Preparation of a biologically active rat prolactin devoid of antidiuretic activity.

Rat prolactin (NIH rPRL-B2) was purified using Sephadex chromatography and isoelectric focusing. SDS-gel electrophoresis of the original material showed a major band of 23K daltons, as well as several minor bands; the purified prolactin had a single 23K band. The original material contained 33 pg of immunoreactive vasopressin per 0.1 micrograms of material; vasopressin was not detectable in the purified material by either RIA or bioassay. The purified preparation had complete biological activity in a mammary gland bioassay and a receptor binding assay.

Type-4 pili and meningococcal adhesiveness.

The ability to interact with non-phagocytic cells is a crucial virulence attribute of the meningococcus. Pili play a major role in this process and are the only means yet discovered by which capsulated bacteria may adhere to cells. Pilus-mediated adhesion is a two-step process which requires (i) the expression of the adhesin PilC1 and (ii) the expression of an appropriate pilin variant. Some pilin variants have the ability to modify the degree of adhesiveness through the formation of bundles of pili which increases bacteria-bacteria interactions.

Vascular structure in the forearm and calf after 6 months of angiotensin converting enzyme inhibition in elderly hypertensive subjects with left ventricular hypertrophy.

OBJECTIVE: To investigate the effects of angiotensin converting enzyme inhibition on the structure of resistive arteries assessed from minimal vascular resistance in the forearm and the calf and on left ventricular mass index of elderly hypertensive subjects with left ventricular hypertrophy. DESIGN AND METHODS: We evaluated 23 elderly patients [12 women and 11 men, aged 70 +/- 1 years (mean +/- SEM)] with essential hypertension assessed with ambulatory blood pressure monitoring and left ventricular hypertrophy before and at the end of 6 months' treatment with quinapril. Minimal vascular resistance was calculated as the ratio of mean arterial pressure to regional blood flow measured upon restoration of circulation after 13 min of ischaemia combined with exercise and taken as an index of resistive vessel structure (i.e. media:lumen ratio). RESULTS: Daytime ambulatory blood pressure had decreased from 164 +/- 2/95 +/- 1 to 147 +/- 3/86 +/- 2 mmHg (P < 0.001) and left ventricular mass index decreased from 138 +/- 4 to 120 +/- 5 g/m2 (P < 0.001) at the end of treatment. Minimal vascular resistance in the forearm had decreased from 3.1 +/- 0.3 to 2.4 +/- 0.2 mmHg/ml per 100 ml per min (P < 0.01) whereas we observed no change in minimal vascular resistance in the calf after treatment (4.6 +/- 0.7 versus 4.2 +/- 0.4 mmHg/ml per 100 ml per min, NS). The decrease in minimal vascular resistance in the forearm was correlated significantly to the fall in 24 h ambulatory mean arterial pressure (r = 0.58, P < 0.01). Changes in left ventricular mass index were not correlated to those in ambulatory blood pressure or to those in minimal vascular resistance in the forearm. CONCLUSIONS: A 6-month reduction in blood pressure under quinapril treatment was associated with decreases in left ventricular hypertrophy and in minimal vascular resistance in the forearm of elderly hypertensive patients. Absence of structural changes in leg vasculature could be related to the greater arterial pressure prevalent in the lower limbs while patients stood upright and, consequently, a proportionately smaller decrease in blood pressure, as well as greater structural changes and fibrous damage than those of the upper limbs.

[The Coordination of service plans.]. / La coordination des plans de services.

Several American laws have been passed to co-ordinate various levels of health services. The aim is to help patients reintegrate their community, particularly for those physically and intellectually handicapped patients, people suffering from severe psychological problems and elderly who are increasingly non self-sufficient. Certain aspects of health care, such as a person's real needs, the resources within a person's community, along with consistent and adequate services, must be considered if we are to overcome the downside of both budget constraints and such a complex health service network. This article reviews the author's ten-year experience in the field of mental health in the U.S.A.

Describing ancient horizontal gene transfers at the nucleotide and gene levels by comparative pathogenicity island genometrics.

