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D A Cooksey

Publications and source records attributed to D A Cooksey.

At least 19 recordsLinked to original sources
PubMed2006

In vitro activities of antibiotics and antimicrobial peptides against the plant pathogenic bacterium Xylella fastidiosa.

AIMS: The objective of this study was to evaluate the effectiveness of antibiotics and antimicrobial peptides against 10 strains of Xylella fastidiosa. METHODS AND RESULTS: The minimal inhibitory concentration (MIC) of 12 antibiotics and 18 antimicrobial peptides were determined by agar dilution tests and growth inhibition assays. Antibiotics with the lowest MIC for X. fastidiosa strains were gentamicin, tetracycline, ampicillin, kanamycin, and novobiocin, chloramphenicol, and rifampin. Plate growth inhibition assays showed that four of the antimicrobial peptides (Magainin 2, Indolicidin, PGQ, and Dermaseptin) were toxic to all X. fastidiosa strains. CONCLUSION: All X. fastidiosa strains were sensitive to several groups of antibiotics, and minor differences in sensitivity to several antimicrobial peptides were observed among strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that antibiotics and antimicrobial peptides have some activity against the pathogen, X. fastidiosa and may have application in protecting plants from developing Pierce's disease.

Anti-Bacterial Agents↗
PubMed2006

Detection and differentiation of Xylella fastidiosa strains acquired and retained by glassy-winged sharpshooters (Hemiptera: Cicadellidae) using a mixture of strain-specific primer sets.

Xylella fastidiosa Wells is a bacterial pathogen that causes a variety of plant diseases, including Pierce's disease (PD) of grapevine, almond leaf scorch, alfalfa dwarf, citrus variegated chlorosis, and oleander leaf scorch (OLS). Numerous strains of this pathogen have been genetically characterized, and several different strains occur in the United States. The dominant vector in southern California is the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). The high mobility of this insect, and its use of large numbers of host plant species, provides this vector with ample exposure to multiple strains of X. fastidiosa during its lifetime. To learn more about the ability of this vector to acquire, retain, and transmit multiple strains of the pathogen, we developed a polymerase chain reaction (PCR)-based method to detect and differentiate strains of X. fastidiosa present in individual glassy-winged sharpshooter adults. Insects were sequentially exposed to plants infected with a PD strain in grapevine and an OLS strain in oleander. After sequential exposure, a few insects tested positive for both strains (7%); however, in most cases individuals tested positive for only one strain (29% PD, 41% OLS). In transmission studies, individual adults transmitted either the PD or OLS strain of the pathogen at a rate (39%) similar to that previously reported after exposure to a single strain, but no single individual transmitted both strains of the pathogen. PD and OLS strains of X. fastidiosa remained detectable in glassy-winged sharpshooter, even when insects were fed on a plant species that was not a host of the strain for 1 wk.

Animals↗
PubMed2001

Chromosomal locus for cadmium resistance in Pseudomonas putida consisting of a cadmium-transporting ATPase and a MerR family response regulator.

Pseudomonads from environmental sources vary widely in their sensitivity to cadmium, but the basis for this resistance is largely uncharacterized. A chromosomal fragment encoding cadmium resistance was cloned from Pseudomonas putida 06909, a rhizosphere bacterium, and sequence analysis revealed two divergently transcribed genes, cadA and cadR. CadA was similar to cadmium-transporting ATPases known mostly from gram-positive bacteria, and to ZntA, a lead-, zinc-, and cadmium-transporting ATPase from Escherichia coli. CadR was related to the MerR family of response regulators that normally control mercury detoxification in other bacterial systems. A related gene, zntR, regulates zntA in E. coli, but it is not contiguous with zntA in the E. coli genome as cadA and cadR were in P. putida. In addition, unlike ZntA and other CadA homologs, but similar to the predicted product of gene PA3690 in the P. aeruginosa genome, the P. putida CadA sequence had a histidine-rich N-terminal extension. CadR and the product of PA3689 of P. aeruginosa also had histidine-rich C-terminal extensions not found in other MerR family response regulators. Mutational analysis indicated that cadA and cadR are fully responsible for cadmium resistance and partially for zinc resistance. However, unlike zntA, they did not confer significant levels of lead resistance. The cadA promoter was responsive to Cd(II), Pb(II), and Zn(II), while the cadR promoter was only induced by Cd(II). CadR apparently represses its own expression at the transcriptional level. However, CadR apparently does not repress cadA. Homologs of the cadmium-transporting ATPase were detected in many other Pseudomonas species.

