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PubMed2001

Staphylococcus aureus Cap5O has UDP-ManNAc dehydrogenase activity and is essential for capsule expression.

The Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) has a repeating unit composed of (-->4)-3-O-acetyl-beta-D-ManNAcA-(1-->4)-alpha-L-FucNAc (1-->3)-beta-D-FucNAc-(1-->)(n). Sixteen chromosomal genes (cap5A through cap5P) are involved in the synthesis of CP5. We recently demonstrated that Cap5P, a 2-epimerase, catalyzes the conversion of UDP-N-acetyl glucosamine (UDP-GlcNAc) to UDP-N-acetylmannosamine (UDP-ManNAc). In this study, we show that UDP-ManNAc is oxidized to UDP-N-acetylmannosaminuronic acid (UDP-ManNAcA) by a UDP-ManNAc dehydrogenase encoded by S. aureus cap5O. We expressed Cap5O in Escherichia coli and purified the recombinant protein. The UDP-ManNAc dehydrogenase activity of purified Cap5O was assessed by incubating Cap5P and UDP-GlcNAc (to produce UDP-ManNAc), together with Cap5O, NAD(+), and a reducing agent. Enzymatic activity was quantitated indirectly by measuring the increase in absorbance at 340 nm resulting from NADH formation. The product of the reaction was confirmed as UDP-ManNAcA by gas chromatography-mass spectroscopy. A cap5O mutation, created by deletion of 727 bp in the 5' end of the gene, was introduced by allelic replacement into S. aureus Reynolds, rendering it CP5 negative. Mice inoculated intravenously or subcutaneously with the wild-type strain Reynolds had greater numbers of S. aureus recovered from their kidneys (P = 0.019) or their subcutaneous abscesses (P = 0.0018), respectively, than did animals inoculated with the cap5O mutant. The results of this study indicate that S. aureus cap5O is essential for capsule production and that capsule promotes staphylococcal virulence in mouse models of abscess formation.

Animals↗
PubMed2001

Generation and characterization of a defined mutant of Streptococcus suis lacking suilysin.

A defined allelic-replacement mutant of the sly gene, encoding a thiol-activated cytolysin, from a European isolate of Streptococcus suis serotype 2 was generated and characterized. Unlike the parental strain, it is nonhemolytic, noncytotoxic for cultured macrophage-like cells, avirulent in a mouse infection model, yet only slightly attenuated in a porcine model of systemic infection.

Animals↗
PubMed2001

Expression of pls, a gene closely associated with the mecA gene of methicillin-resistant Staphylococcus aureus, prevents bacterial adhesion in vitro.

The pls gene, coding for a large surface protein of methicillin-resistant Staphylococcus aureus, was cloned from a strain which adheres poorly to several mammalian proteins. The structure of pls revealed three distinct repeat regions, one of which was a serine-aspartate repeat characteristic of the Clf-Sdr family of surface proteins in staphylococci. The lengths of the repeat regions varied in different clinical strains and could be used as epidemiological markers. pls was found to be closely associated with the mecA gene by pulsed-field gel electrophoresis analysis of SmaI-digested DNA. A pls mutant constructed by allele replacement adhered well to immobilized fibronectin and immunoglobulin G, in contrast to the parental strain, suggesting that Pls could have a role in preventing adhesion at some stages during an infection.

Adhesins, Bacterial↗
PubMed2001

PerR controls oxidative stress resistance and iron storage proteins and is required for virulence in Staphylococcus aureus.

The Staphylococcus aureus genome encodes three ferric uptake regulator (Fur) homologues: Fur, PerR, and Zur. To determine the exact role of PerR, we inactivated the gene by allelic replacement using a kanamycin cassette, creating strain MJH001 (perR). PerR was found to control transcription of the genes encoding the oxidative stress resistance proteins catalase (KatA), alkyl hydroperoxide reductase (AhpCF), bacterioferritin comigratory protein (Bcp), and thioredoxin reductase (TrxB). Furthermore, PerR regulates transcription of the genes encoding the iron storage proteins ferritin (Ftn) and the ferritin-like Dps homologue, MrgA. Transcription of perR was autoregulated, and PerR repressed transcription of the iron homeostasis regulator Fur, which is a positive regulator of catalase expression. PerR functions as a manganese-dependent, transcriptional repressor of the identified regulon. Elevated iron concentrations produced induction of the PerR regulon. PerR may act as a peroxide sensor, since addition of external hydrogen peroxide to 8325-4 (wild type) resulted in increased transcription of most of the PerR regulon, except for fur and perR itself. The PerR-regulated katA gene encodes the sole catalase of S. aureus, which is an important starvation survival determinant but is surprisingly not required for pathogenicity in a murine skin abscess model of infection. In contrast, PerR is not necessary for starvation survival but is required for full virulence (P < 0.005) in this model of infection. PerR of S. aureus may act as a redox sentinel protein during infection, analogous to the in vitro activities of OxyR and PerR of Escherichia coli and Bacillus subtilis, respectively. However, it differs in its response to the metal balance within the cell and has the added capability of regulating iron uptake and storage.

