. Since the general features of chloroplast transit peptides could be observed in this amino-terminal extension, we propose a chloroplast localization for the cDNA-encoded enzyme. Southern blot analysis suggested that cystathionine -lyase is encoded by a single copy gene in A. thaliana.     

Cloning and expression of the Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in Escherichia coli

A gene coding for the endo--1,3-1,4-glucanase of B. circulans ATCC21367 was cloned into Escherichia coli. The cloned enzyme hydrolyzed lichenan or barley -glucan to produce 3-O--cellobiosyl-d-glucose as a main product but was inactive with carboxymethyl cellulose, laminarin and xylan. The enzyme, M _r=28 kDa, remained within the cytoplasm of E. coli. A 771 bp open reading frame was in the 2 kb PstI fragment of the recombinant plasmid pLL200K. The deduced protein sequence consists of 257 amino acids and has a putative signal peptide of 26 amino acids. The amino acid sequence of the endo--1,3-1,4-glucanase showed 68 and 51% homology to previously reported endo--1,3-1,4-glucanases from Bacillus strain N-137 and B. brevis, respectively.     

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions ( to ) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.     

Sequence analysis of cyclodextrin glycosyltransferase from the alkaliphilic Bacillus agaradhaerens

The gene encoding an alkaline active cyclodextrin glycosyltransferase (CGTase) from the alkaliphilic B. agaradhaerens LS-3C was cloned and sequenced. It encodes a mature polypeptide of 679 amino acids with a molecular mass of 76488 Da. The deduced amino acid sequence of the mature CGTase revealed 99 and 95% identity to the CGTase sequences from the other B. agaradhaerens strains, DSM 8721^T and 9948, respectively. The next closest identity was of 59% with B. clarkii enzyme. CGTases from B. agaradhaerens, B. clarkii, and B. firmus/lentus formed a phylogenetically separated cluster from the other CGTases of Bacillus spp. origin. A number of usually conserved residues in the CGTases were found to be replaced in the sequence of B. agaradhaerens enzyme. The sequence analysis indicated the enzyme to be close to the so-called `intermediary enzymes' in the -amylase family.     

Nucleotide sequence of an endo-β-1,4-glucanase gene fromBacillus subtilis CK-2

The gene encoding endo--1,4-glucanase inBacillus subtilis CK-2 was cloned intoEscherichia coli DH5, and the nucleotide sequence determined. The 1500 bp gene encodes a protein of 499 amino-acid residues with a calculated molecular mass of 55 261, and is equipped with a typicalB. subtilis signal peptide. Nucleotide sequence comparison revealed only 2 basepairs deviation between this gene and the endo--1,4-glucanase gene ofB. subtilis PAP115, and 93% to 95% homology was found between the amino acid sequences of these enzymes and otherB. subtilis endo--1,4-glucanases. Regions of similarity were also observed between the carboxy-terminal part of these enzymes and the part of theB. lautus PL236celA enzyme constituting the cellulose-binding domain.     

Cloning and characterization of a secY homolog from Chlamydia trachomatis

Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc- ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50 195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SceY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.     

The glucose kinase gene of Streptomyces coelicolor and its use in selecting spontaneous deletions for desired regions of the genome

Summary A number of deletions in the glucose kinase (glk) region of the Streptomyces coelicolor chromosome were found among spontaneous glk mutants. The deletions were identified by probing Southern blots of chromosomal DNA from glk mutants with cloned glk DNA. The deletions ranged in size from 0.3 kb to greater than 2.9 kb. When cloned glk DNA was introduced on a C31 phage vector into a glk mutant that contained a deletion of the entire homolgous chromosomal glk region, glucose kinase activity was detected in extracts of these cells. The entire coding information for at least a subunit of glucose kinase is there-fore present on the cloned glk DNA. The 0.3 kb glk chromosomal deletion was used to demonstrate that transfer of chromosomal glk mutations on the the C31::glk phage could occur by recombination in vivo. Since glk mutations frequently arise from deletion events, a method was devised for inserting the cloned glk DNA at sites in the chromosome for which cloned DNA is available, and thus facilitating the isolation of deletions in those DNA regions. C31::glk vectors containing a deletion of the phage att site cannot lysogenize S. coelicolor recipients containing a deletion of the glk chromosomal gene unless these phages contain S. coelicolor chromosomal DNA. In such lysogens, the glk gene becomes integrated into the chromosome by homologous recombination directed by the chromosomal insert on the phage DNA. In appropriate selective conditions, mutants which contain deletions of the glk gene that extend into the adjacent host DNA can be easily isolated. This method was used to insert glk into the methylenomycin biosynthetic genes, and isolate derivatives with deletions of host DNA from within the prophage into the adjacent host DNA. Phenotypic and Southern blot analysis of the deletions showed that there are no genes essential for methylenomycin biosynthesis for at least 13 kb to the left of a region concerned with negative regulation of methylenomycin biosynthesis. Many of the deletions also removed part of the C31 prophage.     

