On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers.

Blastocystis spp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects. In silico analysis of the whole genome sequence of Blastocystis subtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified in Blastocystis subtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to either Blastocystis culture supernatants or each recombinant protease. Both Blastocystis culture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis of Blastocystis by increasing intestinal cell permeability.

Enterohemorrhagic Escherichia coli infection has donor-dependent effect on human gut microbiota and may be antagonized by probiotic yeast during interaction with Peyer's patches.

Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens responsible for serious infections ranging from mild diarrhea to hemorrhagic colitis and life-threatening complications. Shiga toxins (Stxs) are the main virulence factor of EHEC. The antagonistic effect of a prophylactic treatment with the probiotic strain Saccharomyces cerevisiae against EHEC O157:H7 was investigated using complementary in vitro human colonic model and in vivo murine ileal loop assays. In vitro, the probiotic treatment had no effect on O157:H7 survival but favorably influenced gut microbiota activity through modulation of short-chain fatty acid production, increasing acetate production and decreasing that of butyrate. Both pathogen and probiotic strains had individual-dependent effects on human gut microbiota. For the first time, stx expression was followed in human colonic environment: at 9 and 12 h post EHEC infection, probiotic treatment significantly decreased stx mRNA levels. Besides, in murine ileal loops, the probiotic yeast specifically exerted a trophic effect on intestinal mucosa and inhibited O157:H7 interactions with Peyer's patches and subsequent hemorrhagic lesions. Taken together, the results suggest that S. cerevisiae may be useful in the fight against EHEC infection and that host associated factors such as microbiota could influence clinical evolution of EHEC infection and the effectiveness of probiotics.

Molecular subtyping of Blastocystis spp. using a new rDNA marker from the mitochondria-like organelle genome.

Blastocystis spp. are common anaerobic intestinal protozoa found in both human and animals. They are characterized by a high genetic diversity with at least 17 subtypes (STs) that have been described on the basis of a 600 bp 'barcoding region' from the 18S rDNA gene. However, analysis of the recently sequenced genome of a Blastocystis ST7 isolate (strain B) revealed the presence of multiple variable copies of the 18S rDNA gene, with 17 completely assembled copies. Comparison of the barcoding region from these 17 copies allowed us to classify the 18S rDNA sequences into 6 clusters, each cluster containing identical sequences. Surprisingly, 4 of these clusters had the highest homology with 18S rDNA sequences from 2 other Blastocystis ST7 isolates referred as QQ98-4 and H. These results suggest that the 18S rDNA gene is not the marker of choice to discriminate between strains within STs. In the present study, we identified a single-copy subtyping rDNA marker in the genome of the mitochondria-like organelles (MLOs). Using a partial sequence of the MLO rDNA, we successfully subtyped 66 isolates from both human and animals belonging to Blastocystis ST1 to ST10. Our results also indicate that this mitochondrial marker could be useful to detect co-infections by different isolates of a same ST.

[Development of a real-time PCR assay for quantitative diagnosis of Toxoplasma gondii after allogeneic bone marrow transplantation]. / Développement d'une technique de PCR quantitative en temps réel dans le diagnostic de la toxoplasmose chez des patients greffés de moelle osseuse.

AIMS OF THE STUDY: A sensitive, rapid and specific diagnostic method is essential for the diagnosis of toxoplasmosis in immunocompromised hosts or in congenital infection. We report the development of a real-time PCR assay for quantitative diagnosis of toxoplasmosis with competitive internal amplification control. This PCR was applied after allogeneic stem cell transplantation to estimate the frequency of reactivation. METHODS AND PATIENTS: Primers and Taqman probe (FAM-BHQ1) were designed to amplify the 529 bp element of T. gondii. The internal amplification control was developed by cloning a fragment of Arabidopsis thaliana DNA flanked by sequences specific for T. gondii 529 bp element. A Taqman probe specific for the competitive internal control was designed and tested (YY-BHQ1). We determined the repeatability and reproducibility of the method. A prospective study was performed on adults who received an allogeneic hematopoietic stem cell transplantation. After transplantation, patients were monitored once per week during the first 100 days; they were then monitored once every 2 weeks until day 180 after transplantation (i.e., day +180). RESULTS: A total of 451 samples from 40 patients were tested. Twenty-five patients had both positive toxoplasmosis serology and an adequate chemoprophylaxis. One sample from one patient was found positive. The rate of reactivation in the population of this study is 4%. CONCLUSION: A monitoring by T. gondii PCR should be realized weekly for patients receiving an allogeneic hematopoietic stem cell transplantation, without an adequate chemoprophylaxis.

