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Characterization of pXV10A, a Copper Resistance Plasmid in Xanthomonas campestris pv. vesicatoria.

The efficacy of copper bactericides for control of Xanthomonas campestris pv. vesicatoria in eastern Oklahoma tomato fields was evaluated. Copper bactericides did not provide adequate control, and copper-resistant (Cu) strains of the pathogen were isolated. The Cu genes in these strains were located on a large indigenous plasmid designated pXV10A. The host range of pXV10A was investigated; this plasmid was efficiently transferred into 8 of 11 X. campestris pathovars. However, the transfer of pXV10A to other phytopathogenic genera was not detected. DNA hybridization experiments were performed to characterize the Cu genes on pXV10A. A probe containing subcloned Cu genes from X. campestris pv. vesicatoria E3C5 hybridized to pXV10A; however, a subclone containing Cu genes from P. syringae pv. tomato PT23 failed to hybridize to pXV10A. Further DNA hybridization experiments were performed to compare pXV10A with pXvCu plasmids, a heterogenous group of Cu plasmids present in strains of X. campestris pv. vesicatoria from Florida. These studies indicated that the Cu genes on pXV10A and pXvCu plasmids share nucleotide sequence homology and may have a common origin. Further experiments showed that these plasmids are distinctly different because pXV10A did not contain sequences homologous to IS476, an insertion sequence present on pXvCu plasmids.

Journal Article↗

Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae.

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cur Smr), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cus Smr strains contained a 68-kb conjugative plasmid. Cur Sms strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Smr genes from the broad-host-range plasmid RSF1010. The Smr determinant was subsequently cloned from a 68-kb Cur Smr plasmid designated pPSR1. A restriction map detailing the organization of the homologous Smr genes from pPSR1 and RSF1010 and cloned Smr genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cur genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cur plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.

Copper↗

Similarity between Copper Resistance Genes from Xanthomonas campestris and Pseudomonas syringae.

Plasmid-borne copper resistance genes from copper-resistant strains of Xanthomonas campestris pv. vesicatoria from California, Florida, and Oklahoma shared structural similarities. A strain of X. campestris pv. campestris also contained plasmid-borne copper resistance genes similar to the resistance genes from X. campestris pv. vesicatoria. Furthermore, a region of the copper resistance genes from X. campestris pv. vesicatoria 07882 hybridized with copA, the first gene of the copper resistance operon (cop) of Pseudomonas syringae pv. tomato. A copper-inducible protein of similar size to CopA was detected by Western blot (immunoblot) analysis from the wild-type strain 07882 and from the cloned copper resistance genes of 07882 introduced into a copper-sensitive strain of X. campestris pv. vesicatoria. A low level of hybridization was observed with chromosomal DNA from other xanthomonads when the copper resistance genes from strain 07882 were used as probes.

Journal Article↗

Genetic and plasmid diversity within natural populations of Pseudomonas syringae with various exposures to copper and streptomycin bactericides.

We examined the genetic and plasmid diversity within natural populations of Pseudomonas syringae isolated from three ornamental pear nurseries in eastern Oklahoma. The bactericide spray regimen differed at each nursery; copper and streptomycin, only copper, and no bactericides were applied at nurseries I, II, and III respectively. Resistance to copper (Cur) and resistance to streptomycin (Smr) were determined for 1,938 isolates of P. syringae; isolates from nurseries I and II were generally Cur Sms; whereas most isolates from nursery III were Cus Sms. The plasmid profiles of 362 isolates were determined, and six, one, seven, and four plasmid profiles were obtained for Cur, Smr, Cur Smr, and Cus Sms isolates, respectively. All Smr plasmids contained sequences homologous to the strA and strB Smr genes from broad-host-range plasmid RSF1010 and were associated with Smr transposon Tn5393. Plasmids were placed into two groups on the basis of hybridization to the oriV and par sequences from pOSU900, a cryptic plasmid in P. syringae pv. syringae. A total of 100 randomly chosen P. syringae isolates from nurseries I and III were analyzed for genetic diversity by using the arbitrarily primed PCR (AP-PCR) technique. An analysis of chromosomal genotypes by AP-PCR revealed a high degree of genetic diversity among the isolates, and the results of this analysis indicated that the isolates could be clustered into two distinct groups. The plasmid profiles were specific to isolates belonging to particular AP-PCR groups. Within each AP-PCR group, identical plasmid profiles were produced by isolates that had different chromosomal genotypes, implying that plasmid transfer has played an important role in the dissemination of Cur and Smr within the populations studied.

