- Volume 148, Issue 2, 2002
Volume 148, Issue 2, 2002
- Mini-Review
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- Microbiology Comment
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- Research Paper
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Brucella abortus strain 2308 produces brucebactin, a highly efficient catecholic siderophore
More LessThe GenBank accession number for the sequence reported in this paper is AF361942.
Brucella abortus is known to produce 2,3-dihydroxybenzoate (2,3-DHBA) and to use this catechol as a siderophore to grow under iron-limited conditions. In this study a mutant (BAM41) is described that is deficient in siderophore production by insertion of Tn5 in the virulent B. abortus strain 2308. This mutant was unable to grow on iron-deprived medium and its growth could not be restored by addition of 2,3-DHBA. Production of catecholic compounds by both the Brucella mutant and parental strains under iron-deprivation conditions was assayed by TLC. Two catecholic substances were identified in the supernatant of the parental strain 2308. The faster migrating spot showed the same retention factor (R f) as that of purified 2,3-DHBA. The mutant BAM41 overproduced 2,3-DHBA, but failed to form the slower migrating catechol. This defect could only be complemented by the addition of the slow-migrating catechol from strain 2308. The genomic region containing Tn5 in BAM41 was cloned and the position of the transposon was determined by nucleotide sequencing. The sequence revealed that the insertion had occurred at a gene with homology to Escherichia coli entF, a locus involved in the late steps of the biosynthesis of the complex catecholic siderophore enterobactin. Intracellular survival and growth rates of the B. abortus wild-type and entF mutant strains in mouse-derived J774 macrophages were similar, indicating that production of this siderophore was not essential in this model of infection. It is concluded that B. abortus synthesizes a previously unknown and highly efficient catecholic siderophore, different from 2,3-DHBA, for which the name brucebactin is proposed.
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‘Streptomyces nanchangensis’, a producer of the insecticidal polyether antibiotic nanchangmycin and the antiparasitic macrolide meilingmycin, contains multiple polyketide gene clusters
More LessSeveral independent gene clusters containing varying lengths of type I polyketide synthase genes were isolated from ‘Streptomyces nanchangensis’ NS3226, a producer of nanchangmycin and meilingmycin. The former is a polyether compound similar to dianemycin and the latter is a macrolide compound similar to milbemycin, which shares the same macrolide ring as avermectin but has different side groups. Clusters A–H spanned about 133, 132, 104, 174, 122, 54, 37 and 59 kb, respectively. Two systems were developed for functional analysis of the gene clusters by gene disruption or replacement. (1) Streptomyces phage ϕC31 and its derived vectors can infect and lysogenize this strain. (2) pSET152, an Escherichia coli plasmid with ϕC31 attP site, and pHZ1358, a Streptomyces–Escherichia coli shuttle cosmid vector, both carrying oriT from RP4, can be mobilized from E. coli into NS3226 by conjugation. pHZ1358 was shown to be generally useful for generating mutant strains by gene disruption and replacement in NS3226 as well as in several other Streptomyces strains. A region in cluster A (∼133 kb) seemed to be involved in nanchangmycin production because replacement of several DNA fragments in this region by an apramycin resistance gene [aac3(IV)] gave rise to nanchangmycin non-producing mutants.
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Analysis of a C-methyltransferase gene (aviG1) involved in avilamycin biosynthesis in Streptomyces viridochromogenes Tü57 and complementation of a Saccharopolyspora erythraea eryBIII mutant by aviG1
More LessThe GenBank accession number for the sequence reported in this paper is AF333038.
Streptomyces viridochromogenes Tü57 is the principal producer of avilamycin A. aviG1, a putative methyltransferase gene, was detected in the avilamycin biosynthetic gene cluster. To determine the function of aviG1, a targeted gene inactivation experiment was performed. The resulting chromosomal mutant, carrying an in-frame deletion in aviG1, was deficient in avilamycin production. aviG1 was used to complement an eryBIII mutant of the erythromycin A producer Saccharopolyspora erythraea [Gaisser, S., Bohm, G. A., Doumith, M., Raynal, M. C., Dhillon, N., Cortes, J. & Leadlay, P. F. (1998) R12 . Mol Gen Genet 258, 78–88]. The presence of erythromycin A in the culture supernatant of the complemented mutant indicated that L-mycarose biosynthesis could be restored and that AviG1 could take over the function of the C-methyltransferase EryBIII.
