Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity

In the plasmid pUC8ksgA7, the coding region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region, the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1-Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 makes beta-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl1-Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning, sequence analysis and heterologous expression of a beta-mannanase gene from Bacillus subtilis Z-2

Mannanase, an extracellular enzyme catalyzes the hydrolysis of hemicelluloses to produce oligosaccharides, has a potential to be applied in food industries. In this study, a mannanase gene from B. subtilis Z-2 was isolated through PCR screening of a genomic DNA library. The nucleotide sequence of the mannanase gene, man, contained an open reading frame of 1.080 bp, which codes for a deduced 26 amino-acid signal peptide and a mature protein with the deduced molecular mass of 38 KDa. The man gene can both be expressed heterologously into the periplasm in plasmid pET22b(+) containing intact signal peptide (pET-NdeI18) and the pelB signal peptide of the pET22b(+)vector (pET-NcoI3). The Escherichia coli BL21 (DE3) containing pET-NcoI3 secreted about twice as much mannanase as that harboring pET-NdeI18. In E. coli DH5alpha, expression of man was under the control of the lac promoter in the pRK415 vector and was much more effective when the Shine-Dalgamo (SD) sequence was changed from GGGGAG to AAGGAG and the start codon was changed from TTG to ATG, respectively. These results suggest that genetic modification of the SD sequence and start codon is practical for high-level expression of mannanase in different bacterial strains.     

Nucleotide sequence of the Bacillus anthracis edema factor gene (cya): a calmodulin-dependent adenylate cyclase

The nucleotide sequence of the Bacillus anthracis edema factor (EF) gene (cya), which encodes a calmodulin-dependent adenylate cyclase, has been determined. EF is part of the tripartite protein exotoxin of B. anthracis. An ATG start codon, immediately upstream from codons which specify the first 15 amino acids (aa) of EF, was preceded by an AAAGGAGGT sequence which is its probable ribosome-binding site. Starting at this ATG codon, there was a continuous 2400-bp open reading frame which encodes the 800-aa EF-precursor protein with a Mr of 92,464. The mature, secreted protein (767 aa; Mr 88,808) was preceded by a 33-aa signal peptide which has characteristics in common with leader peptides for other secreted proteins of the Bacillus species. A consensus amino acid sequence (Gly-X-X-X-X-Gly-Lys-Ser,X = any aa), which was part of the presumed ATP binding site for EF, was also present. The codon usage of the EF gene reflected the high A + T (71%) base composition for its DNA. B. anthracis EF was not related to the Escherichia coli or yeast adenylate cyclases, but was related to the Bordetella pertussis calmodulin-dependent adenylate cyclase.     

Molecular analysis of the Corynebacterium glutamicum transketolase gene

Transketolase is important in production of the aromatic amino acids in Corynebacterium glutamicum. The complete nucleotide sequence of the C. glutamicum transketolase gene has been identified. The DNA-derived protein sequence is highly similar to the transketolase of Mycobacterium tuberculosis, taxonomically related to C. glutamicum. The alignment of the N-terminus regions between both transketolases showed TTG to be the most probable start codon. Potential ribosomal binding and promoter regions were situated upstream from the TTG. The deduced amino acid sequence consists of 700 residues with a calculated molecular mass of 75 kDa, and contains all amino acid residues involved in cofactor and substrate binding in the well-characterized yeast transketolase sequence.     

The role of bases upstream of the Shine-Dalgarno region and in the coding sequence in the control of gene expression in Escherichia coli: translation and stability of mRNAs in vivo

A range of translational initiation regions (TIR) was created by combining synthetic DNA fragments derived from the atpB-atpE intercistronic sequence of Escherichia coli with the cDNA sequence encoding mature human interleukin 2 (IL-2), the E. coli fnr gene, or an fnr::lacZ gene fusion. Both the overall rates of gene expression and the relative concentrations and stabilities of the corresponding mRNA species were estimated in strains bearing the constructs on plasmids. These measurements served as the basis for analyses of the relationship between the structure of the TIR and the true rates of translation that it promotes. The constructs involving the IL-2 cDNA were predicted to allow much less stable secondary structure within the TIR than those involving the N-terminal region of the fnr gene. Thus by combining one set of upstream sequences with two different types of N-terminal coding sequence, it was possible to distinguish between the respective influences of primary and secondary structure upon initiation. The data indicate that in the presence of a given Shine-Dalgarno (SD)/start codon combination, the decisive factor for translational initiation efficiency is the stability of base pairing involving, or in the vicinity of, this region. The sequences contributing to this secondary structure can be many bases upstream of the SD region and/or downstream of the start codon. There was no indication that the specific base sequence upstream of the SD region could, other than to the extent that it contributed to the local secondary structure, significantly influence the efficiency of translational initiation.     

