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PubMed2003

Mgr, a novel global regulator in Staphylococcus aureus.

The virulence determinants of Staphylococcus aureus are coordinately controlled by several unlinked chromosomal loci. Here, we report the identification of CYL5614, derived from strain Becker, with a mutation that affects the expression of type 8 capsular polysaccharide (CP8), nuclease, alpha-toxin, coagulase, protease, and protein A. This novel locus, named mgr, was linked by transposon Tn917 and mapped by three-factorial transduction crosses. The region containing the mgr locus was cloned and sequenced. Deletion mutagenesis and genetic complementation showed that the locus consisted of one gene, mgrA. Interestingly, mgrA-null mutants exhibited a phenotype opposite to that of CYL5614. This was due to a T-to-C mutation upstream of mgrA that resulted in a four- to eightfold increase in mgrA transcription in strain CYL5614. Thus, these results indicate that mgrA is an activator of CP8 and nuclease but a repressor of alpha-toxin, coagulase, protease, and protein A. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the mgr locus profoundly affected extracellular protein production, suggesting that the locus may regulate many other genes as well. The translated MgrA protein has a region of significant homology, which includes the helix-turn-helix DNA-binding motif, with the Escherichia coli MarR family of transcriptional regulators. Northern slot blot analyses suggested that mgr affected CP8, alpha-toxin, nuclease, and protein A at the transcriptional level.

Amino Acid Sequence↗
PubMed1994

Genetic analysis of Escherichia coli O9 rfb: identification and DNA sequence of phosphomannomutase and GDP-mannose pyrophosphorylase genes.

Subcloning, transposon insertion, and deletion analysis revealed that the Escherichia coli O9 rfb region is about 12 kb in size. The region encodes at least seven polypeptides of 89, 74, 55, 50, 44, 41 and 39.5 kDa. Southern hybridization analysis of rfb regions of E. coli O8 and O9, and Klebsiella O3 and O5 serotypes (all of these O polysaccharides are mannose homopolymers and the structures of the repeating unit of E. coli O9 and Klebsiella O3 are identical) showed that a central region specific for E. coli O9 and Klebsiella O3 is flanked by two regions common to all four. Complementation experiments using strains with known defects and specific tests for the enzymic activity showed that the 50 and 55 kDa polypeptides, encoded by the common region, are phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP), respectively. Nucleotide sequencing of the region revealed the presence of two genes, rfbK and rfbM, analogous to the corresponding genes of Salmonella typhimurium. In E. coli O9, rfbK and rfbM encode proteins of 460 amino acids (50,809 Da) and 471 amino acids (52,789 Da). The amino acid sequence of GMP was conserved in RfbMs of E. coli O7 and Salmonella groups B, C1 and C2, CpsB of S. typhimurium, AlgA of Pseudomonas aeruginosa, and XanB of Xanthomonas campestris. The phylogenetic trees of PMM and GMP were different in topology and in the evolutionary distances from ancestors.

Amino Acid Sequence↗
PubMed2001

Complete nucleotide sequence of a Staphylococcus aureus exfoliative toxin B plasmid and identification of a novel ADP-ribosyltransferase, EDIN-C.

The complete nucleotide sequence of pETB, a 38.2-kb Staphylococcus aureus plasmid encoding the exfoliative toxin B (ETB), was determined. A total of 50 open reading frames were identified on the plasmid genome and, among these, 32 showed sequence similarity to known proteins. pETB contains three copies of IS257, which divide the pETB genome into three regions: (i) a cadmium resistance operon-containing region, (ii) a lantibiotic production gene-containing region, and (iii) the remaining part where genes for plasmid replication and/or maintenance are dispersed. In the third region, genes of various kinds of functions are present among the replication- and maintenance-related genes. They include two virulence-related genes, the etb gene and a gene encoding a novel ADP-ribosyltransferase closely related to EDIN, which belongs to the C3 family of ADP-ribosyltransferases modifying Rho GTPases. They also include genes for a cell wall-anchoring surface protein and a phage resistance protein. Based on the determined sequence of pETB, the genome structures of etb-bearing plasmids (ETB plasmids) from various clinical isolates were analyzed by the PCR scanning method. The data indicate that, although the ETB plasmids are highly heterogeneous in genome size, the fundamental genome organization is well conserved. The size variation of the plasmid is mainly attributed to defined regions which may be hot spots for gene shuffling.