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Osmotic regulation of the Streptomyces lividans thiostrepton-inducible promoter, ptipA
More LessTranscriptional activation of the thiostrepton-inducible promoter, ptipA, in Streptomyces lividans is mediated by TipAL. This transcriptional activator belongs to the MerR/SoxR family that characteristically binds an operator sequence located between the −10 and −35 hexamers normally occupied by RNA polymerase. As for the Escherichia coli merT promoter, the ptipA hexamers are separated by a long 19 bp spacer and hence a topological transition of the DNA is likely to be a requisite for alignment with RNA polymerase. Growth conditions that could facilitate this conformational change were investigated using transcriptional fusions of ptipA with reporter genes. Adjustment of growth medium osmolarity led to increased and prolonged TipAL-dependent expression, both with and without the inducer, thiostrepton. These effects correlated with increases in negative DNA supercoiling. Moreover, an inability to induce the promoter with thiostrepton in strain TK64 was corrected by increasing the concentration of osmolyte, compensating for an apparent reduced level of negative DNA supercoiling in the strain. Prolonging the time of activation of tipA in the wild-type by manipulating growth conditions revealed that mycelial autolysis could be induced by thiostrepton in 4-d-old cultures.
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Streptomyces spp. contain class Ia and class II ribonucleotide reductases: expression analysis of the genes in vegetative growth
The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this paper are AJ224870, AJ276618, AJ277778, AJ295338 and AJ295339.
Genes encoding two ribonucleotide reductases (RNRs) were identified in members of the genus Streptomyces. One gene, nrdJ, encoded an oligomeric protein comprising four identical subunits each with a molecular mass of ∼108 kDa. The activity of this protein depended on the presence of 5′-deoxyadenosylcobalamine (coenzyme B12), establishing it as a class II RNR. The Streptomyces clavuligerus nrdJ gene was cloned, using internal peptide sequences from the purified protein, and was found to encode a polypeptide of 961 aa. Molecular phylogenetic analysis showed that the S. clavuligerus class II RNR shares significant similarity with most other bacterial and archaeal class II RNRs. Two other genes, nrdA and nrdB, were initially identified in the Streptomyces coelicolor genome database in unannotated ORFs as encoding a class Ia RNR. Southern analysis demonstrated that the nrdAB genes were present in different Streptomyces spp. The S. coelicolor nrdAB genes were cloned and expressed in Escherichia coli, and the recombinant proteins were shown to represent a class I RNR. It was shown, using quantitative real-time PCR, that the S. clavuligerus class Ia and class II RNR genes were differentially transcribed during vegetative growth. The copy number of the class II nrdJ transcripts was approximately constant throughout the exponential phase of vegetative growth (3–5×105 copies per 400 ng total RNA after reverse transcription). In contrast, the copy number of the class Ia nrdAB transcripts was some 10- to 20-fold less than that of nrdJ in the early-exponential growth phase (2·8×104 copies), and decreased markedly at the mid-exponential (4×103 copies) and late-exponential phases (1·1×103 copies) of growth. A possible role for the involvement of two RNRs during vegetative growth is discussed.
