Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysG region of the Escherichia coli K-12 chromosome.

The DNA sequence and derived amino-acid sequence of a 5618-base region in the 74-min area of the Escherichia coli chromosome has been determined in order to locate the structural gene, nirB, for the NADH-dependent nitrite reductase and a gene, cysG, required for the synthesis of the sirohaem prosthetic group. Three additional open reading frames, nirD, nirE and nirC, were found between nirB and cysG. Potential binding sites on the NirB protein for NADH and FAD, as well as conserved central core and interface domains, were deduced by comparing the derived amino-acid sequence with those of database proteins. A directly repeated sequence, which includes the motif -Cys-Xaa-Xaa-Cys-, is suggested as the binding site for either one [4Fe-4S] or two [2Fe-2S] clusters. The nirD gene potentially encodes a soluble, cytoplasmic protein of unknown function. No significant similarities were found between the derived amino-acid sequence of NirD and either NirB or any other protein in the database. If the nirE open reading frame is translated, it would encode a 33-amino-acid peptide of unknown function which includes 8 phenylalanyl residues. The product of the nirC gene is a highly hydrophobic protein with regions of amino-acid sequence similar to cytochrome oxidase polypeptide 1.

Transcriptional control of the cysG gene of Escherichia coli K-12 during aerobic and anaerobic growth.

The 74-min region of the Escherichia coli chromosome includes five open reading frames of known sequence. The first and last of these genes, nirB and cysG, are transcribed in the same direction and both are essential for NADH-dependent nitrite reductase activity. The functions of the other genes, nirD, nirE and nirC, which are located between nirB and cysG, are unknown. The nirB gene is transcribed from a promoter which is anaerobically induced, expression being dependent on the transcription activator protein, Fnr. Here we show that the nirD, nirE, nirC and cysG genes are also expressed from the nirB promoter. After subcloning cysG, a second promoter was located less than 100 bases upstream of cysG. Two groups of transcription start points separated by 40 bases were detected in this region by S1 mapping. Rates of transcription from the isolated cysG promoter were the same during aerobic growth and anaerobic growth in the presence or absence of nitrite. However, when the nirB gene and its promoter were cloned back upstream from the cysG promoter, the rate of transcription was higher during anaerobic growth than during aerobic growth and was further induced by nitrite. These increases were totally dependent on a functional fnr gene and were shown by S1 mapping experiments to be due to transcriptional read-through from the Fnr-dependent nirB promoter. No promoter activity was associated with DNA fragments between the BamHI site located within the N-terminal coding region of the nirB gene and the cysG promoter located at the C-terminus of nirC.

Molecular analysis of the Haemophilus influenzae type b pilin gene.

A Haemophilus influenzae DNA library was prepared in the vector lambda EMBL3, and recombinant phage were screened for the pilin gene (pil) using a synthetic oligonucleotide. Southern blot analysis of the positive clones revealed a 2.5kb PstI/PvuI fragment that hybridized with the oligonucleotide probe. This fragment was subcloned into pBR322 and sequenced. The nucleotide sequence disclosed an open reading frame of 653 bases. The deduced amino acid sequence corresponded with the known amino acid sequence of the purified pilin protein. Primer extension analysis using total RNA from piliated H. influenzae cells delineated a start site for the gene, -10 and -35 promoter regions, and a ribosome-binding site. No transcripts were seen with the RNA derived from a non-piliated strain. Southern blots of DNA from a number of H. influenzae strains revealed homology with the pil structural gene. DNA from a non-piliated strain of H. influenzae also hybridized with the pil probe. Transcriptional and translational studies were performed in Escherichia coli with plasmids containing: (i) the pil gene on the 2.5 kb PstI/PvuI fragment, (ii) the pil gene fused to the phoA gene, and (iii) the pil gene present on a 12.2 kb insert containing extensive H. influenzae DNA flanking the pil gene. The results suggest that the H. influenzae pil gene is expressed in Escherichia coli, but from a promoter other than the one used in H. influenzae.

Tn7 inserts in both orientations at a single chromosomal location and apparently forms cointegrates in Pasteurella multocida.