Amino Acid Sequence↗
PubMed1990

Identification of a new gene, molR, essential for utilization of molybdate by Escherichia coli.

A mutation in a new gene, molR, prevented the synthesis in Escherichia coli of molybdoenzymes, including the two formate dehydrogenase isoenzymes, nitrate reductase and trimethylamine-N-oxide reductase. This phenotype was suppressed by supplementing the media with molybdate. Thus, the molR mutant was phenotypically similar to previously described chlD mutants, thought to be defective in molybdate transport. The molR gene is located at 65.3 min in the E. coli chromosome, in contrast to the chlD gene, which maps at 17 min and thus can be readily distinguished. The molR gene is also cotransducible with a hitherto unidentified gene essential for the production of 2-oxoglutarate from isocitrate, designated icdB (located at 66 min). The molR mutant strain SE1100 also failed to produce the hydrogenase component of formate hydrogenlyase (HYD3) in molybdate-unsupplemented media. The amount of molybdate required by strain SE1100 for the production of parental levels of formate hydrogenlyase activity was dependent on the growth medium. In Luria-Bertani medium, this value was about 100 microM, and in glucose-minimal medium, 1.0 microM was sufficient. In low-sulfur medium, this value decreased to about 50 nM. The addition of sulfate or selenite increased the amount of molybdate needed for the production of formate hydrogenlyase activity. These data suggest that in the absence of the high-affinity molybdate transport system, E. coli utilizes sulfate and selenite transport systems for transporting molybdate, preferring sulfate transport over the selenite transport system.

Bacteriophage lambda↗
PubMed1997

Identification and characterization of ssb and uup mutants with increased frequency of precise excision of transposon Tn10 derivatives: nucleotide sequence of uup in Escherichia coli.

A Lac+ papillation assay was used to identify mutants (tex) of Escherichia coli that exhibit an increased frequency of precise excision of a lacZ::Tn10dKan insertion. Three tex strains had suffered mutations in the gene (ssb) encoding the essential single-stranded DNA-binding protein SSB, which resulted in the following alterations in the 177-residue protein: G4D; L10F, P24S; and V102M. The phenotypes of these ssb mutants indicated that they were largely unaffected in other functions mediated by SSB, such as DNA replication, recombination, and repair. Strains with multicopy ssb+ exhibited a decreased frequency of Tn10dKan precise excision. Three other tex mutants had insertion mutations in the locus designated uup at 21.75 min on the linkage map. The nucleotide sequence of uup was determined, and the gene was inferred to encode a 625-amino-acid hydrophilic protein that belongs to the superfamily of ABC-domain proteins (with two pairs of the Walker A and B motifs), which are postulated to be involved in coupling ATP hydrolysis with other biological processes. The uup gene product shares extensive homology with the deduced sequences of two proteins of Haemophilus influenzae. The uup gene is also situated immediately upstream of (and is transcribed in the same direction as) the paraquat-inducible SoxRS-regulated pqi-5 gene, two reported promoters for which are situated within the uup coding sequence.

ATP-Binding Cassette Transporters↗
PubMed1996

Effect of regulatory protein levels on utilization of starch by Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, appears to utilize starch by first binding the polymer to its surface and then translocating it into the periplasmic space. Several genes that encode enzymes or outer membrane proteins involved in starch utilization have been identified. These have been called sus genes, for starch utilization system. Previous studies have shown that sus structural genes are regulated at the transcriptional level and their expression is induced by maltose. We report here the identification and characterization of a gene, susR, which appears to be responsible for maltose-dependent regulation of the sus structural genes. The deduced amino acid sequence of SusR protein had a helix-turn-helix motif at its carboxy-terminal end, and this region had highest sequence similarity to the corresponding regions of known transcriptional activators. A disruption in susR eliminated the expression of all known sus structural genes, as expected if susR encoded an activator of sus gene expression. The expression of susR itself was not affected by the growth substrate and was not autoregulated, suggesting that binding of SusR to maltose might be the step that activates SusR. Three susR-controlled structural genes, susA, susB, and susC, are located immediately upstream of susR. These genes are organized into two transcriptional units, one containing susA and another containing susB and susC. susA was expressed at a lower level than susBC, and susA expression was more sensitive to the gene dosage of susR than was that of the susBC operon. An unexpected finding was that increasing the number of copies of susR in B. thetaiotaomicron increased the rate of growth on starch. This effect could be due to higher levels of susA expression. Whatever the explanation, the level of SusR in the cell appears to be a limiting factor for growth on starch.