Amino Acid Sequence↗
PubMed2002

The regulator PerR is involved in oxidative stress response and iron homeostasis and is necessary for full virulence of Streptococcus pyogenes.

Ferric uptake regulator (Fur) and Fur-like proteins form an important family of transcriptional regulators in many bacterial species. In this work we have characterized a Fur-like protein, the peroxide regulator PerR, in an M1 serotype of Streptococcus pyogenes. To determine the role of PerR in S. pyogenes, we inactivated the gene by allelic replacement. PerR-deficient bacteria showed 48% reduction of (55)Fe incorporation from the culture medium. Transcriptional analysis revealed that mtsA, encoding a metal-binding protein of an ABC transporter in S. pyogenes, was transcribed at lower levels than were wild-type cells. Although total iron accumulation was reduced, the growth of the mutant strain was not significantly hampered. The mutant showed hyperresistance to hydrogen peroxide, and this response was induced in wild-type cells by growth in aerobiosis, suggesting that PerR acts as an oxidative stress-responsive repressor. PerR may also participate in the response to superoxide stress, as the perR mutant was more sensitive to the superoxide anion and had a reduced transcription of sodA, which encodes the sole superoxide dismutase of S. pyogenes. Complementation of the mutation with a functional perR gene restored (55)Fe incorporation, response to peroxide stress, and transcription of both mtsA and sodA to levels comparable to those of wild-type bacteria. Finally, the perR mutant was attenuated in virulence in a murine air sac model of infection (P < 0.05). These results demonstrate that PerR is involved in the regulation of iron homeostasis and oxidative stress responses and that it contributes to the virulence of S. pyogenes.

ATP-Binding Cassette Transporters↗
PubMed2003

Aae, an autotransporter involved in adhesion of Actinobacillus actinomycetemcomitans to epithelial cells.

The periodontal pathogen Actinobacillus actinomycetemcomitans possesses myriad virulence factors, among them the ability to adhere to and invade epithelial cells. Recent advances in the molecular manipulation of this pathogen and the sequencing of strain HK 1651 (http://www.genome.ou.edu/act.html) have facilitated examination of the genetics of its interaction with epithelial cells. The related gram-negative organism, Haemophilus influenzae, possesses autotransporter adhesins. A search of the sequence database of strain HK 1651 revealed a homologue with similarity in the pore-forming domain to that of the H. influenzae autotransporter, Hap. A. actinomycetemcomitans mutants deficient in the homologue, Aae, showed reduced binding to epithelial cells. A method for making A. actinomycetemcomitans SUNY 465 transiently resistant to spectinomycin was used with conjugation to generate an isogenic aae mutant. An allelic replacement mutant was created in the naturally transformable A. actinomycetemcomitans strain ATCC 29523. Lactoferrin, an important part of the innate host defense system, protects against bacterial infection by bactericidal and antiadhesion mechanisms. Lactoferrin in human milk removes or cleaves Hap and another autotransporter, an immunoglobulin A1 protease, from the surface of H. influenzae, thereby reducing their binding to epithelial cells. Human milk whey had similar effects on Aae from A. actinomycetemcomitans ATCC 29523 and its binding to epithelial cells; however, there was little effect on the binding of SUNY 465. A difference in the genetic structure of aae in the two strains, apparently due to the copy number of a 135-base repeated sequence, may be the cause of the differential action of lactoferrin. aae is the first A. actinomycetemcomitans gene involved in adhesion to epithelial cells to be identified.

Adhesins, Bacterial↗
PubMed2003

Combined contributions of streptolysin O and streptolysin S to virulence of serotype M5 Streptococcus pyogenes strain Manfredo.