Adenosine Triphosphatases↗
PubMed2000

Genes expressed in Pseudomonas putida during colonization of a plant-pathogenic fungus.

In vivo expression technology (IVET) was employed to study colonization of Phytophthora parasitica by a biological control bacterium, Pseudomonas putida 06909, based on a new selection marker. The pyrB gene, which encodes aspartate transcarbamoylase, an enzyme used for pyrimidine biosynthesis, was cloned from P. putida 06909. A pyrB-disrupted mutant did not grow in pyrimidine-deficient media unless it was complemented with pyrBC' behind an active promoter. Thirty clones obtained from P. putida 06909 that were expressed on fungal hyphae but not on culture media were isolated by IVET based on the promoterless transcriptional fusion between pyrBC' and lacZ. Nineteen of these clones were induced during late-stage bacterial growth in vitro, while 11 of the clones were expressed only when they were inoculated onto fungal hyphae. Restriction analysis of these 11 clones revealed that there were five unique clones. Sequence analyses of three of the five unique clones showed that the 3' ends of the clones fused to pyrB were similar to genes encoding diacylglycerol kinase (DAGK), bacterial ABC transporters, and outer membrane porins. The sequences of the two other clones were not similar to the sequences of any of the genes in the database used. A LuxR family response regulator was found upstream of DAGK, and a LysR family response regulator was found upstream of the ABC transporter. The location of the inducible promoter of two clones suggested that DAGK and the ABC transporter are induced and may play a role in colonization of the fungus P. parasitica by P. putida 06909.

Aspartate Carbamoyltransferase↗
PubMed1996

A chromosomal locus required for copper resistance, competitive fitness, and cytochrome c biogenesis in Pseudomonas fluorescens.

A chromosomal locus required for copper resistance and competitive fitness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil. Sequence analysis of this locus revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cycL, and cycH, with the closest similarity being to the aeg-46.5(yej) region of the Escherichia coli chromosome. The proposed functions of these genes in other bacteria include the binding, transport, and coupling of heme to apocytochrome c in the periplasm of these Gram-negative bacteria. Putative heme-binding motifs were present in the predicted products of cycK and cycL, and TipB contained a putative disulfide oxidoreductase active site proposed to maintain the heme-binding site of the apocytochrome in a reduced state for ligation of heme. Tn3-gus mutagenesis showed that expression of the genes was constitutive but enhanced by copper, and confirmed that the genes function both in copper resistance and production of active cytochrome c. However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, were required for resistance to copper.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of CopR, a transcriptional activator protein that binds to a conserved domain (cop box) in copper-inducible promoters of Pseudomonas syringae.

The copper resistance (cop) operon promoter (Pcop) of Pseudomonas syringae is copper-inducible, and requires the regulatory genes copRS. Sequence analysis revealed that CopR has significant homology with other known activator proteins from bacterial two-component regulatory systems. In the present study we characterized Pcop and its interaction with CopR. We found that crude protein extracts from copper-resistant and -sensitive strains of P. syringae contain a Pcop-specific DNA-binding protein. We hypothesized that this DNA-binding protein was the product of copR. A 27-kDa protein, which corresponded to the predicted copR product, was expressed from this gene in Escherichia coli. CopR was purified, and the first eight amino acids were sequenced to confirm its relationship to copR. Specific binding of purified CopR to the plasmid-borne Pcop and the chromosomally encoded cop homolog promoter (PcopH), identified in this report, was demonstrated using specific and non-specific promoter competitors in DNA mobility shift assays. DNAse I footprinting identified a conserved CopR binding region (cop box) on Pcop and PcopH. The cop box contains an inverted repeat within a stretch of 16 bp, which shares approximately 75% identity with the PhoB binding region from several phosphate regulon gene promoters in E. coli. Primer extension analysis identified the transcriptional initiation site of Pcop 59 bp 5' to the translational start site of copA, and the transcriptional initiation site of PcopH 88 bp 5' to the translational start site of the chromosomal homolog of copA. The cop box was localized to between positions -54 and -35 relative to the transcriptional initiation site of Pcop and PcopH. Deletion analysis of Pcop delimited copper-inducible activity to a 104-bp region. Pcop and PcopH do not share a sequence consensus with other characterized promoters from P. syrinagae or E. coli. The results presented delineate important regions on two copper-inducible promoters form P. syringae.

