Characterization of FapR, a positive regulator of expression of the 987P operon in enterotoxigenic Escherichia Coli

Expression of the 987P gene cluster is activated by the adjacent IS1 element of an ST_pa transposon. Nucleotide sequence analysis of the 987P-DNA region contiguous with this IS1 element revealed the presence of an open reading frame designated fapR, encoding a basic protein of 260 amino acid residues with a molecular mass of 30349 Daltons. The gene product, FapR, possesses similarity to a number of positive regulators of gene expression: VirF, Rns, AppY and EnvY. Moreover, a 43-amino-acid residue sequence in the C-terminal part of FapR is similar to the C-terminal domain of AraC, RhaR, and RhaS. Expression of fapR is dependent on the adjacent IS1 element. The FapR protein appears to be required for activation of the silent promoter of the fimbrial subunit gene, fapC     

Cloning,expression, and functional characterization of the Mycobacterium tuberculosis secA gene

To better understand the protein secretion mechanisms involved in the growth and pathogenesis of Mycobacterium tuberculosis, we examined the secA gene from M. tuberculosis (tbsecA; cosmid sequence accession No. z95121.gb_ba). We generated plasmids containing the full-length tbsecA gene or a fusion containing the 5' sequence from the M. tuberculosis secA gene and the remainder from the Escherichia coli secA gene and evaluated the ability of each construct to complement the defective SecA protein in E. coli MM52ts when grown at the non-permissive temperature. The full-length tbsecA gene was unable to compensate for the temperature-sensitive defect, whereas E. coli MM52ts that has been transformed with plasmid pMF8TB226 containing a chimeric secA gene was able to grow at 42 degrees C. This work confirms that the topography of SecA and its ATP binding sites are highly conserved, whereas its membrane insertion domains are species specific.     

Genetic analysis of superoxide dismutase, the 23 kilodalton antigen of Mycobacterium tuberculosis

The gene encoding a 23 kilodalton protein antigen has been cloned from Mycobacterium tuberculosis by screening of a recombinant DNA library with monoclonal antibodies. The product of the gene has been identified as the superoxide dismutase (SOD) of M. tuberculosis on the basis of sequence comparison and by expression of the recombinant protein in a functionally active form. The derived amino acid sequence of M. tuberculosis SOD reveals a close similarity to manganese-containing SODs from other organisms, in spite of the fact that previous studies using the purified enzyme have identified iron as the preferred metal ion ligand. SOD is present in the extracellular fluid of logarithmic-phase cultures of M. tuberculosis, but the structural gene is not preceded by a signal peptide sequence. Insertion of the M. tuberculosis SOD gene into a novel shuttle vector demonstrated the mycobacteria but is ineffective in Escherichia coli.     

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.     

Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG.

A gene encoding a protein homologous to the periplasmic ABC phosphate binding receptor PstS from Escherichia coli was cloned and sequenced from a lambda gt11 library of Mycobacterium tuberculosis by screening with monoclonal antibody 2A1-2. Its degree of similarity to the E. coli PstS is comparable to those of the previously described M. tuberculosis phosphate binding protein pab (Ag78, Ag5, or 38-kDa protein) and another M. tuberculosis protein which we identified recently. We suggest that the three M. tuberculosis proteins share a similar function and could be named PstS-1, PstS-2, and PstS-3, respectively. Molecular modeling of their three-dimensional structures using the structure of the E. coli PstS as a template and their inducibility by phosphate starvation support this view. Recombinant PstS-2 and PstS-3 were produced and purified by affinity chromatography. With PstS-1, these proteins were used to demonstrate the specificity of three groups of monoclonal antibodies. Using these antibodies in flow cytometry and immunoblotting analyses, we demonstrate that the three genes are expressed and their protein products are present and accessible at the mycobacterial surface as well as in its culture filtrate. Together with the M. tuberculosis genes encoding homologs of the PstA, PstB, and PstC components we cloned before, the present data suggest that at least one, and possibly several, related and functional ABC phosphate transporters exist in mycobacteria. It is hypothesized that the mycobacterial gene duplications presented here may be a subtle adaptation of intracellular pathogens to phosphate starvation in their alternating growth environments.     

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.     