Serotyping, stx2 subtyping, and characterization of the locus of enterocyte effacement island of shiga toxin-producing Escherichia coli and E. coli O157:H7 strains isolated from the environment in France.

Twenty-seven Shiga toxin-producing Escherichia coli (STEC) strains were isolated from 207 stx-positive French environmental samples. Ten of these strains were positive for stx(1), and 24 were positive for stx(2) (10 were positive for stx(2vh-a) or stx(2vh-b), 19 were positive for stx(2d), and 15 were positive for stx(2e)). One strain belonged to serotype O157:H7, and the others belonged to serogroups O2, O8, O11, O26, O76, O103, O113, O121, O141, O166, and O174. The environment is a reservoir in which new clones of STEC that are pathogenic for humans can emerge.

Stx2 subtyping of Shiga toxin-producing Escherichia coli isolated from cattle in France: detection of a new Stx2 subtype and correlation with additional virulence factors.

At least 11 Stx2 variants produced by Shiga toxin-producing Escherichia coli (STEC) isolated from patients and animals have been described. The Stx2 subtyping of STEC isolated from healthy cows positive for stx(2) (n = 104) or stx(2) and stx(1) (n = 63) was investigated. Stx2vh-b, Stx2 (renamed Stx2-EDL933), and Stx2vh-a were the subtypes mostly detected among the bovine isolates (39.5, 39, and 25.5%, respectively). Stx2e was not present, and subtypes included in the Stx2d group (Stx2d-OX3a, Stx2d-O111, and Stx2d-Ount) were found infrequently among the isolates examined (8.5%). A combination of two distinct Stx2 subtypes was observed among 23.5% of the strains. For the first time, a combination of three subtypes (Stx2-EDL933/Stx2vh-b/Stx2d and Stx2vh-a/Stx2vh-b/Stx2d) was detected (3.5% of the isolates). In addition, bovine STEC harboring stx(1) and one or two stx(2) genes appeared highly cytotoxic toward Vero cells. A new Stx2 subtype (Stx2-NV206), present among 14.5% of the isolates, showed high cytotoxicity for Vero cells. Two amino acid residues (Ser-291 and Glu-297) important for the activation of Stx2 by human intestinal mucus were conserved on the Stx2-NV206 A subunit. The gene encoding Ehx enterohemolysin was prominent among STEC harboring stx(2)-EDL933 alone (78%) or a combination of stx(2)-EDL933 and stx(2)vh-b (85%). In addition, Stx2-EDL933 and/or Stx2vh-b subtypes were highly associated with other putative virulence factors such as Stx1 and EspP extracellular serine protease, but not with EAST1 enterotoxin.

Heterogeneity of Shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients, cattle, and food samples in central France.

A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like the stx genes or the plasmids (stx(2)-restriction fragment length polymorphism [RFLP], stx(2) gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However, stx(2)-RFLP and stx(2) variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of stx(2)-RFLP, stx(2) variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.

[Verotoxin-producing Escherichia coli infections: study of its prevalence in children in the Auvergne region]. / Les infections à Escherichia coli producteurs de vérotoxines: étude de la prévalence chez l'enfant dans la région Auvergne.

Verotoxin producing Escherichia coli (VTEC) have been associated with disease outbreaks of diarrhea hemorrhagic colitis and hemolytic-uremic syndrome in humans. Contamination occurs mainly by ingestion of beef and dairy products, but water and person to person transmission have also been described. Most of the clinical signs are due to the production of Stx1 and/or Stx2 Shiga toxins, also called verotoxins. Other virulence factors include enterohemolysin, and the product of the eae gene, intimin, involved in the attaching and effacing adherence phenotype. The predominant serotype is O157:H7, but VTEC strains of more than one hundred serotypes can cause human disease. In order to determine the prevalence of VTEC infections among children in the central part of France, stool samples from hospitalized children were examined for stx1 and stx2 genes by using a polymerase chain reaction (PCR) technique. From October 1997 to September 1998, 658 stool samples were analysed: among them 19 (3%) were stx-PCR positive. Only 8 children out of 19 had diarrhea, and for 5 of them, an enteric pathogen other than VTEC was isolated. VTEC strains were isolated from 10 samples: most of the isolates did not produce verotoxins at a high level, and they did not belong to serotypes associated with pathogenicity, which might explain the absence of relationship between VTEC isolation and pathogenicity in our study.