Base Sequence↗

Effects of Suspended Particulates on the Frequency of Transduction among Pseudomonas aeruginosa in a Freshwater Environment.

Transduction has been shown to play a significant role in the transfer of plasmid and chromosomal DNA in aquatic ecosystems. Such ecosystems contain a multitude of environmental factors, any one of which may influence the transduction process. It was the purpose of this study to show how one of these factors, particulate matter, affects the frequency of transduction. In situ transduction rates were measured in lake water microcosms containing either high or low concentrations of particulate matter. The microcosms were incubated in a freshwater lake in central Oklahoma. Transduction frequencies were found to be enhanced as much as 100-fold in the presence of particulates. Our results suggest that aggregations of bacteriophages and bacterial cells are stimulated by the presence of these suspended particulates. This aggregation increases the probability of progeny phages and transducing particles finding and infecting new host cells. Consequently, both phage production and transduction frequencies increase in the presence of particulate matter.

Journal Article↗

Biodegradation of benzene by halophilic and halotolerant bacteria under aerobic conditions.

A highly enriched halophilic culture was established with benzene as the sole carbon source by using a brine soil obtained from an oil production facility in Oklahoma. The enrichment completely degraded benzene, toluene, ethylbenzene, and xylenes within 1 to 2 weeks. Also, [14C]benzene was converted to 14CO2, suggesting the culture's ability to mineralize benzene. Community structure analysis revealed that Marinobacter spp. were the dominant members of the enrichment.

Aerobiosis↗

Infection exclusion of the rickettsial pathogen anaplasma marginale in the tick vector Dermacentor variabilis.

Anaplasma marginale is a tick-borne, rickettsial cattle pathogen that is endemic in several areas of the United States. Recent studies (J. de la Fuente, J. C. Garcia-Garcia, E. F. Blouin, J. T. Saliki, and K. M. Kocan, Clin. Diagn. Lab. Immunol. 9:658-668, 2002) demonstrated that infection of cultured tick cells and bovine erythrocytes with one genotype of A. marginale excluded infection with other genotypes, a phenomenon referred to as infection exclusion. The present study was undertaken to confirm the phenomenon of infection exclusion of A. marginale genotypes in a tick vector, Dermacentor variabilis. Only one genotype of A. marginale (Virginia isolate) was detected by PCR in ticks that fed first on a calf infected with a Virginia isolate and second on a calf infected with an Oklahoma isolate. These studies demonstrate that infection exclusion of A. marginale genotypes also occurs in naturally infected ticks, as well as in cattle and cultured tick cells, and results in establishment of only one genotype per tick.

Anaplasmataceae↗

SFS, a novel fibronectin-binding protein from Streptococcus equi, inhibits the binding between fibronectin and collagen.

The obligate parasitic bacterium Streptococcus equi subsp. equi is the causative agent of strangles, a serious disease of the upper respiratory tract in horses. In this study we have, using shotgun phage display, cloned from S. equi subsp. equi and characterized a gene, called sfs, encoding a protein termed SFS, representing a new type of fibronectin (Fn)-binding protein. The sfs gene was found to be present in all 50 isolates of S. equi subsp. equi tested and in 41 of 48 S. equi subsp. zooepidemicus isolates tested. The sfs gene is down-regulated during growth in vitro compared to fnz, a previously characterized gene encoding an Fn-binding protein from S. equi subsp. zooepidemicus. Sequence comparisons revealed no similarities to previously characterized Fn-binding proteins, but high scores were obtained against collagen. Besides similarity due to the high content of glycine, serine, and proline residues present in both proteins, there was a nine-residue motif present both in collagen and in the Fn-binding domain of SFS. By searching the Oklahoma S. pyogenes database, we found that this motif is also present in a potential cell surface protein from S. pyogenes. Protein SFS was found to inhibit the binding between Fn and collagen in a concentration-dependent way.