The nucleotide sequence of a regulatory gene present on a plasmid in an enterotoxigenic Escherichia coli strain of serotype O167:H5

A DNA fragment that can functionally substitute for cfaD, the positive regulatory gene involved in expression of CFA/I fimbriae, has recently been cloned from an Escherichia coli strain of serotype O167:H5 that produces CS5 fimbriae. Nucleotide sequence determination showed that the fragment contained a gene, csvR (Coli Surface Virulence factor Regulator) homologous to the cfaD gene, which encoded for a protein of 301 amino acid residues. The csvR gene was found to be located between two different insertion sequences. Comparison of the amino acid sequence of the CsvR and CfaD proteins showed that CsvR is 34 amino acid residues longer at the C-terminus and, in the sequence, it also contains an insertion of two amino acid residues. The similarity between CfaD and Rns, the positive regulator of CS1 and CS2 expression, is much higher (97%) than between CsvR and CfaD (87%). This is reflected by the fact that the level of expression of CFA/I fimbriae induced by CsvR is not as high as when expression is induced by CfaD or Rns.     

A new subfamily of short bacterial adenylate kinases with the Mycobacterium tuberculosis enzyme as a model: A predictive and experimental study.

The adk gene from Mycobacterium tuberculosis codes for an enzyme of 181 amino acids. A sequence comparison with 52 different forms of adenylate kinases (AK) suggests that the enzyme from M. tuberculosis belongs to a new subfamily of "short" bacterial AKs. The recombinant protein, overexpressed in Escherichia coli, exhibits a low catalytic activity and an unexpectedly high thermal stability (Tm = 64.8 degrees C). Based on various spectroscopic data, on the known three-dimensional structure of the AK from E. coli and on secondary structure predictions for various sequenced AKs, we propose a structural model for AK from M. tuberculosis (AKmt). Proteins 1999;36:238-248.     

Expression and purification of immunologically reactive DPPD, a recombinant Mycobacterium tuberculosis skin test antigen, using Mycobacterium smegmatis and Escherichia coli host cells

DPPD is a Mycobacterium tuberculosis recombinant antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both guinea pigs and humans infected with M. tuberculosis. In addition, earlier clinical studies with DPPD suggested that this molecule could improve the specificity of the tuberculin skin test, which is used as an important aid for the diagnosis of tuberculosis. However, these studies could only be performed with DPPD engineered as a fusion molecule with another Mycobacterium spp. protein because no expression of DPPD could be achieved as a single molecule or as a conventional fusion protein in any commercial system. Although recombinant fusion proteins are in general suitable for several biological studies, they are by definition not ideal for studies involving highly purified and defined polypeptide sequences. Here, we report two alternative approaches for the expression of immunologically reactive recombinant genuine DPPD. The first approach used the rapidly growing, nonpathogenic Mycobacterium smegmatis as host cells transformed with the pSMT3 plasmid vector containing the full-length DPPD gene. The second approach used Escherichia coli transformed with the pET-17b plasmid vector containing the DPPD gene engineered in a three-copy fusion manner in tandem with itself. Though at low levels, expression and purification of immunologically reactive DPPD in M. smegmatis could be achieved. More abundant expression and purification of DPPD as a homo-trimer molecule was achieved in E. coli (> or =2 mg/L of bacterial broth cultures). Interestingly, expression could only be achieved in host cells transformed with the DPPD gene containing its leader peptide. However, the expressed proteins lacked the leader sequence, which indicates that processing of the M. tuberculosis DPPD gene was accurately achieved and necessary in both M. smegmatis and E. coli. More importantly, the delayed type hypersensitivity reactions elicited by purified molecules in guinea pigs infected with M. tuberculosis were indistinguishable from that elicited by purified protein derivative. Because the DPPD gene is present only in the tuberculosis-complex organisms of the Mycobacterium genus, these highly purified molecules should be helpful in identifying individuals sensitized with tubercle bacilli.     

Characterization of the katG gene encoding a catalase-peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis.

The isoniazid susceptibility of Mycobacterium tuberculosis is mediated by the product of the katG gene which encodes the heme-containing enzyme catalase-peroxidase. In this study, the chromosomal location of katG has been established and its nucleotide sequence has been determined so that the primary structure of catalase-peroxidase could be predicted. The M. tuberculosis enzyme is an 80,000-dalton protein containing several motifs characteristic of peroxidases and shows strong similarity to other bacterial catalase-peroxidases. Expression of the katG gene in M. tuberculosis, M. smegmatis, and Escherichia coli was demonstrated by Western blotting (immunoblotting). Homologous genes were detected in other mycobacteria, even those which are naturally insensitive to isoniazid.     

Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis

Unraveling of microbial genome data has indicated that two distantly related truncated hemoglobins (trHbs), HbN and HbO, might occur in many species of slow-growing pathogenic mycobacteria. Involvement of HbN in bacterial defense against NO toxicity and nitrosative stress has been proposed. A gene, encoding a putative HbN homolog with conserved features of typical trHbs, has been identified within the genome sequence of fast-growing mycobacterium, Mycobacterium smegmatis. Sequence analysis of M. smegmatis HbN indicated that it is relatively smaller in size and lacks N-terminal pre-A region, carrying 12-residue polar sequence motif that is present in HbN of M. tuberculosis. HbN encoding gene of M. smegmatis was expressed in E. coli as a 12.8kD homodimeric heme protein that binds oxygen reversibly with high affinity (P50 approximately 0.081 mm Hg) and autooxidizes faster than M. tuberculosis HbN. The circular dichroism spectra indicate that HbN of M. smegmatis and M. tuberculosis are structurally similar. Interestingly, an hmp mutant of E. coli, unable to metabolize nitric oxide, exhibited very low NO uptake activity in the presence of M. smegmatis HbN as compared to HbN of M. tuberculosis. On the basis of cellular heme content, specific nitric oxide dioxygenase (NOD) activity of M. smegmatis HbN was nearly one-third of that from M. tuberculosis. Additionally, the hmp mutant of E. coli, carrying M. smegmatis HbN, exhibited nearly 10-fold lower cell survival under nitrosative stress and nitrite derived reactive nitrogen species as compared to the isogenic strain harboring HbN of M. tuberculosis. Taken together, these results suggest that NO metabolizing activity and protection provided by M. smegmatis HbN against toxicity of NO and reactive nitrogen is significantly lower than HbN of M. tuberculosis. The lower efficiency of M. smegmatis HbN for NO detoxification as compared to M. tuberculosis HbN might be related to different level of NO exposure and nitrosative stress faced by these mycobacteria during their cellular metabolism.     

结核分枝杆菌4种抗原的基因克隆、表达、纯化和抗原性初步检测

目的:克隆38kD、ESAT-6、CFP10和MPT64等4种结核分枝杆菌抗原基因,利用大肠杆菌表达系统分别表达重组蛋白,纯化并初步评价其抗原性。方法:通过PCR方法从结核分枝杆菌H37Rv株基因组中扩增38kD、ESAT-6、CFP10和MPT64抗原的基因,连接入pBVIL1表达载体,在大肠杆菌HB101株中进行表达,以间接ELISA方法初步评价其抗原性。结果:获得了结核分枝杆菌抗原38kD、ESAT-6、CFP10和MPT64的基因,并在大肠杆菌中进行了高效表达,初步验证所纯化获得的抗原具有良好的抗原性。结论:pBVIL1表达载体可以高效表达多种结核分枝杆菌抗原,38kD、ESAT-6和CFP10抗原均可作为结核病血清学诊断的候选抗原。     

Evidence for horizontal transfer of the EcoT38I restriction-modification gene to chromosomal DNA by the P2 phage and diversity of defective P2 prophages in Escherichia coli TH38 strains

A DNA fragment carrying the genes coding for a novel EcoT38I restriction endonuclease (R.EcoT38I) and EcoT38I methyltransferase (M.EcoT38I), which recognize G(A/G)GC(C/T)C, was cloned from the chromosomal DNA of Escherichia coli TH38. The endonuclease and methyltransferase genes were in a head-to-head orientation and were separated by a 330-nucleotide intergenic region. A third gene, the C.EcoT38I gene, was found in the intergenic region, partially overlapping the R.EcoT38I gene. The gene product, C.EcoT38I, acted as both a positive regulator of R.EcoT38I gene expression and a negative regulator of M.EcoT38I gene expression. M.EcoT38I purified from recombinant E. coli cells was shown to be a monomeric protein and to methylate the inner cytosines in the recognition sequence. R.EcoT38I was purified from E. coli HB101 expressing M.EcoT38I and formed a homodimer. The EcoT38I restriction (R)-modification (M) system (R-M system) was found to be inserted between the A and Q genes of defective bacteriophage P2, which was lysogenized in the chromosome at locI, one of the P2 phage attachment sites observed in both E. coli K-12 MG1655 and TH38 chromosomal DNAs. Ten strains of E. coli TH38 were examined for the presence of the EcoT38I R-M gene on the P2 prophage. Conventional PCR analysis and assaying of R activity demonstrated that all strains carried a single copy of the EcoT38I R-M gene and expressed R activity but that diversity of excision in the ogr, D, H, I, and J genes in the defective P2 prophage had arisen.