MOTIVATION: Lateral gene transfer is a major mechanism contributing to bacterial genome dynamics and pathovar emergence via pathogenicity island (PAI) spreading. However, since few of these genomic exchanges are experimentally reproducible, it is difficult to establish evolutionary scenarios for the successive PAI transmissions between bacterial genera. Methods initially developed at the gene and/or nucleotide level for genomics, i.e. comparisons of concatenated sequences, ortholog frequency, gene order or dinucleotide usage, were combined and applied here to homologous PAIs: we call this approach comparative PAI genometrics. RESULTS: YAPI, a Yersinia PAI, and related islands were compared with measure evolutionary relationships between related modules. Through use of our genometric approach designed for tracking codon usage adaptation and gene phylogeny, an ancient inter-genus PAI transfer was oriented for the first time by characterizing the genomic environment in which the ancestral island emerged and its subsequent transfers to other bacterial genera.

Role of glycosylation at Ser63 in production of soluble pilin in pathogenic Neisseria.

Pilus-mediated adhesion is essential in the pathogenesis of Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC). Pili are assembled from a protein subunit called pilin. Pilin is a glycoprotein, and pilin antigenic variation has been shown to be responsible for intrastrain variability with respect to the degree of adhesion in both MC and GC. In MC, high-adhesion pilins are responsible for the formation of bundles of pili which bind bacteria and cause them to grow as colonies on infected monolayers. In this work, we selected MC and GC pilin variants responsible for high and low adhesiveness and introduced them into the other species. Our results demonstrated that a given pilin variant expressed an identical phenotype in either GC or MC with respect to bundling and adhesiveness to epithelial cells. However, the production of truncated soluble pilin (S pilin) was consistently more abundant in GC than in MC. In the latter species, the glycosylation of pilin at Ser63 was shown to be required for the production of a truncated monomer of S pilin. In order to determine whether the same was true for GC, we engineered various pilin derivatives with an altered Ser63 glycosylation site. The results of these experiments demonstrated that the production of S pilin in GC was indeed more abundant when pilin was posttranslationally modified at Ser63. However, nonglycosylated variants remained capable of producing large amounts of S pilin. These data demonstrated that for GC, unlike for MC, glycosylation at Ser63 is not required for S-pilin production, suggesting that the mechanisms leading to the production of S pilin in GC and MC are different.

Effect of potassium ion on the phosphorus-31 nuclear magnetic resonance spectrum of the pyridoxal 5'-phosphate cofactor of Escherichia coli D-serine dehydratase.

31P NMR studies were undertaken to determine how potassium ion increases the cofactor affinity of Escherichia coli D-serine dehydratase, a model pyridoxal 5'-phosphate requiring enzyme that converts the growth inhibitor D-serine to pyruvate and ammonia. Potassium ion was shown to promote the appearance of a second upfield shifted cofactor 31P resonance at 4.0 ppm (pH 7.8, 25 degrees C), that increased in area at the expense of the resonance at 4.4 ppm observed in the absence of K+. Na+ antagonized the K+ promoted appearance of the second resonance. These observations suggest that K+ and Na+ stabilize conformational states that differ with respect to O-P-O bond angle, conformation, and/or hydrogen bonding of the phosphate group. An analysis of the dependence of the relative intensities of the two resonances on the K+ concentration yielded a value of ca. 10 mM for the equilibrium constant for dissociation of K+ from D-serine dehydratase. The chemical shift difference between the two resonances indicated that the K+-stabilized and Na+-stabilized forms of the enzyme interconvert at a frequency less than 16 s-1 at pH 7.8, 25 degrees C.

The glycine-rich region of Escherichia coli D-serine dehydratase. Altered interactions with pyridoxal 5'-phosphate produced by substitution of aspartic acid for glycine.