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Nuclease activities and cell death processes associated with the development of surface cultures of Streptomyces antibioticus ETH 7451
More LessThe presence and significance of developmentally regulated nucleases in Streptomyces antibioticus ETH 7451 has been studied in relation to the lytic processes occurring during differentiation. The cell-death processes have been followed in surface cultures by a propidium iodide viability assay. This has allowed the visualization of dead (membrane-damaged, red fluorescent) and live (membrane-intact, green fluorescent) mycelium during development, and has facilitated the analysis of the role of nucleases in these processes. A parallel activity-gel analysis showed the appearance of 20–22 kDa, 34 kDa and 44 kDa nucleases, the latter appearing only when aerial mycelium is formed. The appearance of these nucleases shows a remarkable correlation with the death process of the mycelium during differentiation and with chromosomal DNA degradation. The 20–22 kDa enzymes are possibly related to the lytic phenomena taking place in the vegetative substrate mycelium before the emergence of the reproductive aerial mycelium, whereas the function of the 44 kDa nuclease seems to be related to the sporulation step. The 20–22 kDa nucleases require Ca2+ for activity and are inhibited by Zn2+. The nucleases are loosely bound to the cell wall from where they can be liberated by simple washing. Conceivably, these enzymes work together and co-ordinate to achieve an efficient hydrolysis of DNA from dying cells. The results show that the biochemical reactions related with the lytic DNA degradation during the programmed cell death are notably conserved in Streptomyces. Some of the features of the process and the biochemical characteristics of the enzymes involved are analogous to those taking place during the DNA fragmentation processes in eukaryotic apoptotic cells.
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Kinetics of binding, uptake and degradation of live fluorescent (DsRed) bacteria by Dictyostelium discoideum
More LessThe kinetics of binding, uptake and degradation of bacteria by vegetative Dictyostelium amoeba using Escherichia coli expressing the recombinant fluorescent protein DsRed have been characterized. There are significant advantages to using DsRed-expressing bacteria for phagocytosis assays. Stable expression of the fluorescent protein, DsRed, provides living bacteria with a bright internal fluorescent signal that is degradable in the phagolysosomal pathway. Unlike assays with chemically labelled bacteria or latex beads, the bacteria are alive and possess a natural, unaltered external surface for receptor interaction. Dictyostelium cells rapidly bind and phagocytose DsRed bacteria. Pulse–chase experiments show that the signal derived from DsRed is degraded with a half-life of approximately 45 min. To distinguish internalized bacteria from those bound to the surface, an assay was developed in which sodium azide was used to release surface-bound particles. Surprisingly, surface particle release appears to be independent of myosin II function. Using this assay it was shown that the uptake of bacteria into cells is extremely rapid. After 1 min incubation, 20% of the signal is derived from internalized bacteria. The proportion of the signal from internalized bacteria increases gradually and reaches 50% at steady state. This assay will be useful in investigations of the molecular machinery of phagocytosis and post-internalization vesicle trafficking.
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Physical and genetic map of the Lactobacillus sakei 23K chromosome
The GenBank accession numbers for the sequences reported in this paper are given in Table 2 and the legend to Fig. 3.
The Lactobacillus sakei 23K chromosome was analysed by pulsed-field gel electrophoresis after digestion with the restriction enzymes AscI, NotI and SfiI. The chromosome size was estimated to be 1845±80 kb. The use of I-CeuI, specific for rrn genes encoding 23S rRNAs, showed that seven rrn loci were present, on 40% of the chromosome. The seven rrn clusters were mapped and their orientation was determined, allowing the position of the replication origin to be estimated. Partial I-CeuI digestions were used to construct a backbone and the different restriction fragments obtained with AscI, NotI and SfiI were assembled to a physical map by Southern hybridization. Eleven L. sakei gene clusters previously identified were mapped, as well as 25 new loci located randomly on the chromosome and 11 regions flanking the rrn gene clusters. A total of 47 clusters were thus mapped on L. sakei chromosome. The new loci were sequenced, allowing the identification of 73 complete or incomplete coding sequences. Among these 73 new genes of L. sakei, the function of 36 could be deduced from their similarity to known genes described in databases. However, 10 genes had no homologues, 10 encoded proteins similar to proteins of unknown function and 17 were similar to hypothetical proteins.
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A high-molecular-mass cell-surface protein from Lactobacillus reuteri 1063 adheres to mucus components
More LessThe GenBank accession number for the sequence reported in this paper is AF120104.