Adhesins, Bacterial↗

Identification and molecular analysis of rough-colony-specific outer membrane proteins of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a gram-negative bacterium isolated from the human mouth, has been implicated in the pathogenesis of early-onset periodontitis. Primary isolates cultured from subgingival plaque exhibit an adherent, rough colony phenotype which spontaneously converts to a nonadherent, smooth phenotype upon in vitro subculture. The rough colony variant produces abundant fimbriae and autoaggregates, while the smooth colony variant is planktonic and produces scant fimbriae. To begin to understand the significance of colony variation in biofilm formation by A. actinomycetemcomitans, outer membrane protein profiles of four isogenic rough and smooth colony variants were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two proteins with relative molecular masses of 43 and 20 kDa were expressed by the rough colony variants exclusively. Expression of these proteins was not found to be dependent on growth phase, oxygen tension, or type of complex medium. N-terminal amino acid sequences of these proteins obtained by Edman degradation were compared with sequences from the University of Oklahoma A. actinomycetemcomitans genome database. Two contiguous open reading frames (ORFs) encoding proteins having sequence homology with these proteins were identified. The 43-kDa protein (RcpA [rough colony protein A]) was similar to precursor protein D of the general secretion pathway of gram-negative bacilli, while the 20-kDa protein (RcpB [rough colony protein B]) appeared to be unique. The genes encoding these proteins have been cloned from A. actinomycetemcomitans 283 and sequenced. A BLASTX (gapped BLAST) search of the surrounding ORFs revealed homology with other fimbria-related proteins. These data suggest that the genes encoding the 43-kDa (rcpA) and 20-kDa (rcpB) proteins may be functionally related to each other and to genes that may encode fimbria-associated proteins.

Aggregatibacter actinomycetemcomitans↗

Antigenic variation of Anaplasma marginale: major surface protein 2 diversity during cyclic transmission between ticks and cattle.

The rickettsial pathogen Anaplasma marginale expresses a variable immunodominant outer membrane protein, major surface protein 2 (MSP2), involved in antigenic variation and long-term persistence of the organism in carrier animals. MSP2 contains a central hypervariable region of about 100 amino acids that encodes immunogenic B-cell epitopes that induce variant-specific antibodies during infection. Previously, we have shown that MSP2 is encoded on a polycistronic mRNA transcript in erythrocyte stages of A. marginale and defined the structure of the genomic expression site for this transcript. In this study, we show that the same expression site is utilized in stages of A. marginale infecting tick salivary glands. We also analyzed the variability of this genomic expression site in Oklahoma strain A. marginale transmitted from in vitro cultures to cattle and between cattle and ticks. The structure of the expression site and flanking regions was conserved except for sequence that encoded the MSP2 hypervariable region. At least three different MSP2 variants were encoded in each A. marginale population. The major sequence variants did not change on passage of A. marginale between culture, acute erythrocyte stage infections, and tick salivary glands but did change during persistent infections of cattle. The variant types found in tick salivary glands most closely resembled those present in bovine blood at the time of acquisition of infection, whether infection was acquired from an acute or from a persistent rickettsemia. These variations in structure of an expression site for a major, immunoprotective outer membrane protein have important implications for vaccine development and for obtaining an improved understanding of the mechanisms of persistence of ehrlichial infections in humans, domestic animals, and reservoir hosts.

Anaplasma↗

Competitive relationship between protocatechuic acid and p-aminosalicylic acid for a cellular transport mechanism.