Complete nucleotide sequence of the Escherichia coli recB gene.

The complete nucleotide sequence of the Escherichia coli recB gene which encodes a subunit of the ATP-dependent DNase, Exonuclease V, has been determined. The proposed coding region for the RecB protein is 3543 nucleotides long and would encode a polypeptide of 1180 amino acids with a calculated molecular weight of 133,973. The start of the recB coding sequence overlaps the 3' end of the upstream ptr gene, and the recB termination codon overlaps the initiation codon of the downstream recD gene, suggesting that these genes may form an operon. No sequences which reasonably fit the consensus for an E. coli promoter could be identified upstream of the proposed recB translational start. The predicted RecB amino acid sequence contains regions of homology with ATPases, DNA binding proteins and DNA repair enzymes.     

Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum

The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined. The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli. The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria. The derived amino acid sequence corresponds to a protein of Mr 40,439. Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data. A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E. coli. Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism. However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.     

Nucleotide sequence and analysis of the lethal factor gene (lef) from Bacillus anthracis

The nucleotide sequence of the Bacillus anthracis lethal factor (LF) gene (lef) has been determined. LF is part of the tripartite protein exotoxin of B. anthracis along with protective antigen (PA) and edema factor (EF). The apparent ATG start codon, which is located immediately upstream from codons which specify the first 16 amino acids (aa) of the mature secreted LF, is preceded by an AAAGGAG sequence, which is its probable ribosome-binding site. This ATG codon begins a continuous 2427-bp open reading frame which encodes the 809-aa LF-precursor protein with an Mr of 93,798. The mature secreted protein (776 aa; Mr 90,237) was preceded by a 33-aa signal peptide which has characteristics in common with leader peptides for other secreted proteins of the Bacillus species. The codon usage of the LF gene reflects its high (70%) A + T content. The N-terminus of LF (first 300 aa) shared extensive homology with the N-terminus of the anthrax EF protein. Since LF and EF each bind PA at the same site, these homologous regions probably represent their common PA-binding domains.     

Identification and characterization of the products from the traJ and traY genes of plasmid R100.

The nucleotide sequence of part of the tra region of R100 including traJ and traY was determined, and the products of several tra genes were identified. The nucleotide sequence of traJ, encoding a protein of 223 amino acids, showed poor homology with the corresponding segments of other plasmids related to R100, but the deduced amino acid sequences showed low but significant homology. The first four amino acids at the N-terminal region of the TraJ protein were not essential for positive regulation of expression of traY, the first gene of the traYZ operon. The nucleotide sequence of traY shows that this gene may use TTG as the initiation codon and that it encodes a protein of 75 amino acids. Analysis of the traY gene product, which was obtained as the fusion protein with beta-galactosidase, showed that the N-terminal region of the product has an amino acid sequence identical to that deduced from the assigned frame but lacks formylmethionine. traY of plasmid F, which encodes a larger protein than the TraY protein of R100, is thought to use ATG as an initiation codon. However, a TTG initiation codon was found in the preceding region of the previously assigned traY coding frame of F. Interestingly, when translation of traY of F was initiated from TTG, the amino acid sequence homologous to the TraY protein of R100 appeared in tandem in the TraY protein of F. This may suggest that traY of F has undergone duplication of a gene like the traY gene of R100.     

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.     

The downstream box: an efficient and independent translation initiation signal in Escherichia coli.

The downstream box (DB) was originally described as a translational enhancer of several Escherichia coli and bacteriophage mRNAs located just downstream of the initiation codon. Here, we introduced nucleotide substitutions into the DB and Shine-Dalgarno (SD) region of the highly active bacteriophage T7 gene 10 ribosome binding site (RBS) to examine the possibility that the DB has an independent and functionally important role. Eradication of the SD sequence in the absence of a DB abolished the translational activity of RBS fragments that were fused to a dihydrofolate reductase reporter gene. In contrast, an optimized DB at various positions downstream of the initiation codon promoted highly efficient protein synthesis despite the lack of a SD region. The DB was not functional when shifted upstream of the initiation codon to the position of the SD sequence. Nucleotides 1469-1483 of 16S rRNA ('anti-downstream box') are complementary to the DB, and optimizing this complementarity strongly enhanced translation in the absence and presence of a SD region. We propose that the stimulatory interaction between the DB and the anti-DB places the start codon in close contact with the decoding region of 16S rRNA, thereby mediating independent and efficient initiation of translation.