A gene from Lactobacillus reuteri 1063 encoding a cell-surface protein, designated Mub, that adheres to mucus components in vitro has been cloned and sequenced. The deduced amino acid sequence of Mub (358 kDa) shows the presence of 14 approximately 200 aa repeats and features typical for other cell-surface proteins of Gram-positive bacteria. Fusion proteins consisting of different repeats of Mub and the maltose-binding protein (MBP) were produced. These proteins adhered to pig mucus components, with molecular masses ranging from <0·1 to >2 MDa, to pig gastric mucin and to hen intestinal mucus. The binding of Mub to mucus components occurred in the pH range 3–7·4, with maximum binding at pH 4–5 and could be partly inhibited by the glycoprotein fetuin. Affinity-purified antibodies against recombinant Mub were used in immunofluorescence microscopy to demonstrate the presence of Mub on the cell surface of strain 1063. By using the antibodies in a Western blot analysis, Mub could also be detected in the growth medium. The results implicate Mub as a cell-surface protein that is involved in Lactobacillus interactions with mucin and in colonization of the digestive tract.
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Phage-display antibody detection of Chlamydia trachomatis-associated antigens
More LessA phage-displayed human single-chain Fv antibody library (6·7×109 members) was used to select probes specific to components associated with the surface of Chlamydia trachomatis elementary bodies (EBs). Each of 15 antibodies was characterized by ELISA, dot-blot, immunoblot and immunocytochemistry, resulting in the identification of several new chlamydial components associated with the surface of EBs. In addition, six antibodies were specific for host-cell components associated with the surface of EBs. While phage display has been used effectively to produce specific antibodies for purified components, these data show that this technology is suitable for selection of specific probes from complex antigens such as the surface of a microbial pathogen.
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Genotyping of axenic and non-axenic isolates of the genus Prochlorococcus and the OMF-‘Synechococcus’ clade by size, sequence analysis or RFLP of the Internal Transcribed Spacer of the ribosomal operon
The GenBank accession numbers for the ITS amplicon sequences reported in this paper are AF387607 (PCC 9511T), AF387610 (PCC 6307), AF387608 (PCC 7001) and AF387609 (PCC 7941).
PCR amplicons of the Internal Transcribed Spacer (ITS) of the rrn operon of three axenic OMF (oceanic, marine and freshwater) strains of ‘Synechococcus’ (WH7803, PCC 7001 and PCC 6307, respectively) differ greatly in length from that of the axenic Prochlorococcus marinus subsp. pastoris PCC 9511T, although these four cyanobacteria cluster relatively closely in phylogenetic trees inferred from 16S rRNA gene sequences. The ITSs of three strains (PCC 9511T, PCC 6307 and PCC 7001) were sequenced and compared with those available for strains Prochlorococcus MED4 (CCMP 1378) and MIT9313 from genome sequencing projects. In spite of large differences in length, sequence and mean DNA base composition, conserved domains important for transcriptional antitermination and folding of the rRNA transcripts were identified in all ITSs. A new group-specific primer permitted ITS amplification even with non-axenic isolates of Prochlorococcus and one OMF-‘Synechococcus’ strain. Prochlorococcus isolates of the high-light-adapted clade (HL) differed from representatives of the low-light-adapted clade (LL) by the length of their ITS. Restriction fragment length polymorphism (RFLP) of the ITS amplicons revealed three subclusters among the HL strains. Size, sequence data and RFLP of the ITS amplicons will therefore be valuable markers for the identification of different Prochlorococcus genotypes and for their discrimination from other cyanobacterial relatives with which they often co-exist in oceanic ecosystems.
