Background: Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the causative agent of furunculosis, a bacterial septicaemia of salmonid fish. While other species of Aeromonas are opportunistic pathogens or are found in commensal or symbiotic relationships with animal hosts, A. salmonicida subsp. salmonicida causes disease in healthy fish. The genome sequence of A. salmonicida was determined to provide a better understanding of the virulence factors used by this pathogen to infect fish.

Although the structural gene for diphtheria toxin, tox, is carried by a family of closely related corynebacteriophages, the regulation of lox expression is controfle, to a large extent, by its bacterial host Corynebacleium diphtheriae. Optimal yields oftox gene products are obtained only when iron becomes the growth-rate-limiting substrate. Previous studies suggest that regulation oftox expression Is mediated through an iron-binding aporepressor. To facilitate molecular cloning of the tox regulatory element from genomic libraries of C. diphtheriae, we constructed a tox promoter/operator (toxPO)-acZ transcriptional fusion in Escherichia coli strain DH5a. We report the molecular cloning and nucleic acid sequence of a diphtheria tox iron-dependent regulatory element, dxR, and demonstrate that expression of f-galactosidase from the toxPO-4acZ fusion is regulated by dlxR-encoded protein in an iron-sensitive manner. In addition, we show that expression of the toxPO-acZ fusion is not affected by the E. coUl ironregulatory protein Fur and that the dlxR protein does not inhibit expression offur-regulated outer-membrane proteins.Diphtheria toxin is synthesized by Corynebacterium diphtheriae lysogenic for one of a family of corynebacteriophages that carries the structural gene for the toxin, tox (1, 2). Optimal yields of tox gene products have long been known to be obtained only from C. diphtheriae grown under conditions where iron becomes the growth-rate-limiting substrate (3, 4). In 1936, Pappenheimer and Johnson (5) showed that adding iron in low concentration to the growth medium inhibited the production of diphtheria toxin. Both biochemical and genetic evidence support the hypothesis that the corynebacteriophage tox gene is regulated by a corynebacterial-determined iron-binding repressor as postulated by . This model predicted an aporepressor that in the presence of iron forms a complex; this complex then binds to the tox operator and blocks transcription. Under conditions of iron limitation, the iron-repressor complex dissociates, derepressing the tox gene.The nucleic acid base sequence of tox revealed a 9-basepair (bp) inverted repeat that overlaps the " -10" region of the promoter (13). Because many operators exhibit dyad symmetry and are positioned near their respective promoters, this region was designated the putative tox operator.We here describe the genetic construction of an Escherichia coli host strain that carries a chromosomal diphtheria tox promoter/operator (toxPO)-lacZ transcriptional fusion in single copy. Because this strain constitutively expresses j-galactosidase and is phenotypically blue on 5-bromo-4-chloro-3-indolyl /3-D-galactoside (X-Gal)-containing agar medium, we have used it to screen genomic libraries of nontoxinogenic nonlysogenic C. diphtheriae for determinants that repress lacZ expression. We report the molecular cloning and deduced amino acid sequence (25,316 molecular weight) of a diphtheria tox iron-dependent regulatory element, dtxR.* We show that this factor acts as a negati...

Type IV pili play an important role in bacterial adhesion, motility, and biofilm formation. Here we present high-resolution atomic force microscopy (AFM) images of type IV pili from Pseudomonas aeruginosa bacteria. An individual pilus ranges in length from 0.5 to 7 m and has a diameter from 4 to 6 nm, although often, pili bundles in which the individual filaments differed in both length and diameter were seen. By attaching bacteria to AFM tips, it was possible to fasten the bacteria to mica surfaces by pili tethers. Force spectra of tethered pili gave rupture forces of 95 pN. The slopes of force curves close to the rupture force were nearly linear but showed little variation with pilus length. Furthermore, force curves could not be fitted with wormlike-chain polymer stretch models when using realistic persistence lengths for pili. The observation that the slopes near rupture did not depend on the pili length suggests that they do not represent elastic properties of the pili. It is possible that this region of the force curves is determined by an elastic element that is part of the bacterial wall, although further experiments are needed to confirm this.