Streptolysin O (SLO) and streptolysin S (SLS) are potent cytolytic toxins produced by almost all clinical isolates of group A streptococci (GAS). Allele-replacement mutagenesis was used to construct nonpolar (in-frame) deletion mutations in the slo and sagB genes of the serotype M5 GAS strain Manfredo, producing isogenic single and double SLO- and SLS-defective mutants. In contrast to recent reports on SLS-defective insertion mutants (I. Biswas, P. Germon, K. McDade, and J. Scott, Infect. Immun. 69:7029-7038, 2001; Z. Li, D. Sledjeski, B. Kreikemeyer, A.Podbielski, and M. Boyle, J. Bacteriol. 181:6019-6027, 1999), none of the mutants described here had notable pleiotropic effects on the expression of other virulence factors examined. Comparison of isogenic parent and mutant strains in various virulence models revealed no differences in their abilities to multiply in human blood or in their 50% lethal doses (LD(50)s) upon intraperitoneal infection of BALB/c mice. A single log unit difference in the LD(50)s of the parent and SLS-defective mutant strains was observed upon infection by the subcutaneous (s.c.) route. Comparisons over a range of infective doses showed that both SLO and SLS contributed to the early stages of infection and to the induction of necrotic lesions in the murine s.c. model. Individually, each toxin made an incremental contribution to virulence that was not apparent at higher infective doses, although the absence of both toxins reduced virulence over the entire dose range examined. Interestingly, in some cases, the contribution of SLO to virulence was clear only from an analysis of the double-mutant strain, highlighting the value of not confining virulence studies to mutant strains defective in the expression of only single virulence factors.

Animals↗
PubMed2004

Essential role for the gtfA gene encoding a putative glycosyltransferase in the adherence of Porphyromonas gingivalis.

Porphyromonas gingivalis, an oral bacterium, might play a role in the pathogenesis or progression of adult periodontitis. In this study, we isolated from P. gingivalis a putative glycosyltransferase gene, designated gtfA, which had a consensus domain for glycosyltransferase in its N terminus. GtfA consisted of 248 amino acids and its predicted molecular mass was 28 kDa; however, as the molecular mass of endogenous GtfA protein was around 40 kDa, this suggested that GtfA had undergone some posttranslational modifications. To reveal the role of the gtfA gene in P. gingivalis, we established gtfA-deficient strains by allelic replacement. Morphologically, gtfA-deficient P. gingivalis lacked mature fimbriae. gtfA-deficient P. gingivalis also showed a very low ability for autoaggregation, and its ability to attach to epithelial cells was severely impaired. Thus, the results indicate that the gtfA gene is required for P. gingivalis autoaggregation as well as attachment to epithelial cells. These results suggest that GtfA might have an important role in the pathogenicity of P. gingivalis by regulating adhesion.

Amino Acid Sequence↗
PubMed2005

Role of SraP, a Serine-Rich Surface Protein of Staphylococcus aureus, in binding to human platelets.

The binding of bacteria to platelets is a postulated central event in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus gordonii is mediated in large part by GspB, a high-molecular-mass cell wall glycoprotein. Although Staphylococcus aureus has a GspB homolog (SraP), little is known about its function. SraP has a calculated molecular mass of 227 kDa and, like GspB, is predicted to contain an atypical N-terminal signal sequence, two serine-rich repeat regions (srr1 and srr2) separated by a nonrepeat region, and a C-terminal cell wall anchoring motif (LPDTG). To assess whether SraP contributes to platelet binding, we compared the binding to human platelets of S. aureus strain ISP479C and of an isogenic variant (strain PS767) in which sraP had been disrupted by allelic replacement. Platelet binding in vitro by PS767 was 47% +/- 17% (mean +/- standard deviation) lower than that of ISP479C (P < 0.001). In addition, a recombinant fragment of SraP containing srr1 and the nonrepeat region was found to bind platelets directly. Binding was saturable, suggesting a receptor-ligand interaction. When tested in a rabbit model of endocarditis, in which each animal was simultaneously infected with ISP479C and PS767 at a ratio of approximately 1:1, the titers of the mutant strain within vegetations were significantly lower than those of the parent strain at 1 and 24 h postinfection. These results indicate that SraP can mediate the direct binding of S. aureus to platelets and that the platelet-binding domain of this glycoprotein is located within its N-terminal region. Moreover, the expression of SraP appears to be a virulence determinant in endovascular infection.

Adhesins, Bacterial↗
PubMed2006

Expression microarray and mouse virulence analysis of four conserved two-component gene regulatory systems in group a streptococcus.