Pasteurella multocida transconjugants isolated after mating with Escherichia coli strains that carry one or the other of two Tn7-containing suicide plasmids, pRKTV5 and pUW964 (pRKTV5::Tn5), were analysed. These plasmids have the ColE1 replication origin and were thus expected to deliver transposons but not be maintained as free replicons in Pasteurella. Five out of six transconjugants selected for acquisition of Tn7 from E. coli (pRKTV5) had simple insertions of the transposon, in either orientation, at a single chromosomal location, while the sixth had pRKTV5 integrated at the same location. By contrast, all of 27 transconjugants selected for acquisition of either Tn7 or Tn5 from E. coli (pUW964) maintained pUW964. Of seven subsequently examined at the molecular level, all had pUW964 (in one case, a deletion derivative) integrated at the same location as the Tn7 insertions obtained with pRKTV5. A copy of Tn7 was present at each boundary between the integrated plasmids (pRKTV5 or pUW964) and the chromosome in each strain. The two copies of Tn7 at either end of an integrated plasmid were either in the same (six cases) or in opposite (two cases) orientations with respect to each other. These seem to be products of replicative transposition by Tn7 but can also derive from conservative mechanisms.

Structural and functional analysis of the mini-circle, a transposable element of Streptomyces coelicolor A3(2).

The mini-circle is a transposable element which is present in Streptomyces coelicolor A3(2) in both free circular and chromosomally integrated linear forms. The nucleotide sequences of the mini-circle and its preferred site of integration in the Streptomyces lividans TK64 chromosome were determined. Three putative open reading frames were identified in the mini-circle sequence. The mini-circle does not appear to cause a target site duplication on transposition and does not have perfect terminal inverted repeats. The observed site-specificity of the mini-circle is not mediated by extensive homology between the element and the chromosomal integration site. Transposition of the mini-circle into the S. lividans chromosome was demonstrated and found to be some two orders of magnitude less efficient than integration of the circular form of the element, suggesting that the circular form of the mini-circle might be a normal intermediate in the transposition process.

Extensive diversity of branched-RNA-linked multicopy single-stranded DNAs in clinical strains of Escherichia coli.

Recently it was shown that a clinical strain of Escherichia coli contains a reverse transcriptase that is essential for the synthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA). We now have examined 113 independent clinical isolates of E. coli for the existence of msDNA and found that 7 strains contained msDNA. Four of them were further analyzed by hybridization analysis, which indicated that three of the msDNAs were different, having little sequence homology. When the reverse transcriptase gene associated with one of these msDNAs was used as a probe, it did not hybridize with chromosomal DNA from the other strains containing msDNA. These results indicate that some clinical E. coli strains carry their own unique msDNA-synthesizing systems; msDNAs produced by these systems have little, if any, sequence homology in their RNA and DNA molecules and the reverse transcriptases required for the production of msDNA also have little sequence similarity. Such extensive diversity of the msDNA-synthesizing systems supports the notion that they were acquired by the E. coli genome late during the evolution of E. coli.

Human milk immunoglobulin A antibodies to Shigella virulence determinants.

Because human milk is thought to protect infants from shigellosis, we evaluated milk for immunoglobulin A to Shigella virulence determinants. Milk was preincubated to remove antibodies unrelated to each locus of interest, using defined Shigella and E. coli hybrids containing known Shigella genetic segments prior to immunoblotting. The milk could not be shown to contain antibodies to chromosomally encoded virulence loci except for the expected antibodies to the products of the histidine locus. However, all the milk samples contained antibodies to antigens encoded by the large virulence plasmid. The finding of these antibodies suggests a possible mechanism by which human milk might protect infants.

Reisolation and characterization of Clostridium longisporum, a ruminal sporeforming cellulolytic anaerobe.

Two strictly anaerobic strains of ruminal cellulolytic bacteria were isolated which are very similar to the original description given for Clostridium longisporum. Vegetative cells were 1 micron wide by 5 to 15 microns long. Subterminal spores were observed only when an insoluble carbon source was provided for growth. Besides cellulose, the organisms fermented cellobiose, glucose, galactose, fructose, mannose, pectin, salicin and sucrose. Xylan and xylose were not fermented. Fermentation products from glucose or alfalfa cell walls included formate, acetate, butyrate, ethanol, H2 and CO2. The GC content was 23% for one strain and 33% for the other. These isolates hydrolyzed cell wall fractions of alfalfa, in particular, hemicellulose, more rapidly and extensively than other ruminal cellulolytic species examined.

