Slow induction of RecA by DNA damage in Mycobacterium tuberculosis.

In mycobacteria, as in most bacterial species, the expression of RecA is induced by DNA damage. However, the authors show here that the kinetics of recA induction in Mycobacterium smegmatis and in Mycobacterium tuberculosis are quite different: whilst maximum expression in M. smegmatis occurred 3-6 h after addition of a DNA-damaging agent, incubation for 18-36 h was required to reach peak levels in M. tuberculosis. This is despite the fact that the M. tuberculosis promoter can be activated more rapidly when transferred to M. smegmatis. In addition, it is demonstrated that in both species the DNA is sufficiently damaged to give maximum induction within the first hour of incubation with mitomycin C. The difference in the induction kinetics of recA between the two species was mirrored by a difference in the levels of DNA-binding-competent LexA following DNA damage. A decrease in the ability of LexA to bind to the SOS box was readily detected by 2 h in M. smegmatis, whilst a decrease was not apparent until 18-24 h in M. tuberculosis and then only a very small decrease was observed.

Identification of some DNA damage-inducible genes of Mycobacterium tuberculosis: apparent lack of correlation with LexA binding.

The repair of DNA damage is expected to be particularly important to intracellular pathogens such as Mycobacterium tuberculosis, and so it is of interest to examine the response of M. tuberculosis to DNA damage. The expression of recA, a key component in DNA repair and recombination, is induced by DNA damage in M. tuberculosis. In this study, we have analyzed the expression following DNA damage in M. tuberculosis of a number of other genes which are DNA damage inducible in Escherichia coli. While many of these genes were also induced by DNA damage in M. tuberculosis, some were not. In addition, one gene (ruvC) which is not induced by DNA damage in E. coli was induced in M. tuberculosis, a result likely linked to its different transcriptional arrangement in M. tuberculosis. We also searched the sequences upstream of the genes being studied for the mycobacterial SOS box (the binding site for LexA) and assessed LexA binding to potential sites identified. LexA is the repressor protein responsible for regulating expression of these SOS genes in E. coli. However, two of the genes which were DNA damage inducible in M. tuberculosis did not have identifiable sites to which LexA bound. The absence of binding sites for LexA upstream of these genes was confirmed by analysis of LexA binding to overlapping DNA fragments covering a region from 500 bp upstream of the coding sequence to 100 bp within it. Therefore, it appears most likely that an alternative mechanism of gene regulation in response to DNA damage exists in M. tuberculosis.

Splicing before import - an intein in a mitochondrially targeted preprotein folds and is catalytically active in the cytoplasm in vivo.

Nuclear-encoded mitochondrial proteins are cytoplasmically synthesized and imported into the organelle. The intein-containing RecA protein of Mycobacterium tuberculosis, with or without the CoxIVp mitochondrial targeting signal (MTS), was used to determine where a protein targeted to mitochondria folds and becomes catalytically active. Analysis of fractions from Saccharomyces cerevisiae cells expressing RecA without the MTS revealed that RecA and intein proteins remained cytoplasmic. With the MTS, most of RecA was directed to mitochondria, while most of the intein remained in the cytoplasm. The intein therefore folds into a catalytically active state in the cytoplasm prior to RecA import into mitochondria.

In vivo splicing and functional characterization of Mycobacterium leprae RecA.

The RecA proteins from Mycobacterium tuberculosis and Mycobacterium leprae contain inteins. In contrast to the M. tuberculosis RecA, the M. leprae RecA is not spliced in Escherichia coli. We demonstrate here that M. leprae RecA is functionally spliced in Mycobacterium smegmatis and produces resistance toward DNA-damaging agents and homologous recombination.

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function.

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX, which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA. The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se.

Determination of DNA sequences required for regulated Mycobacterium tuberculosis RecA expression in response to DNA-damaging agents suggests that two modes of regulation exist.