Amino Acid Sequence↗
PubMed2004

Bacillus subtilis YdiH is a direct negative regulator of the cydABCD operon.

During aerobic respiration, Bacillus subtilis utilizes three terminal oxidases, cytochromes aa3, caa3, and bd. Cytochrome bd is encoded by the cydABCD operon. We report here the first identification of a regulator for the cydABCD operon, YdiH. While working with DeltaresDE mutant strains, we identified colonies which contained suppressor mutations (cmp) which bypassed the requirement for ResD for all phenotypes not associated with cytochrome aa3 or caa3. Mapping identified a class of Tn10 insertions which were close to the cmp locus (Tn10-2) and a second class (Tn10-1) which was inserted in cydD, a gene which appears to be essential to the cmp phenotype. Sequencing of the cmp loci from four independent DeltaresDE cmp isolates yielded four loss-of-function alleles of ydiH, a gene encoding a protein with homology to AT-rich DNA-binding proteins. Additionally, we determined that cytochrome bd was aberrantly expressed in the DeltaresDE cmp background. Together these data led to the hypothesis that YdiH serves as a negative regulator of cydABCD expression, a hypothesis supported by both gel-shift and DNase I footprinting analyses. YdiH protected the cydA promoter region at three 22-bp repeats located in the long 5' untranslated region (193 bp). Induction of the cydABCD operon in a DeltaresDE background showed that expression of the terminal oxidase bd was responsible for the bypass phenotype observed in a DeltaresDE cmp strain, indicating that cytochrome bd expression complemented the loss of cytochromes aa3 and caa3 in the DeltaresDE strain.

Bacillus subtilis↗
PubMed2003

Genetic biodiversity of Mycobacterium tuberculosis complex strains from patients with pulmonary tuberculosis in Cameroon.

We analyzed DNA polymorphisms in 455 Mycobacterium tuberculosis complex isolates from 455 patients to evaluate the biodiversity of tubercle bacilli in Ouest province, Cameroon. The phenotypic and genotypic identification methods gave concordant results for 99.5% of M. tuberculosis isolates (413 strains) and for 90% of Mycobacterium africanum isolates (41 strains). Mycobacterium bovis was isolated from only one patient. Analysis of regions of difference (RD4, RD9, and RD10) proved to be an accurate and rapid method of distinguishing between unusual members of the M. tuberculosis complex. Whereas M. africanum strains were the etiologic agent of tuberculosis in 56% of cases 3 decades ago, our results showed that these strains now account for just 9% of cases of tuberculosis. We identified a group of closely genetically related M. tuberculosis strains that are currently responsible for >40% of smear-positive pulmonary tuberculosis cases in this region of Cameroon. These strains shared a spoligotype lacking spacers 23, 24, and 25 and had highly related IS6110 ligation-mediated (LM) PCR patterns. They were designated the "Cameroon family." We did not find any significant association between tuberculosis-causing species or strain families and patient characteristics (sex, age, and human immunodeficiency virus status). A comparison of the spoligotypes of the Cameroon strains with an international spoligotype database (SpolDB3) containing 11,708 patterns from >90 countries, showed that the predominant spoligotype in Cameroon was limited to West African countries (Benin, Senegal, and Ivory Coast) and to the Caribbean area.

Cameroon↗
PubMed1989

An ice nucleation reporter gene system: identification of inducible pathogenicity genes in Pseudomonas syringae pv. phaseolicola.

We have constructed derivatives of the transposon Tn3 that allow an ice nucleation gene (inaZ) to be used as 'reporter' of the transcriptional activity of genes into which it is inserted. In these derivatives (Tn3-Ice and Tn3-Spice), the lacZYA sequences of transposon Tn3-HoHo1 were replaced with inaZ lacking its native promoter. The ice nucleation activity of virB::inaZ fusions in the correct transcriptional orientation was inducible by acetosyringone, a plant metabolite which activates the vir operon of Agrobacterium tumefaciens Ti plasmids, while fusions in the opposite orientation were unresponsive to the inducer. Tn3-Spice was also used to investigate the expression of a cluster of genes (hrp) which control pathogenicity and hypersensitivity elicited by Pseudomonas syringae pv. phaseolicola. An inducible region was identified which is expressed at low levels in vitro but becomes activated when the bacteria come into contact with the susceptible host, bean. Activation of this region occurred within 2 h post-inoculation and was nearly complete by the time the bacteria began to multiply in the leaf tissue. The inaZ reporter appears to be at least 10(5)-fold more sensitive than lacZ in P.s.phaseolicola. Thus, the inaZ fusion system provides a sensitive, convenient and inexpensive tool for the study of bacterial gene expression, particularly during plant pathogenesis, and should be generally useful as a reporter gene system in Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1999

Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis.