Hubbard, Jerry S. (Oklahoma State University, Stillwater), and Norman N. Durham. Competitive relationship between protocatechuic acid and p-aminosalicylic acid for a cellular transport mechanism. J. Bacteriol. 82:361-369. 1961.-The oxidation of protocatechuic acid by a Flavobacterium is inhibited by p-aminosalicyclic acid regardless of whether the organism is grown on protocatechuic acid or sequentially induced to protocatechuic acid by growth on p-aminobenzoic acid. Depletion of the substrate from the medium by the cell suspension is dependent, within defined limits, on the inhibitor to substrate ratio, and the inhibition can be overcome by addition of excess substrate. However, this competitive effect is not observed in high inhibitor to substrate ratios. p-Aminosalicylic acid did not affect the rate or extent of oxidation, carbon dioxide evolution, or formation of beta-ketoadipic acid during degradation of protocatechuic acid by cell extracts. The results suggest that p-aminosalicylic acid antagonizes the oxidation of protocatechuic acid by the cell suspension by competing with the substrate for a specific transport mechanism, thereby regulating the entry and internal accumulation of the substrate. The lack of a competitive effect in high inhibitor to substrate ratios could be interpreted as an indication that the mechanism for accumulating the substrate may consist of more than one active transport system.

4-Aminobenzoic Acid↗

Cell division in a species of Erwinia III. Reversal of inhibition of cell division caused by D-amino acids, penicillin, and ultraviolet light.

Grula, E. A. (Oklahoma State University, Stillwater) and Mary M. Grula. Cell division in a species of Erwinia. III. Reversal of inhibition of cell division caused by d-amino acids, penicillin, and ultraviolet light. J. Bacteriol. 83:981-988. 1962.-Inhibition of cell division in an Erwinia sp. occurs in the presence of any of six d-amino acids, penicillin, or ultraviolet light. Cell-division inhibition caused by d-amino acids is pH-dependent; however, elongation caused by penicillin occurs over a wide range of pH. Bulging and spheroplast formation in the presence of penicillin occurs only at pH values below 7.6; however, division continues to be inhibited at higher pH levels. Reversal of cell-division inhibition caused by two d-amino acids (phenylalanine and histidine) can be partially overcome by their respective l-isomers. Divalent cations (Zn, Ca, Mn) cause varying amounts of reversal of division inhibition in all systems studied; each system appears to have an individual requirement. All induced division inhibitions, including that caused by penicillin, can be reversed by pantoyl lactone or omega-methylpantoyl lactone. Evidence is presented and discussed concerning the possible importance of pantoyl lactone and divalent cations in terminal steps of the cell-division process in this organism.

Amino Acids↗

Cell division in a species of Erwinia IV. Metabolic blocks in panothenate biosynthesis and their relationship to inhibition of cell division.

Grula, Mary M. (Oklahoma State University, Stillwater) and E. A. Grula. Cell division in a species of Erwinia. IV. Metabolic blocks in pantothenate biosynthesis and their relationship to inhibition of cell division. J. Bacteriol. 83:989-997. 1962.-Four compounds that inhibit cell division in an Erwinia sp., d-serine, d-histidine, d-phenylalanine, and penicillin, decrease the intracellular pantothenate content of Erwinia at culture ages of 10 and 16 hr. In the case of penicillin, it appears to be the result of excessive leakage from long cells; however, with the three d-amino acids, there is a genuine inhibition of synthesis. Among agents tested that reverse the inhibition of division, only pantoyl lactone, and to a lesser extent, omega-methylpantoyl lactone, restore intracellular content of pantothenate. This restoration is considerably less effective with d-serine as a division-inhibiting agent than with the others. Other lactones, l-alpha-alanine, and ammonium chloride are ineffective, or only slightly effective, in restoring pantothenate synthesis. Effects of division-inhibiting compounds and reversing agents upon cellular coenzyme A activity in general parallel their effects on pantothenate synthesis. There is no direct correlation between ability of a compound to reverse cell-division inhibition and ability to restore synthesis of either pantothenic acid or coenzyme A. Evidence is presented that d-serine interferes with the utilization of aspartic acid and also blocks synthesis of pantoic acid. Compounds (including glucose) which tend to produce long cells result in the accumulation of pyruvic acid in the growth medium. Pantoic acid reduces, and pantoyl lactone abolishes completely, this accumulation of pyruvate. Other reversing agents do not abolish the pyruvate accumulation.