The recA gene of Mycobacterium tuberculosis has previously been cloned and sequenced (E. O. Davis, S. G. Sedgwick, and M. J. Colston, J. Bacteriol. 173:5653-5662, 1991). In this study, the expression of this gene was shown to be inducible in response to various DNA-damaging agents by using a transcriptional fusion to the reporter gene encoding chloramphenicol acetyltransferase. A segment of DNA around 300 bp upstream of the coding region was shown to be required for expression. However, primer extension analysis indicated that the transcriptional start sites were 47 and 93 bp upstream of the translation initiation codon. Sequence motifs with homology to two families of Escherichia coli promoters but also with significant differences were located near these proposed transcription start sites. The differences from the E. coli consensus patterns would explain the previously described lack of expression of the M. tuberculosis recA gene from its own promoter in E. coli. In addition, the M. tuberculosis LexA protein was shown to bind specifically to a sequence, GAAC-N4-GTTC, overlapping one of these putative promoters and homologous to the Bacillus subtilis Cheo box involved in the regulation of SOS genes. The region of DNA 300 bp upstream of the recA gene was shown not to contain a promoter, suggesting that it functions as an upstream activator sequence.

Mycobacterial recA is cotranscribed with a potential regulatory gene called recX.

The recA gene of Mycobacterium smegmatis has been cloned and sequenced. The amino acid sequence of the RecA protein is highly homologous to other RecA proteins. Three other potential open reading frames were identified. One of these showed extensive homology to a protein, HypB, involved in the incorporation of nickel into hydrogenases. Another, found downstream of and overlapping recA, was similar to a gene, recX, which has been proposed to play a regulatory role related to recA function. The homology between the M. smegmatis sequence and that of Mycobacterium tuberculosis extended upstream of the recA coding region for 140 bp including a motif identical to the Cheo-box consensus sequence which has been shown to bind LexA. In addition, the transcriptional start sites were found to be identical to those identified previously for M. tuberculosis. Transcriptional fusions to the reporter gene chloramphenicol acetyltransferase (CAT) revealed that recA was DNA-damage inducible and that expression required sequences at some distance from the mapped transcriptional start sites. Although a motif with only one mismatch to the Cheo box was found in the intergenic region between orf1 and orf2 these open reading frames were not DNA-damage inducible, nor was this motif required for regulation of recA expression. Gel retardation assays revealed that the reason for this was that LexA did not bind to this sequence containing a mismatch. Reverse transcription/polymerase chain reaction analysis of M. smegmatis RNA demonstrated that recA and orf3 (recX) are within the same transcriptional unit.

Clinical, pathological and epidemiological aspects of flood plain staggers, a corynetoxicosis of livestock grazing Agrostis avenacea.

Flood plain staggers, a corynetoxicosis of grazing livestock, occurred on flood plains of the Darling river in northern New South Wales between spring 1990 and autumn 1991, associated with the grazing of Agrostis avenacea with diseased inflorescences. Over this period 1722 cattle, 2466 sheep and 11 horses died on 31 farms. Clinical signs were similar in sheep and cattle, being characterised by intermittent episodes of cerebral convulsion superimposed on varying degrees of cerebellar dysfunction. Pathological changes were variable and non-specific, principally reflecting trauma and the generalised nature of the intoxication.

Protein splicing--the lengths some proteins will go to.

We review the recently discovered phenomenon of protein splicing which is the excision of an internal protein sequence at the protein level rather than at the RNA level. The means by which examples of protein splicing have been identified are described, and the similarities of the internally spliced protein products (or inteins) are discussed. Comparisons are made between inteins and group I RNA introns. We describe the evidence supporting excision of intiens by a post-translational autocatalytic reaction of a full length polypeptide precursor, rather than by RNA splicing. An examination is made of some of the proposed mechanism schemes and the supporting them presented.

Molecular genetics of a receptor protein for D-xylose, encoded by the gene xylF, in Escherichia coli.

Mutants of Escherichia coli K12 have been isolated containing either the 'XylF' or the 'XylE' transport systems for D-xylose. This enabled the XylF system containing a D-xylose receptor-binding protein to be associated with high-affinity transport, apparent Km 0.2-4 microM, while the XylE sugar-H+ symporter system exhibited relatively low affinity, apparent Km 63-169 microM. Their apparent Vmax values were similar. Isolation of a xylose transport-negative mutation in a downstream gene, xylG, and a series of transduction experiments enabled the gene order xylB xylA p/o xyl(FG) mtlA to be proposed near 80 min on the E. coli chromosome. The xylose receptor protein (XylF) was highly purified from the periplasm and the sequence of its N-terminal 44 amino acids determined. From this a mixed oligonucleotide (14 mer) was synthesised of corresponding DNA sequence, hybridisation of which to restriction fragments from the ColE I hybrid plasmid, pLC32-9, which conferred xylose transport activity on a xylE- xylF- double mutant, enabled precise location of the xylF gene next to the xylose isomerase gene, xylA (3984 kbp on the Kohara physical map). The DNA sequence of the 2.5 kb Bgl II fragment containing the xylF gene was then determined, which confirmed the above gene order and revealed two divergent operons, probably regulated in a co-ordinate manner, one containing enzymes for catabolism of D-xylose, and the other proteins comprising the XylFG receptor-transport system. Analysis of the 330 amino acid sequence of the XylF receptor protein located the scission point of its leader peptide between Ala23 and Lys24, and revealed significant homologies with the other sugar receptor proteins for ribose (RbsB), galactose (MglB) and arabinose (AraF).