Group A streptococcus (GAS) is a gram-positive human bacterial pathogen that causes diseases ranging from relatively mild epithelial cell surface infections to life-threatening invasive episodes. Much is known about the extracellular molecules that contribute to host-pathogen interactions, but in contrast, far less information is available about regulatory genes that control the expression of individual or multiple GAS virulence factors. The eight GAS genomes that have been sequenced have 12 conserved two-component gene regulatory systems (TCSs), but only 3 of these 12 have been studied in detail. Using an allelic replacement strategy with a nonpolar cassette, we inactivated the response regulator of four TCSs that have only weak homology with TCS genes of known or inferred function in other bacteria. The mutant strains were analyzed by expression microarray analysis at four time points and tested in two mouse infection models. Each TCS influenced expression (directly or indirectly) of 12 to 41% of all chromosomal genes, as assessed by growth in Todd-Hewitt broth and a custom Affymetrix GeneChip. None of the isogenic mutant strains was significantly altered for mouse virulence based on intraperitoneal inoculation. Similarly, compared to the wild-type strain, there was no significant difference in skin lesion size for three of the four mutants. In contrast, the DeltaM5005_Spy_0680 mutant strain produced significantly larger abscesses after subcutaneous inoculation into mice, consistent with a hypervirulence phenotype. The mutant strain had significantly higher in vitro expression of several proven and putative virulence genes, including scpA, encoding a peptidase that inactivates complement protein C5a. Together, the data provide new information about previously uncharacterized GAS TCSs.

Animals↗
PubMed2006

Lesions in teichoic acid biosynthesis in Staphylococcus aureus lead to a lethal gain of function in the otherwise dispensable pathway.

An extensive study of teichoic acid biosynthesis in the model organism Bacillus subtilis has established teichoic acid polymers as essential components of the gram-positive cell wall. However, similar studies pertaining to therapeutically relevant organisms, such as Staphylococcus aureus, are scarce. In this study we have carried out a meticulous examination of the dispensability of teichoic acid biosynthetic enzymes in S. aureus. By use of an allelic replacement methodology, we examined all facets of teichoic acid assembly, including intracellular polymer production and export. Using this approach we confirmed that the first-acting enzyme (TarO) was dispensable for growth, in contrast to dispensability studies in B. subtilis. Upon further characterization, we demonstrated that later-acting gene products (TarB, TarD, TarF, TarIJ, and TarH) responsible for polymer formation and export were essential for viability. We resolved this paradox by demonstrating that all of the apparently indispensable genes became dispensable in a tarO null genetic background. This work suggests a lethal gain-of-function mechanism where lesions beyond the initial step in wall teichoic acid biosynthesis render S. aureus nonviable. This discovery poses questions regarding the conventional understanding of essential gene sets, garnered through single-gene knockout experiments in bacteria and higher organisms, and points to a novel drug development strategy targeting late steps in teichoic acid synthesis for the infectious pathogen S. aureus.

Bacterial Proteins↗
PubMed2006

Species-specific differences in the activity of PrfA, the key regulator of listerial virulence genes.

PrfA, the master regulator of LIPI-1, is indispensable for the pathogenesis of the human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii. PrfA is also present in the apathogenic species Listeria seeligeri, and in this study, we elucidate the differences between PrfA proteins from the pathogenic and apathogenic species of the genus Listeria. PrfA proteins of L. monocytogenes (PrfA(Lm) and PrfA*(Lm)), L. ivanovii (PrfA(Li)), and L. seeligeri (PrfA(Ls)) were purified, and their equilibrium constants for binding to the PrfA box of the hly promoter (Phly(Lm)) were determined by surface plasmon resonance. In addition, the capacities of these PrfA proteins to bind to the PrfA-dependent promoters Phly and PactA and to form ternary complexes together with RNA polymerase were analyzed in electrophoretic mobility shift assays, and their abilities to initiate transcription in vitro starting at these promoters were compared. The results show that PrfA(Li) resembled the constitutively active mutant PrfA*(Lm) more than the wild-type PrfA(Lm), whereas PrfA(Ls) showed a drastically reduced capacity to bind to the PrfA-dependent promoters Phly and PactA. In contrast, the efficiencies of PrfA(Lm), PrfA*(Lm), and PrfA(Li) forming ternary complexes and initiating transcription at Phly and PactA were rather similar, while those of PrfA(Ls) were also much lower. The low binding and transcriptional activation capacities of PrfA(Ls) seem to be in part due to amino acid exchanges in its C-terminal domain (compared to PrfA(Lm) and PrfA(Li)). In contrast to the significant differences in the biochemical properties of PrfA(Lm), PrfA(Li), and PrfA(Ls), the PrfA-dependent promoters of hly (Phly(Lm), Phly(L)(i), and Phly(L)(s)) and actA (PactA(Lm), PactA(L)(i), and PactA(L)(s)) of the three Listeria species did not significantly differ in their binding affinities to the various PrfA proteins and in their strengths to promote transcription in vitro. The allelic replacement of prfA(Lm) with prfA(Ls) in L. monocytogenes leads to low expression of PrfA-dependent genes and to reduced in vivo virulence of L. monocytogenes, suggesting that the altered properties of PrfA(Ls) protein are a major cause for the low virulence of L. seeligeri.