Tuberculosis remains the greatest cause of death worldwide due to a single pathogen. In order to identify the genes required for the pathogenicity of Mycobacterium tuberculosis, a functional genomic approach was developed. A library of signature-tagged transposon mutants of this bacterium was constructed and screened for those affected in their multiplication within the lungs of mice. From 1927 mutants tested, 16 were attenuated for their virulence. The insertions harboured by the selected mutants were mapped on the M. tuberculosis genome and most of the mutated loci appeared to be involved in lipid metabolism or transport across the membrane. Four independent mutations identified a cluster of virulence genes located on a 50 kb chromosomal region. These genes might be involved in the production of phthiocerol and phenolphthiocerol derivatives, a group of molecules restricted to eight mycobacterial species, seven of them being either strict or opportunistic pathogens. The interaction of five mutant strains with mouse bone marrow macrophages was investigated. These five mutants were still able to multiply in this cell type. However, in three cases, there was a growth defect in comparison with the wild-type strain. The other two strains exhibited no clear difference from the virulent strain, MT103, in this model. This study, which is the first global research of virulence factors of M. tuberculosis, opens the way to a better understanding of the molecules that are key players in the interaction of this pathogen with its host.

Animals↗
PubMed1987

Identification of polypeptides encoded by an Escherichia coli locus (hflA) that governs the lysis-lysogeny decision of bacteriophage lambda.

We report the cloning of the Escherichia coli hflA locus, which governs stability of phage lambda cII protein and which has been proposed to encode or regulate a cII-specific protease. The hflA locus was cloned on an 18-kilobase DNA fragment by selecting for plasmids that carry the neighboring purA gene. The boundaries of hflA were delimited by analysis of deletions and insertions constructed in vitro and by use of transposon Tn1000. Maxicell analysis of the proteins encoded by the hflA-containing fragment shows that hflA consists of at least two nonoverlapping genes, hflC and hflK, encoding polypeptides of 37,000 (C) and 46,000 (K) daltons. We observe that insertions into one gene eliminate the corresponding polypeptide and greatly reduce synthesis of the other. We suggest that these two polypeptides (K and C) interact to form a multimeric complex and that free subunits are unstable. We have constructed two types of fusions between hflA and lacZ. One is an hflC-lacZ protein fusion constructed in vitro; the other is an hfl-lacZ operon fusion in which a Mu dX(Apr lac) has inserted into the hflK gene. We have used the operon fusion to infer the direction of transcription of the hflK gene--toward hflC and in the same direction as hflC. Last, we describe evidence that hflA contains an additional gene, hflX, encoding a 50,000-dalton polypeptide.

Autoradiography↗
PubMed1990

Identification of genes affecting production of the adhesion organelle of Caulobacter crescentus CB2.

Transposon (Tn5) mutagenesis was used to identify regions in the genome involved with production, regulation, or attachment to the cell surface of the adhesive holdfast of the freshwater bacterium Caulobacter crescentus CB2. A total of 12,000 independently selected transposon insertion mutants were screened for defects in adhesion to cellulose acetate; 77 mutants were detected and examined by Southern blot hybridization mapping methods and pulsed-field gel electrophoresis. Ten unique sites of Tn5 insertion affecting holdfast function were identified that were clustered in four regions of the genome. Representative mutants of the 10 Tn5 insertion sites were examined by a variety of methods for differences in their phenotype leading to the loss of adhesiveness. Four phenotypes were identified: no holdfast production, production of a smaller or an altered holdfast, production of a holdfast that was unable to remain attached to the cell, and a fourth category in which a possible alteration of the stalk was related to impaired adhesion of the cell. With the possible exception of the last class, no pleiotropic mutants (those with multiple defects in the polar region of the cell) were detected among the adhesion-defective mutants. This was unexpected, since holdfast deficiency is often a characteristic of pleiotropic mutants obtained when selecting for loss of other polar structures. Overall, the evidence suggests that we have identified regions containing structural genes for the holdfast, genes involved with proper attachment or positioning on the caulobacter surface, and possibly regions that regulate the levels of holdfast production.