Prevalence and characterization of Shiga toxin-producing Escherichia coli isolated from cattle, food, and children during a one-year prospective study in France.

During a 1-year survey of Shiga toxin-producing Escherichia coli (STEC) prevalence in central France, 2,143 samples were investigated by PCR for Shiga toxin-encoding genes. A total of 330 (70%) of 471 fecal samples collected from healthy cattle at the Clermont-Ferrand slaughterhouse, 47 (11%) of 411 beef samples, 60 (10%) of 603 cheese samples, and 19 (3%) of 658 stool specimens from hospitalized children with and without diarrhea were positive for the stx gene(s). A STEC strain was isolated from 34% (162 of 471) of bovine feces, 4% (16 of 411) of beef samples, 1% (5 of 603) of cheese samples, and 1.5% (10 of 658) of stool specimens. Of the 220 STEC strains isolated, 34 (15%) harbored the stx(1) gene, 116 (53%) harbored the stx(2) gene, and 70 (32%) carried both the stx(1) and stx(2) genes. However, 32 (14.5%) were not cytotoxic for Vero cells. The eae gene, found in 12 (5%) of the 220 strains, was significantly associated with the stx(1) gene and with isolates from children. Sequences homologous to ehxA were found in 102 (46%) of the 220 strains. Thirteen serotypes, OX3:H2, O113:H21, O113:H4, OX3:H21, O6:H10, OX178:H19, O171:H2, O46:H38, O172:H21, O22:H16, O91:H10, O91:H21, and O22:H8, accounted for 102 (55%) of 186 typeable isolates, and only one strain (0.5% of the 186 STEC isolates from cattle), belonged to the O157:H7 serotype. We showed that the majority of the STEC isolates from cattle, beef, and cheese were not likely to be pathogenic for humans and that the STEC strains isolated from children in this study were probably not responsible for diarrheal disease. Finally, the strains associated with hemolytic-uremic syndrome in the same geographical area were shown to belong to particular subsets of the STEC population found in the bovine reservoir.

Non-O157:H7 Stx2-producing Escherichia coli strains associated with sporadic cases of hemolytic-uremic syndrome in adults.

From August 1996 to May 1997, six verotoxin-producing Escherichia coli (VTEC) strains were isolated from stool specimens of adults suffering from hemolytic-uremic syndrome (HUS). All the isolates were stx2 positive and belonged to different serotypes: O6:H4, O91:H10, O91:H21, O rough:H16, OX3:H-, and O nontypeable: H-. The enterohemolysin (Ehly)-encoding genes were detected in two isolates, and none of the isolates harbors the intimin (Eae)-encoding gene. These findings suggest that stx2-positive non-O157:H7 VTEC is a major cause of HUS in adults and that several sources of pathogens are responsible for local endemic infections.

Adhesive properties and antibiotic resistance of Klebsiella, Enterobacter, and Serratia clinical isolates involved in nosocomial infections.

Intestinal colonization by Klebsiella, Enterobacter, and Serratia (KES) strains is a crucial step in the development of nosocomial infections. We studied the adhesive properties, antibiotic resistance, and involvement in colonization or infection of 103 KES clinical isolates: 30 Klebsiella pneumoniae (29%), 16 Klebsiella oxytoca (15%), 30 Enterobacter aerogenes (29%), 14 Enterobacter cloacae (14%), and 13 Serratia sp. (13%) isolates. Half of them were resistant to several antimicrobial agents, including aminoglycosides and beta-lactam antibiotics. A total of 27 of 30 K. pneumoniae isolates (90%) adhered to the human cell line Intestine-407 (Int-407), while none of the K. oxytoca or E. aerogenes isolates and only 2 of the E. cloacae isolates adhered. Three adhesive patterns were observed for K. pneumoniae: an aggregative adhesion in 57% of the isolates, a diffuse adhesion in only one isolate, and a new pattern, localized adhesion, in 30% of the isolates. While most of the sensitive strains adhered with the aggregative phenotype, the localized pattern was associated with resistant K. pneumoniae isolates producing the CAZ-5 beta-lactamase. Furthermore, 45% of such localized-adhesion isolates were involved in severe infections. The distributions of type 1 and type 3 fimbriae, enteroaggregative E. coli, and cf29, pap, and afa/Dr adhesin-encoding genes were determined by using specific DNA probes. No relationship was found between the adhesive pattern and the production of specific fimbriae, suggesting that several unrecognized adhesive factors are involved. Our study indicates that special adhesive properties associated with resistance to antimicrobial agents could account for the pathogenicity of certain nosocomial strains.