Amino Acid Sequence↗
PubMed2006

Regulation of sulfur assimilation pathways in Burkholderia cenocepacia: identification of transcription factors CysB and SsuR and their role in control of target genes.

Two genes encoding transcriptional regulators involved in sulfur assimilation pathways in Burkholderia cenocepacia strain 715j have been identified and characterized functionally. Knockout mutations in each of the B. cenocepacia genes were constructed and introduced into the genome of 715j by allelic replacement. Studies on the utilization of various sulfur sources by 715j and the obtained mutants demonstrated that one of the B. cenocepacia regulators, designated CysB, is preferentially involved in the control of sulfate transport and reduction, while the other, designated SsuR, is required for aliphatic sulfonate utilization. Using transcriptional promoter-lacZ fusions and DNA-binding experiments, we identified several target promoters for positive control by CysB and/or SsuR--sbpp (preceding the sbp cysT cysW cysA ssuR cluster), cysIp (preceding the cysI cysD1 cysN cysH cysG cluster), cysD2p (preceding a separate cluster, cysD2 cysNC), and ssuDp (located upstream of the ssuDCB operon)--and we demonstrated overlapping functions of CysB and SsuR at particular promoters. We also demonstrated that the cysB gene is negatively controlled by both CysB and SsuR but the ssuR gene itself is not significantly regulated as a separate transcription unit. The function of B. cenocepacia CysB (in vivo and in vitro) appeared to be independent of the presence of acetylserine, the indispensable coinducer of the CysB regulators of Escherichia coli and Salmonella. The phylogenetic relationships among members of the "CysB family" in the gamma and beta subphyla are presented.

Alkanesulfonates↗
PubMed2006

The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth.

The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc(2)155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.

Dose-Response Relationship, Drug↗
PubMed2006

Streptococcus pneumoniae DivIVA: localization and interactions in a MinCD-free context.

To clarify the function of DivIVA in Streptococcus pneumoniae, we localized this protein in exponentially growing cells by both immunofluorescence microscopy and immunoelectron microscopy and found that S. pneumoniae DivIVA (DivIVA(SPN)) had a unique localization profile: it was present simultaneously both as a ring at the division septum and as dots at the cell poles. Double-immunofluorescence analysis suggested that DivIVA is recruited to the septum at a later stage than FtsZ and is retained at the poles after cell separation. All the other cell division proteins that we tested were localized in the divIVA null mutant, although the percentage of cells having constricted Z rings was significantly reduced. In agreement with its localization profile and consistent with its coiled-coil nature, DivIVA interacted with itself and with a number of known or putative S. pneumoniae cell division proteins. Finally, a missense divIVA mutant, obtained by allelic replacement, allowed us to correlate, at the molecular level, the specific interactions and some of the facets of the divIVA mutant phenotype. Taken together, the results suggest that although the possibility of a direct role in chromosome segregation cannot be ruled out, DivIVA in S. pneumoniae seems to be primarily involved in the formation and maturation of the cell poles. The localization and the interaction properties of DivIVA(SPN) raise the intriguing possibility that a common, MinCD-independent function evolved differently in the various host backgrounds.

Bacterial Proteins↗
PubMed1993

The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY.