Bacterial Outer Membrane Proteins↗
PubMed1994

Characterization of pPT23B, the plasmid involved in syringolide production by Pseudomonas syringae pv. tomato PT23.

Avirulence gene D (avrD) in strain PT23 of Pseudomonas syringae pv. tomato (Pst) specifies the production of syringolides, which are elicitors of plant defense reactions. An 83-kb indigenous plasmid (pPT23B) that carries avrD has been mapped and characterized and a putative par region was identified. pPT23B contains a large amount of DNA that is repeated in other native plasmids in PT23. A putative mobile insertion element that occurs on plasmid pPT23A as well as on the chromosome was also identified in strain PT23. New broad-host-range expression vectors that functioned in Pst were constructed for overexpression of the cloned avrD gene and high-level production of the syringolides. Introduction of an avrD overexpression plasmid into PT23 or plasmid-cured strains led to identical syringolide peaks on HPLC with no new peaks observed. These results suggested that neither pPT23B nor other indigenous plasmids in Pst carry additional genes required for syringolide production or metabolism. Pst strains lacking pPT23B were not impaired in virulence on tomato plants.

Bacterial Toxins↗
PubMed1994

Molecular mechanisms of copper resistance and accumulation in bacteria.

An unusual mechanism of metal resistance is found in certain plant pathogenic strains of Pseudomonas syringae that are exposed to high levels of copper compounds used in disease control on agricultural crops. These bacteria accumulate blue Cu2+ ions in the periplasm and outer membrane. At least part of this copper sequestering activity is determined by copper-binding protein products of the copper resistance operon (cop). Potential copper-binding sites of the periplasmic CopA protein show conservation with type-1, type-2, and type-3 copper sites of several eukaryotic multi-copper oxidases. In addition to compartmentalization of copper in the periplasm, two components of the cop operon, copC and copD, appear to function in copper uptake into the cytoplasm. Copper resistance operons related to cop have been described in the related plant pathogen Xanthomonas campestris and in Escherichia coli, but these resistance systems may differ functionally from the Pseudomonas syringae system.

Amino Acid Sequence↗
PubMed1994

Mutations Affecting Hyphal Colonization and Pyoverdine Production in Pseudomonads Antagonistic toward Phytophthora parasitica.

In previous studies, Pseudomonas putida 06909 and Pseudomonas fluorescens 09906 suppressed populations of Phytophthora parasitica in the citrus rhizosphere, suggesting that these bacteria may be useful in biological control of citrus root rot. In this study we investigated the mechanisms of antagonism between the bacteria and the fungus. Both bacteria colonized Phytophthora hyphae and inhibited the fungus on agar media. A hyphal column assay was developed to measure the colonization of bacteria on fungal hyphae and to enrich for colonization-deficient mutants. In this way we identified Tn5 mutants of each pseudomonad that were not able to colonize the hyphae and inhibit fungal growth in vitro. Colonization-deficient mutants were nonmotile and lacked flagella. Survival of nonmotile mutants in a citrus soil was similar to survival of a random Tn5 mutant over a 52-day period. Additional screening of random Tn5 mutants of both pseudomonads for loss of fungal inhibition in vitro yielded two distinct types of mutants. Mutants of the first type were deficient in production of pyoverdines and in inhibition of the fungus in vitro, although they still colonized fungal hyphae. Mutants of the second type lacked flagella and were not able to colonize the hyphae or inhibit fungal growth. No role was found for antibiotic production by the two bacteria in the inhibition of the fungus. Our results suggest that both hyphal colonization and pyoverdine production are important in the inhibition of Phytophthora parasitica by P. fluorescens and P. putida in vitro.