Replacement of glycine by aspartic acid at either of two sites in a conserved, glycine-rich region inactivates the pyridoxal 5'-phosphate-dependent enzyme D-serine dehydratase (DSD) from Escherichia coli. To investigate why aspartic acid at position 279 or 281 causes a loss of activity, we measured the affinity of the G----D variants for pyridoxal 5'-phosphate and a cofactor:substrate analog complex and compared the UV, CD, and fluorescence properties of wild-type D-serine dehydratase and the inactive variants. The two G----D variants DSD(G279D) and DSD (G281D) displayed marked differences from wild-type D-serine dehydratase and from each other with respect to their affinity for pyridoxal 5'-phosphate and for a pyridoxal 5'-phosphate:glycine Schiff base. Compared to the wild-type enzyme, the cofactor affinity of DSD(G279D) and DSD(G281D) was decreased 225- and 50-fold, respectively, and the ability to retain a cofactor:glycine complex was decreased 765- and 1970-fold. The spectral properties of the inactive variants suggest that they form a Schiff base linkage with pyridoxal 5'-phosphate but do not hold the cofactor in a catalytically competent orientation. Moreover, the amount of cofactor aldamine in equilibrium with cofactor Schiff base is increased in DSD(G279D) and DSD(G281D) relative to that in wild-type DSD. Collectively, our findings indicate that introduction of a carboxymethyl side chain at G-279 or G-281 directly or indirectly disrupts catalytically essential protein-cofactor and protein-substrate interactions and thereby prevents processing of the enzyme bound cofactor:substrate complex. The conserved glycine-rich region is thus either an integral part of the D-serine dehydratase active site or conformationally linked to it.

High adhesiveness of encapsulated Neisseria meningitidis to epithelial cells is associated with the formation of bundles of pili.

Pili are indispensable in adhesion of encapsulated Neisseria meningitidis (MC) to eukaryotic cells. Intrastrain variability with respect to the degree of adhesion is the result of pilin antigenic variation. We have localized the region responsible for this variability to the 20-amino-acid hypervariable domain of pilin. The replacement of an aspartic acid, located in the hypervariable region of a low-adhesive variant by a lysine restored high adhesiveness. To assess whether hyperadhesiveness conferred by some pilin variants was related to the generation of a new pilus-associated ligand, high- and low-adhesive variants were purified. In a first step, low- and high-adhesive pilins were fused to maltose binding protein (MBP). These hybrid proteins bound epithelial cells with the same affinity. Truncated MBP pilin fusions identified a cell-binding domain within the 77 residues of the N-terminal end of mature pilin. This region of the protein is common to low- and high-adhesive derivatives used in this work, thus eliminating the possibility that high adhesiveness conferred by some pilin variants was because of the generation of a new pilus-associated ligand. Electron-microscopic examination showed that low-adhesive derivatives expressed long and distinct pili and adhered as single cells. In contrast, pili of derivatives expressing high-adhesive pilins, either wild type or mutagenized from the low-adhesive variant, formed large bundles which bound bacteria and caused them to grow as colonies on infected monolayers. These data demonstrate that aggregative pili promote high adhesiveness of encapsulated MC.

Effects of different training intensities on 24-hour blood pressure in hypertensive subjects.

BACKGROUND: It is generally accepted that physical training decreases blood pressure in hypertensive subjects, but the importance of training intensity has not been established. This study compared the effects of endurance training at different intensities on ambulatory blood pressure and on blood pressure load (percentage of readings above 140/90 and 120/80 mm Hg during the waking and sleeping periods, respectively). METHODS AND RESULTS: Previously sedentary subjects with mild to moderate hypertension were evaluated in a crossover fashion according to a Latin square after a sedentary control period and after training at low and at moderate intensity corresponding to 50% and 70% of maximal oxygen uptake, respectively. Each period lasted 10 weeks. After training at moderate intensity, a higher maximal oxygen uptake was found compared with sedentary values but not after training at low intensity. Both training intensities exerted a similar antihypertensive effect of about 5 mm Hg for systolic and diastolic 24-hour blood pressures. However, training at low intensity reduced blood pressure exclusively during the walking hours, whereas training at a moderate intensity reduced blood pressure only during the evening and sleeping hours. Waking blood pressure load decreased from 66% to 49% after training at low intensity, whereas sleeping blood pressure load decreased from 61% to 34% after training at moderate intensity (both P < .05). CONCLUSIONS: Low- and moderate-intensity training produce similar 24-hour blood pressure reductions, but each training intensity may interfere with different pathogenic effects associated with different blood pressure profiles.

The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion.