Amino Acids↗

Cell division in a species of Erwinia. V. Effect of metabolic inhibitors on terminal division and composition of a "division" medium.

Grula, E. A. (Oklahoma State University, Stillwater) and Mary M. Grula. Cell division in a species of Eriwinia. V. Effect of metabolic inhibitors on terminal division and composition of a "division" medium. J. Bacteriol. 84:492-499. 1962.-Terminal division in Erwinia spp. involves a triggering action and subsequent septum synthesis. It is a metabolic process requiring organic nitrogen and carbon and energy. The process, when triggered by pantoic acid or pantoyl lactone, is inhibited strongly by 2,4-dinitrophenol, hydroxylamine, mitomycin C, and Hg ion and to a lesser degree by cyanide, azide, 5-fluorouracil, and diisopropylfluorophosphate. Ethylenediaminetetraacetic acid completely inhibits division only when calcium ion is the triggering agent. Heating of the cells at 43 C for 10 to 20 min also completely inhibits division. Hydroxylamine, iodoacetate, and mitomycin C cause extensive lysis of growing cells. No evidence has been obtained to demonstrate the need for protein or normal ribonucleic acid synthesis in terminal division. Requirement for intact deoxyribonucleic acid (DNA) or DNA synthesis is questionable; -SH groups are not directly involved. Once triggering has occurred, septum formation appears to require synthesis of at least cell-wall mucopeptide. A "division" medium is reported and discussed.

2,4-Dinitrophenol↗

Tryptophan-niacin relationship in Xanthomonas pruni.

Wilson, R. G. (Oklahoma State University, Stillwater) and L. M. Henderson. Tryptophan-niacin relationship in Xanthomonas pruni. J. Bacteriol. 85:221-229. 1963.-The observation that Xanthomonas pruni, a bacterial pathogen for the peach, requires niacin for growth and can use tryptophan or 3-hydroxyanthranilic acid as a substitute was confirmed. To determine whether niacin is synthesized via the tryptophan-3-hydroxyanthranilic acid pathway, experiments using labeled metabolites were undertaken. Labeled tryptophan, 3-hydroxyanthranilic acid, quinolinic acid, and nicotinic acid were supplied in the basal medium in amounts sufficient to insure maximal growth. Nicotinic and quinolinic acids were isolated from the cells after the growth period. The isotope was incorporated from the first three labeled compounds into niacin with dilutions approximately the same in all cases, ranging from 7.6 to 17.1. The dilution of isotopic niacin was 3.1- to 5.9-fold. Only labeled quinolinic acid gave rise to labeled quinolinic acid in the cell, but this acid gave rise to niacin with 10- to 12-fold reduction in specific activity. The results indicate that if quinolinate participates as an obligatory intermediate in the synthesis of niacin from tryptophan, its concentration within the cell is very small and it does not equilibrate readily with exogenous quinolinate. The results confirm the conclusion, drawn from growth studies, that niacin is needed to permit tryptophan synthesis at a sufficient rate to promote growth. In the absence of an external source of niacin, tryptophan or some of its metabolites can promote growth by acting as precursors of niacin.

Niacin↗

INHIBITION OF MICROBIAL GROWTH AND SEPARATION BY D-SERINE, VANCOMYCIN, AND MITOMYCIN C.