Amplification of the amyE-tmrB region on the chromosome in tunicamycin-resistant cells of Bacillus subtilis

Summary In a class of tunicamycin-resistant mutants (tmrA7) of Bacillus subtilis, the production of extracellular -amylase is increase by about five fold. The tmrA7 characteristics (tunicamycin resistance and hyperproduction of extracellular -amylase) can be transferred to recipient cells by transformation. In the transformants and the original tmrA7 mutant, typical amplification of the region from 4 kb upstream of the amyE gene to the tmrB gene on the chromosome was detected. The repeating unit, 16 kb in size, repeats tandemly about five and ten times in the mutant and transformants, respectively, and the -amylase production is proportional to the copy number of the amyE gene. Simultaneous amplification of the tmrB gene, which is responsible for tunicamycin resistance in the multicopy state, and the -amylase structural gene (amyE) seems to be the cause of the pleiotropy of the tmrA7 mutation.     

Structure and expression of the Chlorobium vibrioforme hemA gene

The green sulfur bacterium, Chlorobium vibrioforme, synthesizes the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate via the RNA-dependent five-carbon pathway. A 1.9-kb clone of genomic DNA from C. vibrioforme that is capable of transforming a glutamyl-tRNA reductase-deficient, ALA-dependent, hemA mutant of Escherichia coli to prototrophy was sequenced. The transforming C. vibrioforme DNA has significant sequence similarity to the E. coli, Salmonella typhimurium, and Bacillus subtilis hemA genes and contains a 1245 base open reading frame that encodes a 415 amino acid polypeptide with a calculated molecular weight of 46174. This polypeptide has over 28% amino acid identity with the polypeptides deduced from the nucleic acid sequences of the E. coli, S. typhimurium, and B. subtilis hemA genes. No sequence similarity was detected, at either the nucleic acid or the peptide level, with the Rhodobacter capsulatus or Bradyrhizobium japonicum hemA genes, which encode ALA synthase, or with the S. typhimurium hemL gene, which encodes glutamate-1-semialdehyde aminotransferase. These results establish that hemA encodes glutamyl-tRNA reductase in species that use the five-carbon ALA biosynthetic pathway. A second region of the cloned DNA, located downstream from the hemA gene, has significant sequence similarity to the E. coli and B. subtilis hemC genes. This region contains a potential open reading frame that encodes a polypeptide that has high sequence identity to the deduced E. coli and B. subtilis HemC peptides. hemC encodes the tetrapyrrole biosynthetic enzyme, porphobilinogen deaminase, in these species. Preliminary evidence was obtained for the existence of a 3.0-kb polycistronic meassge that includes the hemA sequence, in exponentially growing C. vibrioforme cells. Results of condon usage analysis for the C. vibrioforme hemA gene indicate that green sulfur bacteria are more closely related to purple nonsulfur bacteria than to enteric bacteria. Sequences corresponding to a polyadenylation signal and a poly(A) attachment site were found immediately downstream from the 3 end of the hemA open reading frame.     

Induction of a Streptomyces cacaoi β-lactamase gene cloned in S. lividans

Summary The previously cloned class A -lactamase gene (bla) of Streptomyces cacaoi was shown to be inducible by -lactam compounds in the host organism S. lividans. A regulatory region of 2.75 kb was identified and the nucleotide sequence determined. It contained four open reading frames (ORFs) of which only two were complete and required for induction. ORF1-ORF2 exerted a positive regulatory effect on the expression of bla. Inactivation of ORF1 or of ORF2 resulted not only in the loss of induction, but also in a 30- to 60-fold decrease in the basal (non-induced) level of -lactamase production. ORF1 codes for a DNA-binding protein related to the AmpR repressor/activator, which controls the expression of ampC (class C -lactamase) genes in several Enterobacteria.     

Close genetic relationship between Nitrobacter hamburgensis nitrite oxidoreductase and Escherichia coli nitrate reductases

The nitrite oxidoreductase (NOR) from the facultative nitrite-oxidizing bacterium Nitrobacter hamburgensis X14 was investigated genetically. In order to develop a probe for the gene norB, the N-terminal amino acid sequence of the NOR -subunit (NorB) was determined. Based on that amino acid sequence, an oligo-nucleotide was derived that was used for the identification and cloning of gene norB. Sequence analysis of DNA fragments revealed three adjacent open reading frames in the order norA, norX, norB. The DNA sequences of norX and norB represented complete genes while the open reading frame of norA was truncated by the cloning site. The deduced amino acid sequence of protein NorB contained four cysteine clusters with striking homology to those of iron-sulfur centers of bacterial ferredoxins. NorB shares significant sequence similarity to the -subunits (NarH, NarY) of the two dissimilatory nitrate reductases (NRA, NRZ) of Escherichia coli. Additionally, the derived amino acid sequence of the truncated open reading frame of norA showed striking resemblance to the -subunits (NarG, NarZ) of the E. coli nitrate reductases.     

Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.