The ability of Neisseria meningitidis (MC) to interact with cellular barriers is essential to its pathogenesis. With epithelial cells, this process has been modeled in two steps. The initial stage of localized adherence is mediated by bacterial pili. After this phase, MC disperse and lose piliation, thus leading to a diffuse adherence. At this stage, microvilli have disappeared, and MC interact intimately with cells and are, in places, located on pedestals of actin, thus realizing attaching and effacing (AE) lesions. The bacterial attributes responsible for these latter phenotypes remain unidentified. Considering that bacteria are nonpiliated at this stage, pili cannot be directly responsible for this effect. However, the initial phase of pilus-mediated localized adherence is required for the occurrence of diffuse adherence, loss of microvilli, and intimate attachment, because nonpiliated bacteria are not capable of such a cellular interaction. In this work, we engineered a mutation in the cytoplasmic nucleotide-binding protein PilT and showed that this mutation increased piliation and abolished the dispersal phase of bacterial clumps as well as the loss of piliation. Furthermore, no intimate attachment nor AE lesions were observed. On the other hand, PilT- MC remained adherent as piliated clumps at all times. Taken together these data demonstrate that the induction of diffuse adherence, intimate attachment, and AE lesions after pilus-mediated adhesion requires the cytoplasmic PilT protein.

NMR spectra of exchangeable protons of pyridoxal phosphate-dependent enzymes.

We have recorded 1H NMR spectra in H2O for exchangeable protons of four pyridoxal phosphate-dependent enzymes: D-serine dehydratase, aspartate aminotransferase, tryptophan: indole-lyase and glutamate decarboxylase. The molecular masses range from 48-250 kDa. In every case there are downfield peaks which are lost when the apoenzyme is formed. In most cases some peaks shift in response to interactions with substrates and inhibitors and with changes in pH. We associate one downfield resonance with the proton on the ring nitrogen of the coenzyme and others with imidazole groups that interact with coenzyme or substrates. The chemical shift for the coenzyme-bound proton differs for free enzyme, substrate Schiff base or quinonoid forms.

Contribution of a conserved arginine near the active site of Escherichia coli D-serine dehydratase to cofactor affinity and catalytic activity.

We have employed site-directed mutagenesis to investigate the contribution of a conserved arginyl residue to the catalytic activity and cofactor affinity of D-serine dehydratase, a model pyridoxal 5'-phosphate (vitamin B6) enzyme. Replacement of R-120 in the active site peptide of D-serine dehydratase by L decreased the affinity of the enzyme for pyridoxal 5'-phosphate by 20-fold and reduced turnover by 5-8-fold. kappa cat displayed modified substrate alpha-deuterium isotope effects and altered dependence on both temperature and pH. Analysis of the pH rate profiles of DSD and the R-120----L variant indicated that R-120 interacts electrostatically with catalytically essential ionizable groups at the active site of wild type D-serine dehydratase. The decrease in cofactor affinity observed for DSD(R120L) was not accompanied by significant perturbations in the UV, CD, or 31P NMR spectrum of the holoenzyme, suggesting that the contribution of R-120 to pyridoxal phosphate affinity may be indirect or else involve an interaction with a cofactor functional group other than the 5'-phosphoryl moiety. The properties of two other site-directed variants of D-serine dehydratase indicated that the pyridoxal 5'-phosphate:K-118 Schiff base was indifferent to a small change in the shape of the side chain at position 117 (I-117----L), whereas replacement of K-118 by H resulted in undetectable levels of enzyme. A poor ability to bind cofactor may have rendered DSD(K118H) susceptible to intracellular proteolysis.

Disruption of active site interactions with pyridoxal 5'-phosphate and substrates by conservative replacements in the glycine-rich loop of Escherichia coli D-serine dehydratase.