Characterisation of methicillin-resistant Staphylococcus aureus isolates by restriction endonuclease digestion of chromosomal DNA.

Clinical isolates of Staphylococcus aureus from the London Hospital were characterised by genetic analysis of antibiotic-resistance determinants and by restriction endonuclease digestion of chromosomal DNA and compared with isolates from elsewhere in the UK. Restriction enzyme digestion of chromosomal DNA confirmed that a single strain of methicillin-resistant S. aureus (MRSA) persists at the London Hospital, although its antibiotic-resistance profile and plasmid carriage are not constant. Methicillin-sensitive isolates, on the other hand, each had readily distinguishable and unique DNA restriction patterns. The DNA restriction digest pattern of the London Hospital MRSA isolates was identical to that of "epidemic" (E) MRSA isolates from the Thames regions. By contrast, other MRSA isolates had DNA restriction patterns which differed from those of EMRSA isolates and from each other. These results confirm the discriminatory value of restriction pattern analysis as a typing method.

Mapping of Escherichia coli chromosomal Tn5 and F insertions by pulsed field gel electrophoresis.

A low resolution Not I physical map of Escherichia coli was recently constructed. In this report we demonstrated that this map can be used to map Tn5 and F insertions physically. The transposon, Tn5, contains Not I recognition sequences in its IS50 sequences. F plasmid contains an unmapped Not I site. Hence, the location of Tn5 and F in the chromosome can be mapped by identifying the location of the introduced Not I sites using pulsed field gel electrophoresis. The physical mapping of genetically mapped Tn5 insertions confirm the previously constructed Not I map and helps align the E. coli physical and genetic maps. The use of Tn5 can assist the construction of both physical and genetic maps for microorganisms lacking such maps. Variations on this approach will facilitate physical mapping with a wide variety of organisms, enzymes, and genetic elements.

Analysis and genetic manipulation of Shigella virulence determinants for vaccine development.

Shigellosis is a major public health problem in developing countries. Current epidemics of Shigella dysenteriae serotype 1 strains are particularly serious and are characterized by high mortality rates. A high proportion of the isolates are resistant to many of the antibiotics currently in use in these countries, a feature which seriously compromises clinical treatment of the infections. Efficacious vaccines are thus urgently needed. Basic studies on Shigella virulence factors, infections in laboratory models, and host responses has led to the development of several strategies for the production of vaccines. All of these are live oral vaccines involving bacteria capable of at least limited survival in the animal intestine and of carrying selected antigens to the mucosal immune system. One type of vaccine involves non-pathogenic shigellae, attenuated either by introduction of a requirement for aromatic amino acids (aroD) or by loss of the large plasmid that specifies bacterial invasion of the mucosal epithelium. S. dysenteriae 1 strains under development as vaccines need to be engineered to eliminate high level Shiga toxin production, and a rapid and effective method to achieve this was recently elaborated. The second type of vaccine is represented by hybrid strains consisting of a carrier organism, such as an attenuated Salmonella or an Escherichia coli K-12 strain carrying the Shigella invasion plasmid, and the selected foreign antigen that it produces, in all cases so far the Shigella O antigen polysaccharide.

Relaxation of DNA torsional tension in defined domains of bacterial chromosomes in vivo.

A procedure is described for selectively relaxing the DNA torsional tension in defined regions of the chromosome of living bacterial cells. Regions of the chromosomal DNA labelled with bromodeoxyuridine are selectively nicked by irradiation of the cells with long-wavelength ultraviolet light and then trimethylpsoralen residues are photobound to the chromosome in vivo. It is demonstrated that the rate of photobinding to the bromouridine-labelled parts of the chromosomes declines relative to the unlabelled parts of the same chromosomes as nicks are introduced into the former regions. The maximal difference in photobinding rates is that expected for the difference between relaxed and negatively supercoiled DNA. Analysis of the number of DNA breaks required for minimizing the photobinding rates permits a calculation of the number of domains of supercoiling per Bacillus subtilis chromosome.

Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene.