Journal Article↗
PubMed1993

Copper uptake and resistance in bacteria.

Copper ions are essential for bacteria but can cause a number of toxic cellular effects if levels of free ions are not controlled. Investigations of copper-resistant bacteria have revealed several mechanisms, mostly plasmid-determined, that prevent cellular uptake of high levels of free copper ions. However, these studies have also revealed that bacteria apparently have efficient chromosomally encoded systems for uptake and management of trace levels of copper. This review will explore the relationship of copper uptake systems to resistance mechanisms and the possibility that copper resistance has evolved directly through modification of chromosomal copper uptake genes.

Amino Acid Sequence↗
PubMed1993

Role of copper resistance in competitive survival of Pseudomonas fluorescens in soil.

A copper-resistant strain (09906) of Pseudomonas fluorescens that was isolated from a citrus grove soil is being investigated as a biological control agent for Phytophthora root rot. Since citrus grove soils in California are often contaminated with copper from many years of copper fungicide applications, the role of copper resistance in survival of strain 09906 was investigated. Three copper-sensitive Tn5 mutants were obtained with insertions in different chromosomal DNA regions. These insertions were not in the chromosomal region that hybridized with the copper resistance operon (cop) cloned from Pseudomonas syringae. A copper-sensitive mutant survived as well as the wild type in a sterile loamy sand without added copper, but with 10 and 15 micrograms of CuSO4 added per g of soil, populations of the copper-sensitive mutant were 27- and 562-fold lower, respectively, than that of the wild type after a 25-day period. In a sterilized citrus grove soil, populations of the copper-sensitive mutant and wild-type strain were similar, but in nonsterile citrus soil, populations of the copper-sensitive mutant were 112-fold lower than the wild type after 35 days. These data suggest that copper resistance genes can be important factors in persistence of P. fluorescens in soil contaminated with copper. In addition, these genes appear to play a role in competitive fitness, even in soils with a low copper content.

Agriculture↗
PubMed1993

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↗
PubMed1993

Copper Hypersensitivity and Uptake in Pseudomonas syringae Containing Cloned Components of the Copper Resistance Operon.

Copper resistance in Pseudomonas syringae carrying the copABCD operon is associated with accumulation of copper in the periplasm and outer membrane, apparently as a function of the copper-binding activities of the copABC gene products. However, no specific function for copD has been determined. In this study, P. syringae cells containing copCD or copBCD cloned behind the lac promoter were hypersensitive to copper. An increased accumulation of copper was measured in cells containing several combinations of cop genes that included copC and copD. Our data suggest that CopC, a periplasmic copper-binding protein, and CopD, a probable inner membrane protein, may function together in copper uptake.

Journal Article↗
PubMed1993

Characterization of chromosomal homologs of the plasmid-borne copper resistance operon of Pseudomonas syringae.

Copper-resistant and copper-sensitive strains of Pseudomonas syringae, as well as many other pseudomonads, contain chromosomal DNA homologous to the plasmid-borne copper resistance operon (copABCD). cop homologs were cloned from the chromosome of P. syringae pv. tomato PT12.2, which had an elevated level of resistance to copper compared with typical copper-sensitive strains of other P. syringae pathovars and showed an unusually high frequency of spontaneous mutation to high levels of copper resistance. Two chromosomal cop homolog regions were cloned. Homolog 1 hybridized with copA and copB, and homolog 2 hybridized with copA, copB, copC, and the copper-responsive regulatory genes copRS. Homolog 1 had no detectable function when transferred to a copper-sensitive strain of P. syringae. However, homolog 2 conferred the low level of copper resistance observed with PT12.2 and produced proteins related to CopA and CopC. In addition, homolog 2 conferred a high frequency of mutation to full copper resistance. In a spontaneously mutated derivative of the cloned homolog 2 (pCOPH2R) that conferred copper resistance, an increased level of CopA was observed. pCOPH2R also supported a higher level of transcriptional activity of the cop promoter that was fused to lacZ and provided in trans (pCOP38), suggesting that the spontaneous mutation was regulatory, probably involving the copRS homologs. Homolog 2 was similar but not identical to the plasmid-borne cop operon, and it did not complement site-specific mutations in cop genes.