We have used site-directed mutagenesis to examine the function of three putative active site residues (C278, G279, and G281) of the vitamin B6 enzyme D-serine dehydratase. These residues lie in or adjacent to a conserved glycine-rich loop that is known to interact with the pyridoxal 5'-phosphate cofactor in several B6 enzymes and that resembles the GXGXXG loop of nucleotide-binding sites. The cofactor affinity, catalytic properties, and spectral properties (UV, CD, fluorescence, and 31P NMR) of alanine variants C278A, G279A, and G281A were measured as well as the susceptibility of each variant to thiol modification by 5,5'-dithiobis(2-nitrobenzoic acid). The specific thiols modified in each variant and wild type D-serine dehydratase were identified by amino acid sequencing of labeled tryptic peptides. C278A, G279A, and G281A displayed 10-, 33-, and 22-fold lower affinities for pyridoxal 5'-phosphate than did wild type D-serine dehydratase and turnover numbers with D-serine that were 50, 6, and 60% of normal, respectively. The introduction of a methyl side chain into G281 enhanced catalytic efficiency with the substrates D-threonine, D-allo-threonine, and L-serine, whereas the methyl side chain at position 279 impaired catalysis of all substrates as well as cofactor affinity. The 31P NMR spectrum of D-serine dehydratase was minimally perturbed by the alanine substitutions, consistent with the view that neither G279 nor G281 interacts with the phosphate group of the cofactor (in contrast to the arrangement found in several other B6 enzymes). C311 was the single thiol modified by 5,5'-dithiobis(2-nitrobenzoic acid) in wild type D-serine dehydratase. Two normally inaccessible thiol groups, C233 and C278, were rendered susceptible to modification as a consequence of either G----A substitution, and modification of C278 was associated with inactivation of G279A and G281A. These observations suggest that small perturbations in the glycine-rich loop induce conformational changes spanning a considerable area around the active site.

D-serine dehydratase from Escherichia coli. DNA sequence and identification of catalytically inactive glycine to aspartic acid variants.

We have identified two glycyl residues whose integrity is essential for the catalytic competence of a model pyridoxal 5'-phosphate requiring enzyme, D-serine dehydratase from Escherichia coli. This was accomplished by isolating and sequencing the structural gene from wild type E. coli and from two mutant strains that produce inactive D-serine dehydratase. DNA sequencing indicated the presence of a single glycine to aspartic acid replacement in each variant. The amino acid replacements lie in a glycine-rich region of D-serine dehydratase well removed from pyridoxal 5'-phosphate-binding lysine 118 in the primary structure of the enzyme. The striking effect of these two glycine to aspartic acid replacements on catalytic activity, the conservation of the glycine-rich region in several pyridoxal 5'-phosphate-dependent enzymes that catalyze alpha/beta-eliminations, and the placement of similar glycine-rich sequences in well-characterized active site structures suggest that the glycine-rich region interacts with the cofactor at the active site of the enzyme.

[Primary biliary cirrhosis: therapeutic trial using plasma exchange and immunosuppression. Preliminary results]. / Cirrhose biliaire primitive: essai thérapeutique par échanges plasmatiques et immunosuppression. Résultats préliminaires.

Four women aged 35 to 57 years (m: 49.2) with primary biliary cirrhosis and intractable pruritus had an estimated median survival time according to Christensen between 6 and 50 months (m: 27). They were enrolled in a therapeutic trial associating plasma exchange (PE) and immunosuppressive drugs. During the first 2 months, they received prednisone 15 mg/day, cyclophosphamide 2 mg/kg BW/day and 28 PE (60 ml/kg BW). Pruritus disappeared rapidly. After 2 months, mean levels of bilirubin, alkaline phosphatases, IgM and anti-mitochondrial antibodies dropped by 27 p. 100, 47 p. 100, 50 p. 100 and 85 p. 100 respectively, whereas amino-transferase and gamma-glutamyl-transpeptidase activities were unaltered. Two patients then received supportive therapy only: one was lost to follow-up after one year, the other died 50 months later from liver failure. The third patient received PE only when intractable pruritus reappeared: anti-mitochondrial antibodies, IgM and alkaline phosphatases remained below initial values for 38 months, until successful liver transplantation was performed. The fourth patient was treated on a long term basis by PE twice a month, prednisone 10 mg/day for 3 years and cyclophosphamide 1 mg/kgBW/day for one year only. Her initial estimated survival time was 6 months, but 3 years later she developed portal hypertension; anti-mitochondrial antibody titer was between 0 and 1/80, alkaline phosphatase levels reduced by 80 p. 100 to 70 p. 100; bilirubin level up by 50 p. 100. The mean survival time for the 4 patients exceeded 34 months, results better than those obtained with other kinds of therapy.

Consequences of the loss of O-linked glycosylation of meningococcal type IV pilin on piliation and pilus-mediated adhesion.