Bacterial Adhesion↗
PubMed1997

Detection of Mycobacterium bovis in formalin-fixed, paraffin-embedded tissues of cattle and elk by PCR amplification of an IS6110 sequence specific for Mycobacterium tuberculosis complex organisms.

A presumptive diagnosis of tuberculosis can be made if a tissue has characteristic histopathologic changes and acid-fast organisms. However, definitive diagnosis requires culture and species identification of the causative mycobacterium, a process that takes several weeks to complete. The purpose of work reported here was to determine if formalin-fixed, paraffin-embedded tissues could be tested by polymerase chain reaction (PCR) to provide a more rapid diagnosis of tuberculosis. Nondecalcified tissues from cases of tuberculosis in cattle and elk (Cervus elaphus) were examined. The primers used for PCR amplified a 123-bp fragment of IS6110, an insertion sequence that is specific for organisms in the Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. microti, M. africanum). The PCR test detected this sequence in tissues from 92 of 99 (93%) tuberculosis cases, including 3 of 4 elk. In 80 tissues, the positive results were obtained using material prepared by immersion of paraffin sections in water containing a detergent, followed by alternating boil/freeze cycles. The remaining positive results were obtained with DNA isolated from the crude tissue extracts by proteinase K digestion and phenol/chloroform purification. Accuracy of the IS6110 PCR test was demonstrated by negative test results on 31 tissues that had either nonmycobacterial granulomas or granulomatous lesions caused by other mycobacteria (M. paratuberculosis or M. avium). The findings of this study show that a PCR test usually can provide a rapid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.

Animals↗
PubMed2003

Identification of the gene encoding the sole physiological fumarate reductase in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a Gram-negative, nonfermentative rod with a complex electron transport system which facilitates its ability to use a variety of terminal electron acceptors, including fumarate, for anaerobic respiration. CMTn-3, a mutant isolated by transposon (TnphoA) mutagenesis, can no longer use fumarate as an electron acceptor; it lacks fumarate reductase activity as well as a 65-kDa soluble tetraheme flavocytochrome c. The sequence of the TnphoA-flanking genomic DNA of CMTn-3 did not align to those for fumarate reductase or related electron transport genes from other bacteria. Sequence analysis of the MR-1 genomic database demonstrated that an open reading frame encoding a 65-kDa tetraheme cytochrome c with sequence similarity to the fumarate reductase from S. frigidimarina NCIMB400 was found 8 kb away from the TnphoA-flanking genomic DNA of CMTn-3. PCR analysis demonstrated that a large deletion (>or=9.2 kb and <or=11 kb) of genomic DNA occurred in CMTn-3 as a result of TnphoA insertion. This deletion included at least half of the fumarate reductase gene as well as approximately 8 kb of upstream DNA. Complementation of CMTn-3 with the fumarate reductase gene plus 0.5-kb of upstream DNA restored growth on fumarate. These studies explicitly define the sole physiological fumarate reductase gene from the several possibilities suggested by the genomic sequence of MR-1. Surprisingly, the fumarate reductase gene plus 0.77-kb upstream DNA from S. frigidimarina NCIMB400 did not complement CMTn-3.

DNA Transposable Elements↗
PubMed1994

A functional analysis of the Tn5 transposase. Identification of domains required for DNA binding and multimerization.

A series of deletions were constructed in the 476 amino acid Tn5 transposase in order to assemble an initial domain structure for this protein. The first four amino acids were found to be important for transposition activity but not for DNA binding to the Tn5 outside end (OE). Larger amino-terminal deletions result in the complete loss of transposition in vivo and the concomitant loss of specific DNA binding. Four point mutants and a six base-pair deletion in the amino terminus between residues 20 and 36 were also found to impair DNA binding to the OE. Analysis of a series of carboxy-terminal deletions has revealed that the carboxy terminus may actually mask the DNA binding domain, since deletions to residues 388 and 370 result in a large increase in DNA binding activity. In addition, the carboxy-terminal deletion to residue 370 results in a significant increase in the mobility of the Tnp-OE complex indicative of a change in the oligomeric state of this complex. Further carboxy-terminal deletions beyond residue 370 also abolished DNA binding activity. These results indicate that the first four amino acids of Tnp are important for transposition but not DNA binding, a region between residues 5 and 36 is critical for DNA binding, the wild-type carboxy terminus acts to inhibit DNA binding, and that a region towards the carboxy terminus, defined by residues 370 to 387, is critical for Tnp multimeric interactions.