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Fusobacterium nucleatum supports the growth of Porphyromonas gingivalis in oxygenated and carbon-dioxide-depleted environments
More LessThe authors compared the differences in tolerance to oxygen of the anaerobic periodontopathic bacteria Fusobacterium nucleatum and Porphyromonas gingivalis, and explored the possibility that F. nucleatum might be able to support the growth of P. gingivalis in aerated and CO2-depleted environments. Both micro-organisms were grown as monocultures and in co-culture in the presence and absence of CO2 and under different aerated conditions using a continuous culture system. At steady state, viable counts were performed and the activities of the enzymes superoxide dismutase and NADH oxidase/peroxidase were assayed in P. gingivalis. In co-culture, F. nucleatum was able to support the growth of P. gingivalis in aerated and CO2-depleted environments in which P. gingivalis, as a monoculture, was not able to survive. F. nucleatum not only appeared to have a much higher tolerance to oxygen than P. gingivalis, but a significant increase in its numbers occurred under moderately oxygenated conditions. F. nucleatum might have an additional indirect role in dental plaque maturation, contributing to the reducing conditions necessary for the survival of P. gingivalis and possibly other anaerobes less tolerant to oxygen. Additionally, F. nucleatum is able to generate a capnophilic environment essential for the growth of P. gingivalis.
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A novel paralogous gene family involved in phase-variable flagella-mediated motility in Campylobacter jejuni
More LessFlagella-mediated motility is recognized to be one of the major factors contributing to virulence in Campylobacter jejuni. Motility of this bacterium is known to be phase variable, although the mechanism of such variation remains unknown. C. jejuni genome sequencing revealed a number of genes prone to phase variation via a slipped-strand mispairing mechanism. Many of these genes are hypothetical and are clustered in the regions involved in formation of three major cell surface structures: capsular polysaccharide, lipooligosaccharide and flagella. Among the genes of unknown function, the flagellar biosynthesis and modification region contains seven hypothetical paralogous genes designated as the motility accessory factor (maf) family. Remarkably, two of these genes (maf1 and maf4) were found to be identical and both contain homopolymeric G tracts. Using insertional mutagenesis it was demonstrated that one of the genes, maf5, is involved in formation of flagella. Phase variation of the maf1 gene via slipped-strand mispairing partially restored motility of the maf5 mutant. The maffamily represents a new class of bacterial genes related to flagellar biosynthesis and phase variation. Reversible expression of flagella may be advantageous for the adaptation of C. jejunito the varied in vivo and ex vivo environments encountered during its life cycle, as well in evasion of the host immune response.
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rDNA analyses of planktonic heterocystous cyanobacteria, including members of the genera Anabaenopsis and Cyanospira
More LessThe GenBank accession numbers of the 16S rDNA gene sequences reported in this paper are AY038032–AY038037.
The taxonomic coherence and phylogenetic relationships of 11 planktonic heterocystous cyanobacterial isolates were examined by investigating two areas of the rRNA operon, the 16S rRNA gene (rrnS) and the internal transcribed spacer (ITS) located between the 16S rRNA and 23S rRNA genes. The rrnS sequences were determined for five strains, including representatives of Anabaena flos-aquae, Aphanizomenon flos-aquae, Nodularia sp. and two alkaliphilic planktonic members of the genera Anabaenopsis and Cyanospira, whose phylogenetic position was previously unknown. Comparison of the data with those previously published for individual groups of planktonic heterocystous cyanobacteria showed that, with the exception of members assigned to the genus Cylindrospermopsis, all the planktonic strains form a distinct subclade within the monophyletic clade of heterocystous cyanobacteria. Within this subclade five different phylogenetic clusters were distinguished. The phylogenetic groupings of Anabaena and Aphanizomenon strains within three of these clusters were not always consistent with their generic or specific assignments based on classical morphological definitions, and the high degree of sequence similarity between strains of Anabaenopsis and Cyanospira suggests that they may be assignable to a single genus. Ribotyping and additional studies performed on PCR amplicons of the 16S rDNA or the ITS for the 11 planktonic heterocystous strains demonstrated that they all contain multiple rrn operons and ITS regions of variable size. Finally, evidence is provided for intra-genomic sequence heterogeneity of the 16S rRNA genes within most of the individual isolates.