The gene encoding lipoamide dehydrogenase from Azotobacter vinelandii has been cloned in Escherichia coli. Fragments of 9-23 kb from Azotobacter vinelandii chromosomal DNA obtained by partial digestion with Sau3A were ligated into the BamHI site of plasmid pUC9. E. coli TG2 cells were transformed with the resulting recombinant plasmids. Screening for clones which produced A. vinelandii lipoamide dehydrogenase was performed with antibodies raised against the purified enzyme. A positive colony was found which produced complete chains of lipoamide dehydrogenase as concluded form SDS gel electrophoresis of the cell-free extract, stained for protein or used for Western blotting. After subcloning of the 14.7-kb insert of this plasmid the structural gene could be located on a 3.2-kb DNA fragment. The nucleotide sequence of this subcloned fragment (3134 bp) has been determined. The protein-coding sequence of the gene consists of 1434 bp (478 codons, including the AUG start codon and the UAA stop codon). It is preceded by an intracistronic region of 85 bp and the structural gene for succinyltransferase. A putative ribosome-binding site and promoter sequence are given. The derived amino acid composition is in excellent agreement with that previously published for the isolated enzyme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three-dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483-496], it appears that essential residues in all four domains have been conserved. The enzyme is strongly expressed, although expression does not depend on the vector-encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.

Sequence features of the replication terminus of the Bacillus subtilis chromosome.

The sequence of 1267 nucleotides spanning the replication terminus, terC, of the Bacillus subtilis 168 chromosome has been determined. The site of arrest of the clockwise fork, which defines terC, has been localized to a 30-nucleotide portion (approximately) within this sequence. The arrest site occurs in an A + T-rich region between two open reading frames and very close to one of two imperfect inverted repeats (47-48 nucleotides each) which are separated by 59 nucleotides. The closeness of approach of the arrested clockwise fork to the first imperfect inverted repeat encountered in this region raises the possibility of a role for the inverted repeats in the mechanism of fork arrest.

Molecular cloning of a species-specific DNA probe for Campylobacter jejuni.

Conventional procedures for isolating and identifying Campylobacter jejuni are cumbersome and time-consuming. A simpler approach would be to use DNA probes to identify these organisms. To obtain such probes we cloned chromosomal DNA from C. jejuni into the lambda replacement vector EMBL 4. Recombinant phages were screened for C. jejuni-specific inserts by DNA hybridization using chromosomal DNA from either C. jejuni or C. coli which had been radioactively labelled with 32P. By this means, recombinant phages were identified which hybridized to C. jejuni but not to C. coli DNA. These phages were then subjected to further screening using DNA from other Campylobacter species. Three DNA fragments were identified which hybridized to DNA from eight ATCC (American Type Culture Collection) strains of C. jejuni but not to DNA from C. coli, C. laridis, C. fetus or a variety of other bacterial species. These DNA fragments are suitable for use as specific probes in DNA-based diagnostic tests for C. jejuni infections.

Phase variation in Bordetella pertussis is accompanied by changes in DNA modification.

Pathogenic strains of Bordetella pertussis tend to undergo a phase variation process when propagated in vitro. The phase variants do not express part or all of the virulence factors of the pathogenic strain and are phenotypically stable. We have previously shown that variation involves a non-reversible, non-random process. In an attempt to characterize the molecular changes accompanying phase variation, chromosomal DNA, isolated from B. pertussis and its variants, was digested with a variety of restriction enzymes followed by agarose gel electrophoresis. While variant DNA was digested by all tested enzymes, pathogenic strain DNA was not digested by part of the enzymes, thus suggesting modification of the DNA at specific sites. DNA isolated from reversible, growth medium induced variants, demonstrated sensitivity to digestion identical to that of spontaneous, stable variants. Analysis of the restriction sequences of all the enzymes, which did not digest DNA from pathogenic strains, failed to reveal any common sequence known to be affected by methylation. HPLC and nearest-neighbor analysis showed a 2-fold increase in the level of DNA methylation in the pathogenic strain. It was concluded that (a) the chromosomal DNA in virulent strains of B. pertussis is protected against enzymatic digestion by an as yet unknown modification and (b) variation in B. pertussis may be caused by changes in the modification of the DNA rather than by mutation.