Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from Pseudomonas fluorescens and Pseudomonas aureofaciens. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from P. aeruginosa is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes, phzM and phzS, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of phzS alone in Escherichia coli or in enzymes, pyocyanin-nonproducing P. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine. P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene, phzH, which has a homologue in Pseudomonas chlororaphis, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in P. aeruginosa consisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide.Phenazine compounds produced by fluorescent Pseudomonas species are biologically active metabolites that function in microbial competitiveness (37), the suppression of soilborne plant pathogens (1,11,55,56), and virulence in human and animal hosts (35).The most widely studied phenazine-producing fluorescent pseudomonad is P. aeruginosa, a gram-negative opportunistic pathogen of animals, insects, nematodes, and plants (30,33,35,46). In humans, P. aeruginosa infects immunocompromised, burned, or injured patients and can cause both acute and chronic lung disease. Strains of P. aeruginosa produce a variety of redox-active phenazine compounds, including pyocyanin, phenazine-1-carboxylic acid (PCA), 1-hydroxyphenazine (1-OH-PHZ), and phenazine-1-carboxamide (PCN) (7,52,57).From 90 to 95% of P. aeruginosa isolates produce pyocyanin (52), and the presence of high concentrations of pyocyanin in the sputum of cystic fibrosis patients has suggested that this compound plays a role in pulmonary tissue damage observed with chronic lung infections (64). This idea is supported by several recent studies which demonstrated that pyocyanin contributes in a variety of ways to the pathophysiological effects observed in airways infected by P. aeruginosa. Pyocyanin interferes with the regulation of ion transport, ciliary beat frequency, and mucus secretion in airway epithelial cells by altering the cytosolic concentration of calcium (15). It may interact with endothelium-derived relaxing factor or with nitric oxide (which plays a central role in the control ...
White mold caused by the necrotrophic fungus Sclerotinia sclerotiorum (Lib.) de Bary limits common bean (Phaseolus vulgaris L.) production in temperate climates. Disease resistance has been identified, but breeding is hampered by a paucity of resistance sources and complex inheritance, as numerous quantitative trait loci (QTL) conditioning partial resistance have been found. Our objectives were to characterize the partial white mold resistance found in breeding lines I9365‐31 and VA19 and to construct a comparative linkage map for all the white mold resistance QTL identified to date. Recombinant inbred line (RIL) populations ‘Benton’/VA19 (BV) and ‘Raven’/I9365‐31 (R31) consisting of 79 F6 and 105 F5 RILs, respectively, were evaluated for white mold (WM) reaction in multiple greenhouse and field tests. Two QTL were found in BV, WM2.2 expressed in the greenhouse (R2 = 33%) and the field (13%) and WM8.3 expressed in field (11%) only, and seven were found in R31 (WM2.2, WM4.2, WM5.3, WM5.4, WM6.1, WM7.3, WM8.4), three expressed in greenhouse tests and four in the field, ranging in phenotypic variance from 5 to 52%. These QTL were compared with 26 previously identified QTL, resulting in a comparative linkage map of 35 QTL, which coalesced into 21 distinct regions across nine linkage groups. Four QTL found in R31 were novel. Sequence characterized amplified region markers associated with WM2.2, WM8.3, and WM7.3 QTL were generated. The comparative linkage map provides a framework for integrating and interpreting future QTL studies concerning white mold resistance in common bean.
Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease, which causes significant problems to aquaculture worldwide. A recent study (Soule M, Cain K, LaFrentz S, Call DR [2005] Infect Immun 73:3799-3802) identified two 16S rRNA gene sequence variants (6 base differences) within the variable stem-loop region 3 for F. psychrophilum strains ATCC 49418 and CSF 259-93. That study also hypothesized that F. psychrophilum is composed of at least 2 distinct genetic lineages (I and II) described by a microarray-based comparative genomics study. In the present study, we augmented an existing 16S rDNA microarray to detect both 16S rRNA sequence variants from F. psychrophilum. Subsequent microarray experiments showed that CSF 259-93 hybridized as expected, but ATCC 49418 was positive for both sequence variants. We then developed a PCR-restriction fragment length polymorphism (RFLP) assay (MnlI and MaeIII) to distinguish between the 2 sequences. Gel isolation of PCR-RFLP products, cloning, and sequencing confirmed that ATCC 49418 harbors both 16S rRNA sequences. Microarray experiments showed that 11 of 14 strains from genetic Lineage I harbor both the CSF 259-93 and ATCC 49418 16S rRNA sequence variants, whereas all 15 Lineage II strains were only positive for the CSF 259-93 sequence (p < 0.0001). Elastin hydrolysis and tetracycline resistance were most closely associated with the latter strains (p < 0.0001 and p = 0.024, respectively). These data support the hypothesis that F. psychrophilum is composed of at least 2 distinct genetic lineages that are closely associated with host origin. KEY WORDS: 16S rRNA · Flavobacterium psychrophilum · Bacterial coldwater disease · Elastin hydrolysis · Microarray hybridization · Tetracycline resistance Resale or republication not permitted without written consent of the publisherDis Aquat Org 65: [209][210][211][212][213][214][215][216] 2005 In general, Flavobacterium psychrophilum strains are biochemically homogeneous, with the exception of some variation in the presence or absence of caseinases, gelatinases, and elastases (Bertolini et al. 1994, Lorenzen et al. 1997, Madetoja et al. 2001. Not all strains of F. psychrophilum are capable of causing the same degree of disease (Holt 1987, Madetoja et al. 2002, LaFrentz et al. 2003 and they can be antigenically diverse (Evensen & Lorenzen 1996, Crump et al. 2001, Madetoja et al. 2002. Immunization with killed cells of one strain does not necessarily convey protective immunity from infection by other strains (Holt et al. 1989, Obach & Laurencin 1991.Genetic variation has been assessed between strains using ribotyping, plasmid profiles, randomly amplified polymorphic DNA (RAPD), and PCR-RFLP of specific gene fragments (Chakroun et al. 1997, Chakroun et al. 1998, Madetoja et al. 2001, Izumi et al. 2003. Studies of outbreak strains from Danish trout farms suggest dominance by a single ribotype , Madetoja et al. 2002, but other studies show greater genetic variation across geographic scales (Chakroun et a...
Reciprocal subtractive libraries were prepared for two strains of Flavobacterium psychrophilum, one virulent and the other avirulent in a trout challenge model. Unique clones were sequenced and their distribution assessed among 34 strains. The analysis showed that F. psychrophilum is composed of two genetic lineages, possibly reflecting host specificity.Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease and rainbow trout fry syndrome, both of which affect salmonid fish and impact commercial aquaculture and resource enhancement hatcheries worldwide (12,19,21). Coho salmon (Oncorhynchus kisutch [Walbaum]) and rainbow trout (O. mykiss) are particularly susceptible, although F. psychrophilum also infects other fish species (21). F. psychrophilum strains vary greatly in the ability to establish disease (virulence). For example, one well-studied strain (ATCC 49418 [4,6]) is unable to cause significant mortality (9) while strain CSF 259-93 causes high mortality (16) in a trout challenge model. There are no commercial vaccines available for bacterial coldwater disease, although several research groups have active programs in this area (11,14,20). From these studies we know that not all strains elicit an antibody response that is effective against other strains and consequently there may be considerable genetic variation between strains.The goals of this project were (i) to examine genetic differences between two strains of F. psychrophilum and ATCC 49418 [16]) and (ii) to assess the extent and distribution of genetic variation between other strains relative to geographical source and host species. Strains for the latter assessment were chosen based on availability at the time of this study. All strains were stored at Ϫ80°C and were cultured at 16 to 17°C in tryptone yeast extract salts medium (TYES; 0.4% tryptone, 0.04% yeast extract, 0.05% calcium chloride, 0.05% magnesium sulfate, pH 7.2). Genomic DNA (gDNA) was extracted using the DNeasy tissue kit (QIAGEN, Valencia, CA).We prepared reciprocal suppression subtractive hybridization libraries for the two strains by using the PCR-Select Bacterial Genomic Subtraction Kit (Clontech, Palo Alto, CA) according to the manufacturer's protocol, except that tester and driver gDNAs were restriction enzyme digested with RsaI (supplied with the kit) and DraI (New England Biolabs, Inc,, Beverly, MA) and the hybridization step was at 59°C. Resulting DNA fragments were cloned in pCR2.1 (Invitrogen, Carlsbad, CA), and 576 randomly chosen recombinant clones were used to make a microarray as previously described (8, 10). gDNA (0.5 g) was nick translated for 2 h in the presence of biotindATP (BioNick Labeling System; Invitrogen) and hybridized to the microarray (data not shown). Microarray slides were processed, imaged, and analyzed as described previously (23). Using stringent selection criteria, where the median probe intensity was Ն95% of the maximum pixel intensity in one strain and Յ5% of the maximum pixel intensity in the other strain, we identified...
Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixedgenome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50 Enterococcus isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four Enterococcus species (Enterococcus casseliflavus, E. faecalis, E. hirae, or E. mundtii). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of libraryindependent markers.
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