Durham, Norman N. (Oklahoma State University, Stillwater). Inhibition of microbial growth and separation by d-serine, vancomycin, and mitomycin C. J. Bacteriol. 86:380-386. 1963.-A study was made of the growth and separation inhibition of Pseudomonas fluorescens by vancomycin and mitomycin C and of a Flavobacterium sp. sensitive to mitomycin C and d-serine. Aberrant morphological forms (primarily "boat-shaped" cells and "chain-like" filaments) were observed during growth of the sensitive organism in the presence of the inhibitors. Reversal studies indicated that Mg(++), and to a lesser extent Mn(++), partially reversed vancomycin and mitomycin C growth inhibition during the early stages of growth but did not influence d-serine inhibition. Selected metabolites also showed a tendency to partially reverse mitomycin C and vancomycin inhibition of growth during the initial growth phase, and the differences in reversal trends suggested that d-serine, vancomycin, and mitomycin C produce metabolic lesions at different sites in the cell with respect to growth. Cultivation of the sensitive organism in low concentrations of the inhibitors resulted in a structurally fragile population, indicating formation of a defective cell structure. Mg(++) partially reversed the vancomycin inhibition of separation in these organisms. None of the other metals or metabolites, either singly or in varied combinations, was able to reverse inhibition of separation by the various inhibitors.

Flavobacterium↗

Synthesis of Protocatechuate Oxygenase by Pseudomonas fluorescens in the Presence of Exogenous Carbon Sources.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Synthesis of protocatechuate oxygenase by Pseudomonas fluorescens in the presence of exogenous carbon sources. J. Bacteriol. 90:15-22. 1965.-The addition of glucose, ribose, or fructose (0.45 or 45.0 mumoles/ml) simultaneously with protocatechuic acid shortens the lag period required for synthesis of protocatechuate oxygenase by a washed-cell suspension of Pseudomonas fluorescens. Glucose is readily oxidized and supports growth of P. fluorescens, whereas neither ribose nor fructose readily supports growth. High glucose concentrations (45.0 mumoles/ml) shorten the lag period but lower the total enzyme synthesis. The pH drops during glucose oxidation, and this is accompanied by a decrease in the rate of enzyme synthesis. High glucose concentrations, with adequate buffering, permitted "normal" enzyme synthesis. A decrease in the total enzyme synthesis was not observed in the presence of high concentrations of ribose or fructose. Succinate, pyruvate, acetate, or formate (0.45 mumole/ml) were readily oxidized, but did not shorten the lag period required for synthesis of the enzyme. The data suggest that glucose, ribose, or fructose may serve as a "specific" carbon source (such as ribose-5-phosphate or a similar precursor important in ribonucleic acid synthesis) functional in the synthesis of protocatechuate oxygenase.

Journal Article↗

Correlation of Carbohydrate Catabolism and Synthesis of Macromolecules During Enzyme Synthesis in Pseudomonas fluorescens.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Correlation of carbohydrate catabolism and synthesis of macromolecules during enzyme synthesis in Pseudomonas fluorescens. J. Bacteriol. 90: 23-28. 1965.-Glucose, ribose, and fructose shorten the lag period required for synthesis of protocatechuate oxygenase. Radioactivity from uracil-2-C(14) is incorporated into the hot trichloroacetic acid-soluble fraction after a lag period of approximately 20 min after addition of protocatechuic acid. Addition of glucose or ribose simultaneously with the inducer shortens the lag period to approximately 5 min and increases the rate of uracil incorporation. The inducer must be present to initiate incorporation of radioactivity, and the exogenous carbon source accelerates incorporation but is not sufficient to initiate synthesis by itself. The addition of protocatechuic acid increases the rate and total incorporation of radioactivity from uniformly labeled glucose or ribose-1-C(14) into the hot trichloroacetic acid-soluble fraction. Ribose decreases the incorporation of radioactivity from uniformly labeled glucose into the hot trichloroacetic acid-soluble fraction, and glucose shows a similar effect on incorporation of radioactivity from ribose-1-C(14), indicating the two sugars are serving in the same capacity to enhance enzyme synthesis. Radioactivity from glucose-1-C(14) is not incorporated into the hot trichloroacetic acid-soluble fraction. The results suggest that glucose and ribose shorten the lag period for inducible enzyme formation by serving as a "specific" carbon source for synthesis of macromolecules such as ribonucleic acid.

Journal Article↗