Pili, which are assembled from protein subunits called pilin, are indispensable for the adhesion of capsulated Neisseria meningitidis (MC) to eukaryotic cells. Both MC and Neisseria gonorrhoeae (GC) pilins are glycosylated, but the effect of this modification is unknown. In GC, a galactose alpha-1,3-N-acetyl glucosamine is O-linked to Ser-63, whereas in MC, an O-linked trisaccharide is present between residues 45 and 73 of pilin. As Ser-63 was found to be conserved in pilin variants from different strains, it was replaced by Ala in two MC variants to test the possible role of this residue in pilin glycosylation and modulation of pili function. The mutated alleles were stably expressed in MC, and the proteins they encoded migrated more quickly than the normal protein during SDS-PAGE. As controls, neighbouring Asn-61 and Ser-62 were replaced by an Ala with no effect on electrophoretic mobility. Silver staining of purified pilin obtained from MC after oxidation with periodic acid confirmed the loss of glycosylation in the Ser-63-->Ala pilin variants. Mass spectrometry of HPLC-purified trypsin-digested peptides of pilin and Ser-63-->Ala pilin confirmed that peptide 45-73 has the molecular size of a glycopeptide in the wild type. In strains producing non-glycosylated pilin variants, we observed that (i) no truncated S pilin monomer was produced; (ii) piliation was slightly increased; and (iii) presumably as a consequence, adhesiveness for epithelial cells was increased 1.6- to twofold in these derivatives. In addition, pilin monomers and/or individual pilus fibres, obtained after solubilization of a crude pili preparation in a high pH buffer, were reassociated into insoluble aggregates of pili more completely with non-glycosylated variants than with the normal pilin. Taken together, these data eliminate a major role for pilin glycosylation in piliation and subsequent pilus-mediated adhesion, but they demonstrate that glycosylation facilitates solubilization of pilin monomers and/or individual pilus fibres.

Purification of alpha 1-microglobulin produced by human hepatoma cell lines. Biochemical characterization and comparison with alpha 1-microglobulin synthesized by human hepatocytes.

alpha 1-Microglobulin (alpha 1m) was determined by radio-immunoassay in the supernatants of five human hepatoma cell lines. High amounts of alpha 1m were produced by PLC/PRF/5, intermediate ones by Hep G2 and Hep 3B and very low ones by Malhavu and SK Hepl. alpha 1m isolated from hepatoma cell lines PLC/PRF/5 or Hep G2 supernatants displayed the same physicochemical properties as that purified from human urines: the apparent molecular mass was 26 kDa and the pI from 5.6 to 6.4 as measured after two-dimensional polyacrylamide gel electrophoresis in denaturating conditions; for the native molecule the pI was estimated to be 4.0-4.9. Both urinary and hepatoma alpha 1m migrate as a diffuse band in the alpha zone in agarose gel at pH 8.6 in non-denaturing conditions and present a brown chromophore covalently associated with the molecule. After biosynthetic labelling with [35S]methionine, proteins extracted from hepatoma cell line PLC/PRF/5 and from isolated hepatocytes of human liver were separated by two-dimensional PAGE and transferred to a nitrocellulose membrane. alpha 1m was identified and found to be identical in both cases. However, when compared with the alpha 1m isolated from cell supernatants, less charge heterogeneity but also minor additional spots of higher molecular mass were observed.

The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence.

The Yersinia pseudotuberculosis chromosome contains a seven-gene polycistronic unit (the pmrF operon) whose products share extensive homologies with their pmrF counterparts in Salmonella enterica serovar Typhimurium (S. typhimurium), another Gram-negative bacterial enteropathogen. This gene cluster is essential for addition of 4-aminoarabinose to the lipid moiety of LPS, as demonstrated by MALDI-TOF mass spectrometry of lipid A from both wild-type and pmrF-mutated strains. As in S. typhimurium, 4-aminoarabinose substitution of lipid A contributes to in vitro resistance of Y. pseudotuberculosis to the antimicrobial peptide polymyxin B. Whereas pmrF expression in S. typhimurium is mediated by both the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems, it appears to be PmrA-PmrB-independent in Y. pseudotuberculosis, with the response regulator PhoP interacting directly with the pmrF operon promoter region. This result reveals that the ubiquitous PmrA-PmrB regulatory system controls different regulons in distinct bacterial species. In addition, pmrF inactivation in Y. pseudotuberculosis has no effect on bacterial virulence in the mouse, again in contrast to the situation in S. typhimurium. The marked differences in pmrF operon regulation in these two phylogenetically close bacterial species may be related to their dissimilar lifestyles.