A new fimbrial antigen harbored by CAZ-5/SHV-4-producing Klebsiella pneumoniae strains involved in nosocomial infections.

We purified and characterized a new fimbria termed KPF-28 (Klebsiella pneumoniae fimbria with a fimbrin molecular mass of 28 kDa) involved in K. pneumoniae adherence to the human carcinoma cell line Caco-2. Electron microscopy of bacterial surface protein preparations and immunogold labeling of bacterial cells showed that KPF-28 was a long, thin, and flexible fimbria about 4 to 5 nm in diameter and 0.5 to 2 microm long. The N-terminal amino acid sequence of the KPF-28 major fimbrial subunit showed no homology with type 1 and type 3 pili of K. pneumoniae but showed 61.7% identity with residues 6 to 19 of the N-terminal amino acid sequence of PapA, the Pap major pilus subunit expressed by uropathogenic Escherichia coli strains. Total amino acid content determination showed that the KPF-28 major subunit composition was close to that of the GVVPQ fimbrial family major subunits expressed by pathogenic E. coli strains. The study of the prevalence of KPF-28 among K. pneumoniae strains involved in nosocomial infections revealed that KPF-28 was found in the great majority of the K. pneumoniae strains producing the CAZ-5/SHV-4 extended-spectrum beta-lactamase. As shown by curing and mating experiments, the R plasmid encoding the CAZ-5/SHV-4 enzyme was found to be involved in but not solely responsible for KPF-28 expression. Hybridization experiments using an oligonucleotide probe corresponding to the N-terminal part of the 28-kDa protein revealed that the structural gene encoding the KPF-28 major subunit was localized on this R plasmid. KPF-28 is a putative colonization factor of the human gut, since the ceftazidine-sensitive derivative strain CF914-1C no longer adhered and since the Fab fragments of antibodies raised against KPF-28 inhibited adhesion of K. pneumoniae CF914-1 to the Caco-2 cell line.

Molecular characterization and adhesive properties of CF29K, an adhesin of Klebsiella pneumoniae strains involved in nosocomial infections.

We previously described a CS31A-related protein, CF29K, expressed by Klebsiella pneumoniae strains involved in nosocomial infections. In this study, we cloned and sequenced cf29A, the structural gene of the CF29K protein, and showed that CF29K is an antigenic subtype of CS31A. The CF29K protein was found to be identical to the CS31A-L protein on the basis of biochemical and immunological properties. In contrast, the CS31A-H protein presented a different apparent molecular mass during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a different limited degradation pattern with endopeptidase V8, and a specific conformational epitope. We cloned and sequenced the CS31A-L structural gene and confirmed that CF29K and CS31A-L are identical, but their major subunits differ from ClpG (the CS31A-H subtype major subunit) by one amino acid at position 89 of the mature protein, which is a lysine in CF29K instead of the asparagine in ClpG. Site-directed mutagenesis experiments demonstrated that the biochemical and immunological differences between CS31A-H and CF29K or CS31A-L were dependent only on the amino acid at position 89 of the mature protein. To study the adhesive properties of CS31A-H and CF29K in the same Escherichia coli reference strain, we performed transcomplementation experiments with the cloned CS31A major-subunit structural genes or cloned cf29A gene and the clp accessory genes of the CS31A operon. We showed that CS31A-L, CF29K, and CS31A-H were involved in adhesion to Caco-2 and Int-407 cells but not to HEp-2 cells. Nevertheless, K. pneumoniae strains and corresponding E. coli transconjugants producing CF29K adhered to cultured Caco-2, Int-407, and HEp-2 cells, indicating the expression of another R-plasmid-encoded adhesin that mediated adhesion to HEp-2 cells. The carbohydrate part of the eucaryotic receptor of CF29K and CS31A-H adhesins was investigated by adhesion inhibition experiments with Int-407 cells. Although CS31A and CF29K belong to the K88 adhesin family, the receptor does not contain N-acetyl-D-galactosamine residues but contains N-acetylneuraminic acid and N-acetyl-D-glucosamine.