We report the characterization and determination of 2,6-dichloro-1,4-benzoquinone and three new disinfection byproducts (DBPs): 2,6-dichloro-3-methyl-1,4-benzoquinone, 2,3,6-trichloro-1,4-benzoquinone, and 2,6-dibromo-1,4-benzoquinone. These haloquinones are suspected bladder carcinogens and are likely produced during drinking water disinfection treatment. However, detection of these haloquinones is challenging, and consequently, they have not been characterized as DBPs until recently. We have developed an electrospray ionization tandem mass spectrometry technique based on our observation of unique ionization processes. These chloro- and bromo-quinones were ionized through a reduction step to form [M + H](-) under negative electrospray ionization. Tandem mass spectra and accurate mass measurements of these compounds showed major product ions, [M + H - HX](-), [M + H - HX - CO](-), [M + H - CO](-), and/or X(-) (where X represents Cl or Br). The addition of 0.25% formic acid to water samples was found to effectively stabilize the haloquinones in water and to improve the ionization for analysis. These improvements were rationalized from the estimates of pK(a) values (5.8-6.3) of these haloquinones. The method of tandem mass spectrometry detection, combined with sample preservation, solid phase extraction, and liquid chromatography separation, enabled the detection of haloquinones in chlorinated water samples collected from a drinking water treatment plant. The four haloquinones were detected only in drinking water after chlorination treatment, with concentrations ranging from 0.5 to 165 ng/L, but were not detectable in the untreated water. This method will be useful for future studies of occurrence, formation pathways, toxicity, and control of these new halogenated DBPs.

N-Nitrosodimethylamine (NDMA), a member of a group of probable human carcinogens, has been detected as a disinfection byproduct (DBP) in drinking water supplies in Canada and the United States. To comprehensively investigate the occurrence of possible nitrosamines in drinking water supplies, a liquid chromatography-tandem mass spectrometry technique was developed to detect both thermally stable and unstable nitrosamines. This technique consisted of solid-phase extraction (SPE), liquid chromatography (LC) separation, and tandem quadrupole linear ion trap mass spectrometry (MS/MS) detection. It enabled the determination of sub-ng/L levels of nine nitrosamines. Isotope-labeled N-nitrosodimethylamine-d6 (NDMA-d6) was used as the surrogate standard for determining recovery, and N-nitrosodi-n-propylamine-dl4 (NDPA-dl4) was used as the internal standard for quantification. The method detection limits were estimated to be 0.1-10.6 ng/L, and the average recoveries were 41-111% for the nine nitrosamines; of these, NDMA, N-nitrosopyrrolidine (NPyr), N-nitrosopiperidine (NPip), and N-nitrosodiphenylamine (NDPhA) were identified and quantified in drinking water samples collected from four locations within the same distribution system. In general, the concentrations of these four nitrosamines in this distribution system increased with increasing distance from the water treatment plant, indicating that the amount of formation was greater than the amount of decomposition within this time frame. The identification of NPip and NDPhA in drinking water systems and the distribution profiles of these nitrosamines have not been reported previously. These nitrosamines are toxic, and their presence as DBPs in drinking water may have toxicological relevance.