The ins and outs of protein splicing elements.

Protein splicing involves the removal of an internal protein sequence from a precursor molecule and the ligation of the two flanking sequences to produce a mature protein product, in a post-translational event analogous to the removal of an intron from rRNA. Protein splicing introns, or 'inteins' appear to be a novel type of genetic element capable of mediating gene conversion of an 'intein-less' allele, and hence promoting their own dissemination. The mechanism by which protein splicing is achieved is probably entirely encoded within the internal protein sequence, or intein, and does not require other accessory molecules. Although the concept of protein splicing inteins as selfish genetic elements of no immediate consequence to the host organism has emerged, this interpretation is questioned by recent evidence that in at least one example there appears to have been selection for protein splicing.

Evidence of selection for protein introns in the recAs of pathogenic mycobacteria.

Protein introns are recently discovered genetic elements whose intervening sequences are removed from a precursor protein by an unusual protein splicing reaction. This involves the excision of a central spacer molecule, the protein intron, and the religation of the amino- and carboxy-terminal fragments of the precursor. The recA gene of Mycobacterium tuberculosis contains one such element and we now show that the other major mycobacterial pathogen, Mycobacterium leprae, also possesses a protein intron in its recA, although other mycobacterial recA genes do not. However, these two protein introns are different in size, sequence and location of insertion of their coding sequences into the recAs of M. tuberculosis and M. leprae, indicating that acquisition of the protein introns has occurred independently in the two species, and thus suggesting that there has been selection for splicing in the maturation of RecA in the pathogenic mycobacteria. The M. leprae protein intron provides an example of conditional protein splicing, splicing occurring in M. leprae itself but not when expressed in Escherichia coli, unlike most previously described protein introns. These observations suggest that protein introns may perform a function for their host, rather than being just selfish elements.

Establishing a nurse-managed health center: assuring excellence.

The roles of accreditation, clinical protocols, and quality assurance in maintaining high standards of quality in the nurse-managed health center are discussed. The components of a quality assurance program for the nurse-managed center are presented, including peer chart review, diagnosis-related review, and review of high-risk client encounters. Various methods of generating practice protocols for the nurse-managed center are outlined.

Cloning and sequencing of the gene which encodes the highly inducible acetamidase of Mycobacterium smegmatis.

The acetamidase of Mycobacterium smegmatis NCTC 8159 was purified, and the sequences of its amino-terminus and of two peptides obtained by proteolysis of the protein were obtained. A DNA fragment including the amidase structural gene was cloned in Escherichia coli, using oligonucleotide probes designed on the basis of the peptide sequences and a codon usage table calculated from published sequences of nine protein-antigen-encoding genes of the Mycobacterium tuberculosis complex. Sequence analysis of the cloned DNA revealed that the amidase gene encoded 406 amino acid residues. The nucleotide sequence close to and upstream of the amidase gene contained a probable ribosome-binding site but no identifiable promoter sequences. Three additional potential open-reading frames were found upstream of and very close to the amidase gene, with consensus '-35' and '-10' promoter sites between the first and second of these. It is hoped that the highly inducible expression of the acetamidase gene can be exploited to allow regulated expression of other genes cloned in mycobacteria.

Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence.

The M. tuberculosis recA locus comprises an 85 kd open reading frame but produced 38 kd RecA and 47 kd products in E. coli. No RNA processing was detected; rather, an 85 kd precursor protein was spliced, releasing a 47 kd spacer protein, and joining its terminal fragments to form mature RecA protein. "Spacer" protein was also produced in M. tuberculosis and from a hybrid spacer-LacZ alpha fusion molecule. Mutagenesis at codon wobble positions at one splice junction showed that protein rather than nucleotide sequence determined splicing activity. Other mutants defined additional regions needed for splicing and allowed processing to be followed. Splicing was essential for RecA activity in E. coli. The possibility that splicing is a manifestation of a novel class of genetic element is discussed.

Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product.

A fragment of Mycobacterium tuberculosis DNA containing recA-like sequences was identified by hybridization with the Escherichia coli recA gene and cloned. Although no expression was detected from its own promoter in E. coli, expression from a vector promoter partially complemented E. coli recA mutants for recombination, DNA repair, and mutagenesis, but not for induction of phage lambda. This clone produced a protein which cross-reacts with antisera raised against the E. coli RecA protein and was approximately the same size. However, the nucleotide sequence of the cloned fragment revealed the presence of an open reading frame for a protein about twice the size of other RecA proteins and the cloned product detected by Western blotting (immunoblotting). The predicted M. tuberculosis RecA protein sequence was homologous with RecA sequences from other bacteria, but this homology was not dispersed; rather it was localized to the first 254 and the last 96 amino acids, with the intervening 440 amino acids being unrelated. Furthermore, the junctions of homology were in register with the uninterrupted sequence of the E. coli RecA protein. Identical restriction fragments were found in the genomic DNAs of M. tuberculosis H37Rv and H37Ra and of M. bovis BCG. It is concluded that the ancestral recA gene of these species diversified via an insertional mutation of at least 1,320 bp of DNA. Possible processing mechanisms for synthesizing a normal-size RecA protein from this elongated sequence are discussed.

Analysis of three nodD genes in Rhizobium leguminosarum biovar phaseoli; nodD1 is preceded by noIE, a gene whose product is secreted from the cytoplasm.

In a strain of Rhizobium leguminosarum biovar phaseoli, three copies of the regulatory nodulation gene nodD were identified on the Sym plasmid and sequenced. Two were closely linked to each other and the third was near, but not adjacent, to the nodABC genes. Each of these nodD genes could correct the Nod- defect of a nodD mutant strain of R. leguminosarum biovar viciae on peas. A truncated form of nodD2 could also correct this mutant, indicating that the C-terminus of NodD2 is not needed for inducing activity. Upstream of nodD1 and in the same operon is a newly described gene, noIE, whose product appears to be exported into the periplasm. Close to nodD2 is another gene, noIP, with no known counterpart in other rhizobia. Both noIP and noIE-nodD1 are preceded by 'nod-box' sequences and, in the former case, there appear to be two tandemly repeated nod-box sequences. Mutations in each of the nodD genes and in the noIE and noIP genes did not abolish nodulation or nitrogen fixation on beans.

Identification, sequencing, and expression of Mycobacterium leprae superoxide dismutase, a major antigen.

The gene encoding a major 28-kilodalton antigen of Mycobacterium leprae has now been sequenced and identified as the enzyme superoxide dismutase (SOD) on the basis of the high degree of homology with known SOD sequences. The deduced amino acid sequence shows 67% homology with a human manganese-utilizing SOD and 55% homology with the Escherichia coli manganese-utilizing enzyme. The gene is not expressed from its own promoter in E. coli but is expressed from its own promoter in Mycobacterium smegmatis. The amino acid sequences of epitopes recognized by monoclonal antibodies against the 28-kilodalton antigen have been determined.

Regulatory functions of the three nodD genes of Rhizobium leguminosarum biovar phaseoli.

The three nodD genes of a strain of Rhizobium leguminosarum biovar phaseoli were cloned to study their effects on transcription of themselves and of the nodC genes of biovars phaseoli and viciae. Efficient transcription of nodD1 required nodD1 and was enhanced by exposure of the cells to bean exudate consistent with the presence of a nod-box preceding the noIE-nodD1 operon. Transcription of nodD2 and nodD3 was constitutive. nodC of R. leguminosarum biovar phaseoli was activated by each of the nodD genes of that biovar in the absence of inducers but expression was enhanced in cells grown with bean exudate or the flavonoids genistein or naringenin. A mutant of nodD2, lacking 60 bp at its 3' end, activated nodC in the presence of inducer, but was defective in regulating certain of the nodD genes. The nodC gene of R. leguminosarum biovar viciae responded differently to the various nodD genes of R. leguminosarum biovar phaseoli than did the nodC of the latter biovar.