Escherichia coli strains involved in diarrhea in France: high prevalence and heterogeneity of diffusely adhering strains.

Two hundred sixty-two strains of Escherichia coli isolated from diarrheal stool specimens from infants, children, and adults hospitalized in Clermont-Ferrand, France, were studied to classify them in the previously described pathogenic groups of E. coli involved in diarrheal diseases. A total of 1.5% of them belonged to the enterotoxigenic E. coli pathotype, but none belonged to the enteroinvasive E. coli, enterohemorrhagic E. coli, or enteropathogenic E. coli pathotypes. Seventeen strains (6.5%) exhibited an aggregative pattern of adhesion to HEp-2 cells (EAggEC pathotype), but of these, three (17.6%) did not hybridize with the EAggEC DNA probe. Most of the strains involved in diarrhea belonged to the diffusely adhering E. coli group; 100 strains (38.2%) exhibited a diffuse adhesion (DA) to HEp-2 cells. Only eight strains (8.9%) from controls diffusely adhered to HEp-2 cells. The highly significant difference (P < 0.0001) between DA strains from patients and from controls suggests that the diffusely adhering E. coli strains should be considered pathogens. Only 33 of them (33%) hybridized with the previously described DA DNA probe, and only 2 (2%) hybridized with the AIDA DNA probe. Four different major proteins were observed in the bacterial surface extracts of the 33 strains positive with the DA DNA probe. In addition, 16 strains that diffusely adhered to HEp-2 cells induced a cytotoxic effect on HEp-2 cells that was characterized by pyknosis and lysis of the cytoplasmic membrane. This cytotoxic effect was correlated with the synthesis of a hemolysin. The genes involved in diffuse adhesion to HEp-2 cells were located on conjugative R plasmids in strains that did not hybridize with the DA or AIDA DNA probes.

In vivo DNA-protein interactions at the divergent mercury resistance (mer) promoters. I. Metalloregulatory protein MerR mutants.

Regulation of transcriptional initiation of the Tn21 mercury resistance (mer) operon occurs at the divergent promoter region lying between the structural genes (merTPCAD) and a regulatory gene (merR). During repression, both promoters are negatively regulated by MerR bound to a dyadic operator located between the -10 and -35 hexamers of PTPCAD. Upon Hg(II) induction, MerR activates transcription only from PTPCAD and continues to repress transcription from PR. Using in vivo dimethyl sulfate and KMnO4 footprinting of the merOP region of strains carrying wild-type MerR or MerR mutants, we have dissected the steps in MerR-mediated positive and negative regulation of the divergent mer promoters. The greater sensitivity of primer extension footprinting allowed the resolution of details previously undetectable in vivo. Two MerR mutants unable to bind merOP DNA allow RNA polymerase to form an open complex preferentially at PR. The intensity of the PR open complex is considerably less than that which occurs upon MerR-Hg(II) activation of PTPCAD and considerably greater than that which occurs at PTPCAD when MerR is deleted; this is the first in vivo estimate of the relative strengths of these two promoters. Although retaining the wild-type capacity to sequester RNA polymerase in the closed complex, the four MerR mutants defective in Hg(II) binding do not distort the dyad DNA or foster open complex formation when the inducer is added. Two activation-defective MerR mutants foster closed complex formation as well as wild-type, but they do not distort the dyad DNA or foster open complex formation when the inducer is added, although they are also able to bind Hg(II). Two semiconstitutive inducible MerR mutants differ from each other in that one (which lies nearer the COOH terminus) does distort the dyad center upon Hg(II) induction, whereas the other (which lies in near the center of merR) exhibits no dyad distortion upon induction. Paradoxically, despite their relatively strong semiconstitutive expression of mer-lac transcriptional fusions, neither of these mutants has a detectable open complex in the absence of added Hg(II).