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In vitro reconstructed human epithelia reveal contributions of Candida albicans EFG1 and CPH1 to adhesion and invasion
More LessThe individual and synergistic contributions of two transcription factors, EFG1 and CPH1, have been characterized with regard to adhesion to, and invasion of, human epithelia by Candida albicans. For this purpose two in vitro reconstructed tissue models were developed. A multi-layered model of human epidermis was used to simulate superficial infections of the skin, whereas a reconstructed human intestinal model was used to mimic the first steps of systemic infections. It was shown that C. albicans deleted for both transcription factors CPH1 and EFG1, in contrast to the congenic clinical isolate Sc5314, was neither able to adhere to, nor to penetrate, either of the model systems. A strain deleted for EFG1 alone showed significant reduction in adhesion and was not able to penetrate through the stratum corneum. However, strains deleted for CPH1 showed phenotypes paralleling the phenotypes of the clinical isolate Sc5314. Using different types of multi-layered human tissues reconstructed in vitro the individual contributions of Efg1p and Cph1p to two important virulence factors of C. albicans, namely adhesion and invasion, could be defined.
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The metIC operon involved in methionine biosynthesis in Bacillus subtilis is controlled by transcription antitermination
More LessThere are two major pathways for methionine biosynthesis in micro-organisms. Little is known about these pathways in Bacillus subtilis. The authors assigned a function to the metI (formerly yjcI) and metC (formerly yjcJ) genes of B. subtilis by complementing Escherichia coli metB and metC mutants, analysing the phenotype of B. subtilis metI and metC mutants, and carrying out enzyme activity assays. These genes encode polypeptides belonging to the cystathionine γ-synthase family of proteins. Interestingly, the MetI protein has both cystathionine γ-synthase and O-acetylhomoserine thiolyase activities, whereas the MetC protein is a cystathionine β-lyase. In B. subtilis, the transsulfuration and the thiolation pathways are functional in vivo. Due to its dual activity, the MetI protein participates in both pathways. The metI and metC genes form an operon, the expression of which is subject to sulfur-dependent regulation. When the sulfur source is sulfate or cysteine the transcription of this operon is high. Conversely, when the sulfur source is methionine its transcription is low. An S-box sequence, which is located upstream of the metI gene, is involved in the regulation of the metIC operon. Northern blot experiments demonstrated the existence of two transcripts: a small transcript corresponding to the premature transcription termination at the terminator present in the S-box and a large one corresponding to transcription of the complete metIC operon. When methionine levels were limiting, the amount of the full-length transcript increased. These results substantiate a model of regulation by transcription antitermination.
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Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets
The lack of a convenient high-resolution strain-typing method has hampered the application of molecular epidemiology to the surveillance of bacteria of the Mycobacterium tuberculosis complex, particularly the monitoring of strains of Mycobacterium bovis. With the recent availability of genome sequences for strains of the M. tuberculosis complex, novel PCR-based M. tuberculosis-typing methods have been developed, which target the variable-number tandem repeats (VNTRs) of minisatellite-like mycobacterial interspersed repetitive units (MIRUs), or exact tandem repeats (ETRs). This paper describes the identification of seven VNTR loci in M. tuberculosis H37Rv, the copy number of which varies in other strains of the M. tuberculosis complex. Six of these VNTRs were applied to a panel of 100 different M. bovis isolates, and their discrimination and correlation with spoligotyping and an established set of ETRs were assessed. The number of alleles varied from three to seven at the novel VNTR loci, which differed markedly in their discrimination index. There was positive correlation between spoligotyping, ETR- and VNTR-typing. VNTR-PCR discriminates well between M. bovis strains. Thirty-three allele profiles were identified by the novel VNTRs, 22 for the ETRs and 29 for spoligotyping. When VNTR- and ETR-typing results were combined, a total of 51 different profiles were identified. Digital nomenclature and databasing were intuitive. VNTRs were located both in intergenic regions and annotated ORFs, including PPE (novel glycine-asparigine-rich) proteins, a proposed source of antigenic variation, where VNTRs potentially code repeating amino acid motifs. VNTR-PCR is a valuable tool for strain typing and for the study of the global molecular epidemiology of the M. tuberculosis complex. The novel VNTR targets identified in this study should additionally increase the power of this approach.
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