The Pseudomonas aeruginosa pilG gene, encoding a protein which is involved in pilus production, was cloned by phenotypic complementation of a unique, pilus-defective mutant of strain PAO1. This mutant, designated FA2, although resistant to the pilus-specific phage D3112 was sensitive to the pilus-specific phages B3 and F116L. In spite of the unusual phage sensitivity pattern, FA2 lacked the ability to produce functional polar pili (pil) and was incapable of twitching motility (twt). Genetic analysis revealed that the FA2 pil mutation, designated pilG1, mapped near the met-28 marker located at 20 min and was distinct from the previously described pilT mutation. This map location was confirmed by localization of a 6.2-kb EcoRI fragment that complemented FA2 on the SpeI and DpnI physical map of the P. aeruginosa PAO1 chromosome. A 700-bp region encompassing the pilG gene was sequenced, and a 405-bp open reading frame, with characteristic P. aeruginosa codon bias, was identified. The molecular weight of the protein predicted from the amino acid sequence of PilG, which was determined to be 14,717, corresponded very closely to that of a polypeptide with the apparent molecular weight of 15,000 detected after expression of pilG from the T7 promoter in Escherichia coli. Moreover, the predicted amino acid sequence of PilG showed significant homology to that of the enteric CheY protein, a single-domain response regulator. A chromosomal pilG insertion mutant, constructed by allele replacement of the wild-type gene, was not capable of pilus production or twitching motility but displayed normal flagellum-mediated motility. These results, therefore, suggest that PilG may be an important part of the signal transduction system involved in the elaboration of P. aeruginosa pili.

Amino Acid Sequence↗
PubMed1994

The Salmonella typhimurium katF (rpoS) gene: cloning, nucleotide sequence, and regulation of spvR and spvABCD virulence plasmid genes.

The spv region of Salmonella virulence plasmids is essential for the development of a systemic infection in mice. Transcriptional activation of the spvABCD operon occurs during stationary growth phase and is mediated by the regulatory gene product SpvR. We have previously shown that expression of a spvRAB'-cat fusion in Escherichia coli was dependent on the katF (rpoS) locus which encodes an alternative sigma factor (sigma S). The katF gene from Salmonella typhimurium has been cloned, sequenced, and used to construct Salmonella katF mutants by allelic replacement. Using these mutants, we demonstrated by mRNA and gene fusion analyses that sigma S, in conjunction with SpvR, controls the transcription of the regulatory gene spvR. In a second series of experiments, we sought to clarify the relationship between sigma S and SpvR in the control of spvABCD transcription. It was shown that expression of a transcriptional spvAB'-lacZ fusion could be restored in E. coli and Salmonella katF mutants when spvR was expressed in trans from an exogenous promoter. Moreover, identical spvA mRNA startpoints were detected in katF+ and katF strains. These results indicate that the reduction of spvABCD transcription in katF mutants is mainly due to decreased expression of spvR. Finally, mouse inoculation studies with S. typhimurium katF mutants of both wild-type and virulence plasmid-cured strains suggest that katF contributes to Salmonella virulence via the regulation of chromosomal genes in addition to that of spv genes.

Bacterial Proteins↗
PubMed1995

Identification and analysis of aarP, a transcriptional activator of the 2'-N-acetyltransferase in Providencia stuartii.

The aarP gene has been identified in a search for activators of the 2-N-acetyltransferase [encoded by aac(2')-Ia] in Providencia stuartii. Introduction of aarP into P. stuartii on a multicopy plasmid resulted in a 9.9-fold increase in the accumulation of beta-galactosidase from an aac(2')-lacZ fusion. Northern (RNA) blot analysis demonstrated that this increased aac(2')-Ia expression occurred at the level of mRNA accumulation. The deduced AarP protein was 15,898 Da in size and exhibited significant homology to a number of transcriptional activators in the AraC/XyIS family, including TetD,Rob, MarA, and SoxS. The similarity of AarP to the MarA and SoxS proteins prompted an investigation to determine whether AarP is involved in activation of genes in either the multiple antibiotic resistance (Mar) phenotype or redox stress (SoxRS) system. Introduction of aarP on a multicopy plasmid into either P. stuartii or Escherichia coli conferred a Mar phenotype with higher levels of resistance to tetracycline, chloramphenicol, and ciprofloxacin. Multiple copies of aarP in E. coli also resulted in activation of the endonuclease IV gene (nfo), a gene in the SoxRS regulon of E. coli. The function of aarP in its single-copy state was addressed by using allelic replacement to construct an aarP::Cm disruption, which resulted in a fivefold reduction in the accumulation of aac(2')-Ia mRNA. Analysis of aarP regulation showed that aarP mRNA accumulation was slightly increased by exposure to tetracycline and dramatically increased in cells containing the aarB3 (aar3) mutation, which was previously shown to increase transcription of the aac(2')-Ia gene. (P.N. Rather, E. Oroz, K.J. Shaw, R. Hare, and G. Miller, J. Bacteriol. 175:6492-6498).

Acetyltransferases↗