In vivo DNA-protein interactions at the divergent mercury resistance (mer) promoters. II. Repressor/activator (MerR)-RNA polymerase interaction with merOP mutants.

Transcription of the Tn21 mercury resistance (mer) operon is regulated by MerR which represses and activates the mer structural genes (merTPCAD) in the absence and presence of Hg(II), respectively. The promoter for the structural genes (PTPCAD) is divergently overlapped with the promoter for the regulatory gene (PR), and a dyadic operator lies between the -10 and -35 hexamers of PTPCAD. Using in vivo dimethyl sulfate and KMnO4 footprinting of mutant mer operator-promoter (merOP) DNA to observe MerR and RNA polymerase-mediated interactions with the merOP region, we have identified three distinct domains within the palindromic mer operator. Dyad domain I consists of the outermost bases on the left arm of the operator palindrome whose alteration causes a shift, but apparently not a major loss, in occupancy by MerR, and no decrease in RNA polymerase occupancy. Mutants in dyad domain I are semiconstitutive but support additional Hg(II)-induced open complex formation at PTPCAD. Dyad domain II consists of the four highly conserved inner bases ( ... GTAC ... GTAC ... ) of the seven-base interrupted dyad, alteration of which severely modifies both MerR and RNA polymerase contacts in the promoter region. Mutants in domain II generally allow constitutive open complex formation at PR. One unusual mutant of this group retains most of the wild-type dyad's ability to repress both promoters but is unable to support activation at PTPCAD in response to Hg(II), indicating that MerR undergoes a conformational change and that the required base contacts for activation are different than those for repression. Dyad domain III is tentatively defined by a mutant in the outermost base of the right palindrome arm which is unaffected in either MerR or RNA polymerase occupancy, however, a second lesion within the PTPCAD -10 hexamer of this mutant limits effective open complex formation. Other mutations lying solely within the -10 RNA polymerase recognition hexamer of PTPCAD are similarly competent in both MerR and RNA polymerase binding, but inadequate for open complex formation. One such mutant also affects the overlapping -10 hexamer of PR and results in reduced occupancy by both MerR and RNA polymerase, likely as a result of inefficient transcriptional initiation of merR mRNA. Finally, mutations affecting the -35 hexamer of PTPCAD bind MerR but not RNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)

Sequence and molecular characterization of the ROB-1 beta-lactamase gene from Pasteurella haemolytica.

The ROB-1 beta-lactamase-encoding plasmids from eight Pasteurella and two Haemophilus strains were compared by restriction endonuclease and hybridization analyses. Two types of ROB-1-encoding plasmids, which differed in size, were detected. One (4.1 kb) was found only in Pasteurella strains. The other (4.4 kb) was found in both Haemophilus influenzae and in one of the eight Pasteurella strains examined. These two plasmids shared multiple homologous fragments, suggesting that one was derived from the other. The ROB-1-encoding gene from Pasteurella haemolytica LNPB 51 was cloned and sequenced. An open reading frame of 915 nucleotides was found; it encoded a 305-amino-acid protein. Analysis of this amino acid sequence confirmed that the enzyme was found; it encoded a 305-amino-acid protein. Analysis of this amino acid sequence confirmed that the enzyme is a class A beta-lactamase. It had 32 to 48% homology with other class A enzymes and exhibited several common features of the gram-positive beta-lactamases. The ROB-1 mature protein, however, contained only one cysteine residue at position 123. These results suggest that ROB-1 is a link between beta-lactamases of gram-negative and gram-positive bacteria. An internal 230-bp DraI fragment from ROB-1 hybridized only with plasmid DNA from ROB-1-producing strains. This specific probe could be useful in epidemiological studies.

Genetic determinant of the ROB-1 beta-lactamase in bovine and porcine Pasteurella strains.

The ROB-1 beta-lactamase, previously described in Haemophilus influenzae, has been found in the genus Pasteurella. In three bovine strains of Pasteurella multocida and Pasteurella haemolytica, ROB-1 production was determined by plasmids of 4.4 kilobases. In one porcine strain of Pasteurella aerogenes, the enzyme seems to be chromosomally encoded.