Bacterial Outer Membrane Proteins↗
PubMed1993

A two-component regulatory system required for copper-inducible expression of the copper resistance operon of Pseudomonas syringae.

Specific induction of the copper resistance operon (cop) promoter from Pseudomonas syringae was measured by beta-galactosidase production from a cop promoter-lacZ fusion. Induction of the cop promoter in P. syringae pv. syringae required trans-acting factors from copper resistance plasmid pPT23D, from which cop was originally cloned. Tn5 mutagenesis of pPT23D was used to localize two complementation groups immediately downstream from copABCD. Cloning and sequencing of the DNA in this region revealed two genes, copR and copS, expressed in the same orientation as the cop operon but from a separate constitutive promoter. The amino acid sequence deduced from these genes showed distinct similarities to known two-component regulatory systems, including PhoB-PhoR and OmpR-EnvZ. In addition, CopR showed strong similarity to copper resistance activator protein PcoR from Escherichia coli. Functional chromosomal homologs to copRS activated the cop promoter, in a copper-inducible manner, in copper-resistant or -sensitive strains of P. syringae pv. tomato and other Pseudomonas species. This implies that copper-inducible gene regulation is associated with a common chromosomally encoded function, as well as plasmid-borne copper resistance, in Pseudomonas spp.

Amino Acid Sequence↗
PubMed1992

Accumulation of copper and other metals by copper-resistant plant-pathogenic and saprophytic pseudomonads.

Copper-resistant strains of Pseudomonas syringae carrying the cop operon produce periplasmic copper-binding proteins, and this sequestration outside the cytoplasm has been proposed as a resistance mechanism. In this study, strain PS61 of P. syringae carrying the cloned cop operon accumulated more total cellular copper than without the operon. Several other copper-resistant pseudomonads with homology to cop were isolated from plants, and these bacteria also accumulated copper. Two highly resistant species accumulated up to 115 to 120 mg of copper per g (dry weight) of cells. P. putida 08891 was more resistant to several metals than P. syringae pv. tomato PT23, but this increased resistance was not correlated with an increased accumulation of metals other than copper. Several metals were accumulated by both PT23 and P. putida, but when copper was added to induce the cop operon, there was generally no increase of accumulation of the other metals, suggesting that the cop operon does not contribute to accumulation of these other metals. The exceptions were aluminum for PT23 and iron for P. putida, which accumulated to higher levels when copper was added to the cultures. The results of this study support the role of copper sequestration in the copper resistance mechanism of P. syringae and suggest that this mechanism is common to several copper-resistant Pseudomonas species found on plants to which antimicrobial copper compounds are applied for plant disease control.

Journal Article↗
PubMed1991

Copper resistance in Pseudomonas syringae mediated by periplasmic and outer membrane proteins.

Copper-resistant strains of Pseudomonas syringae pathovar tomato accumulate copper and develop blue colonies on copper-containing media. Three of the protein products of the copper-resistance operon (cop) were characterized to provide an understanding of the copper-resistance mechanism and its relationship to copper accumulation. The Cop proteins, CopA (72 kDa), CopB (39 kDa), and CopC (12 kDa), were produced only under copper induction. CopA and CopC were periplasmic proteins and CopB was an outer membrane protein. Leader peptide sequences of CopA, CopB, and CopC were confirmed by amino-terminal peptide sequencing. CopA, CopB, and CopC were purified from strain PT23.2, and their copper contents were determined. One molecule of CopA bound 10.9 +/- 1.2 atoms of copper and one molecule of CopC bound 0.6 +/- 0.1 atom of copper. The Cop proteins apparently mediate sequestration of copper outside of the cytoplasm as a copper-resistance mechanism.

Amino Acid Sequence↗