Amino Acid Sequence↗
PubMed1988

Saturation mutagenesis of the Tn10-encoded tet operator O1. Identification of base-pairs involved in Tet repressor recognition.

Saturation mutagenesis of Tn10-encoded tet operator O1 was performed by chemical synthesis of 30 sequence variants yielding all possible point mutations of an operator half side. Their effect on Tet repressor binding was scored by an in-vivo repressor titration system. Tet repressor affinities of selected operator mutants were further characterized in vitro by dissociation rate measurements. The O1 sequence spans 19 base-pairs. Out of these, all 18 palindromic base-pairs are involved in Tet repressor recognition. The central base-pair does not contribute to sequence-specific binding of Tet repressor. At position 1 a pyrimidine residue is sufficient for maximal affinity to the repressor. At positions 2, 3 and 4, each mutation reduces repressor binding at least tenfold. Mutations at positions 5, 6, 7, 8 and 9 result in less drastic reductions of Tet repressor binding. Differential effects of mutations at a given position are used to deduce the chemical functions contacted by Tet repressor. The T.A to A.T transversion at position 9 increases Tet repressor affinity slightly, while all other mutations decrease repressor binding. The increased affinity of the wild-type tet operator O2 compared to wild-type O1 results from the addition of two favorable transversions at positions +/- 9 and an unfavorable T.A to C.G transition at position -7. Deletion or palindromic doubling of the central base-pair of the O1 palindrome reveals that the wild-type spacing of both operator half sides is crucial for efficient Tet repressor binding.

Base Sequence↗
PubMed1988

Gene regulation on broad host range plasmid RK2: identification of three novel operons whose transcription is repressed by both KorA and KorC.

The product of the korA gene of broad host range plasmid RK2 is a key transcriptional repressor which regulates not only the expression of the essential replication gene trfA but also its own expression and that of the kilA operon. It has previously been proposed that korA also encodes a positive activator of transcription of the korC gene, which may act as a transcriptional antiterminator. Here we show that the action of korA in relation to korC can be explained entirely through the korA protein's property as a transcriptional repressor. The limited ability of the previously cloned korC gene to suppress kilC on its own is shown to be due to the fact that korC in RK2 is transcribed from the bla promoter of Tn1 which was deleted in the original korC clones. We demonstrate that korA is a second repressor along with korC of three operons, one of which encodes kilC, the other two not having been described previously and serving an as yet unknown function. We have designated these operons kcrA, B and C for KorC-regulated. Putative kilC is designated kcrC. The homology between the expression signals of these operons suggests that they have arisen by duplication. This is confirmed in the case of kcrA and B by the existence of considerable homology between the products of the first ORFs in each of these operons.

Amino Acid Sequence↗
PubMed2004

Identification of Francisella tularensis genes encoding exported membrane-associated proteins using TnphoA mutagenesis of a genomic library.

Francisella tularensis, the causative agent of tularemia, is a highly infectious pathogen of humans and animals, yet little is known about the surface proteins of this organism that mediate mechanisms of pathogenicity. lambdaTnphoA was used to generate random alkaline phosphatase gene fusions in a F. tularensis subsp. tularensis (strain Schu S4) genomic library to identify genes encoding exported extracytoplasmic proteins. Eleven genes encoding membrane-associated proteins were identified by this method and their respective signal peptides were characterized. Three of the genes encoded conserved 'housekeeping' enzymes, while the other eight genes were unique to F. tularensis, encoding proteins with molecular masses ranging from 11 to 78kDa as deduced from the amino acid sequences. Two genes putatively encoded lipoproteins based on the presence of characteristic signal peptidase II cleavage sites. Four selected proteins were found associated with outer membranes from Schu S4 and LVS strains by Western blotting. Indirect immunofluorescence of strain Schu S4 cells also showed evidence of protein localization to the outer membrane. Protein database searches produced significant alignments with proteins from other bacteria involved in carbohydrate transport, lipid metabolism, and cell envelope biogenesis, thereby providing clues for putative functions. These findings demonstrated that TnphoA mutagenesis can be used in conjunction with F. tularensis genome sequence data to provide a foundation for studies to identify and define cellular surface protein virulence factors of this pathogen.

Alkaline Phosphatase↗