The structural gene for diphtheria toxin, tox, is carried by a family of closely related corynebacteriophages; however, the regulation of tox expression is controlled by a Corynebacterium diphtheriae-encoded regulatory element, dtxR. The molecular cloning and sequence analysis of dtxR was recently described. Previous studies have suggested that DtxRmediated regulation of the diphtheria tox operator involves the formation of an iron-repressor complex, which specifically binds to the tox operator. We have expressed and purified DtxR from recombinant Escherichia coli. Immunoblot analysis shows DtxR to be a single Mr 28,000 protein band in both recombinant E. coli and the C7(-) and C7hm723(-) strains of C. diphtheriae. In addition, we demonstrate that the binding of DtxR to a diphtheria tox promoter/operator probe requires the addition of Mn2+ to the reaction mixture; however, binding may be blocked by addition of the chelator 2,2'-dipyridyl, anti-DtxR antiserum, and excess unlabeled probe to the reaction mixture. Deletion of one of the 9-base-pair inverted repeat sequences from the tox operator results in a loss of DtxR binding. The results presented here demonstrate that regulation of diphtheria toxin expression by DtxR requires direct interaction between this regulatory factor and the tox operator in the presence of a divalent heavy metal ion.While the structural gene for diphtheria toxin, tox, is carried by a family of closely related corynebacteriophages, the regulation of tox expression in Corynebacterium diphtheriae is mediated by a bacterial host determinant (1, 2). Boyd et al. (3) have recently reported molecular cloning and DNA sequence analysis of the dtxR gene from genomic libraries of the nonlysogenic nontoxigenic C7(-) strain of C. diphtheriae. DtxR has a deduced molecular weight of 25,316 and has been shown to regulate the expression of .8-galactosidase from a tox promoter/operator (toxPO)-lacZ transcriptional fusion in recombinant Escherichia coli. The DtxR-mediated regulation of tox expression in C. diphtheriae, as well as in recombinant E. coli, has been shown to be dependent on the concentration of iron in the growth medium (3, 4). Schmitt and Holmes (5) and Boyd et al. (6) have recently demonstrated by molecular cloning of an iron-insensitive mutant dtxR allele from the C7hm723(-) strain of C. diphtheriae that DtxR from C7hm723 fails to regulate the expression of f3-galactosidase from toxPO-lacZ transcriptional fusions.The recent studies of Boyd and colleagues (3, 6) and Schmitt and Holmes (5, 7) have provided additional support for the hypothesis that the regulation of tox expression is regulated by a corynebacterial-determined iron-binding repressor as initially postulated by Murphy and Bacha (8). This model predicted DtxR to be an aporepressor, which in the presence of iron would form a complex that binds to the tox operator and blocks transcription. The putative tox operator is a 9-base-pair (bp) inverted repeat sequence that is separated by 9 bp (9-12). The putative operator overlap...

Formation of nine N-nitrosamines has been investigated when seven different source waters representing various qualities were each treated with eleven bench-scale disinfection processes, without addition of nitrosamine precursors. These disinfection treatments included chlorine (OCl-), chloramine (NH2Cl), chlorine dioxide (ClO2), ozone (O3), ultraviolet (UV), advanced oxidation processes (AOP), and combinations. The total organic carbon (TOC) of the seven source waters ranged from 2 to 24 mg x L(-1). The disinfected water samples and the untreated source waters were analyzed for nine nitrosamines using a solid phase extraction and liquid chromatography-tandem mass spectrometry method. Prior to any treatment, N-nitrosodimethylamine (NDMA) was detected ranging from 0 to 53 ng x L(-1) in six of the seven source waters, and its concentrations increased in the disinfected water samples (0-118 ng x L(-1)). N-nitrosodiethylamine (NDEA), N-nitrosomorpholine (NMor), and N-nitrosodiphenylamine (NDPhA) were also identified in some of the disinfected water samples. NDPhA (0.2-0.6 ng x L(-1)) was formed after disinfection with OCl-, NH2Cl, O3, and MPUV/OCl-. NMEA was produced with OCl- and MPUV/OCl-, and NMor formation was associated with O3. In addition, UVtreatment alone degraded NDMA; however, UV/ OCl- and AOP/OCl- treatments produced higher amounts of NDMA compared to UV and AOP alone, respectively. These results suggest that UV degradation or AOP oxidation treatment may provide a source of NDMA precursors. This study demonstrates that environmental concentrations and mixtures of unknown nitrosamine precursors in source waters can form NDMA and other nitrosamines.

The recently described type III secretion system (TTSS) of Aeromonas salmonicida subsp. salmonicida has been linked to virulence in salmonids. In this study, three TTSS effector genes, aexT, aopH or aopO, were inactivated by deletion, as was ascC, the gene encoding the outer-membrane pore of the secretion apparatus. Effects on virulence were assayed by live challenge of Atlantic salmon (Salmo salar). The DascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain.