Donald A. Cooksey

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.

Chromosomal Locus for Cadmium Resistance in Pseudomonas putida Consisting of a Cadmium-Transporting ATPase and a MerR Family Response Regulator

ABSTRACT Pseudomonads from environmental sources vary widely in their sensitivity to cadmium, but the basis for this resistance is largely uncharactarized. 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, regulateszntA in E. coli, but it is not contiguous withzntA in the E. coli genome as cadAand 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 represscadA. Homologs of the cadmium-transporting ATPase were detected in many other Pseudomonas species.

Global Effect of Indole-3-Acetic Acid Biosynthesis on Multiple Virulence Factors of Erwinia chrysanthemi 3937

ABSTRACT Production of the plant hormone indole-3-acetic acid (IAA) is widespread among plant-associated microorganisms. The non-gall-forming phytopathogen Erwinia chrysanthemi 3937 (strain Ech3937) possesses iaaM (ASAP16562) and iaaH (ASAP16563) gene homologues. In this work, the null knockout iaaM mutant strain Ech138 was constructed. The IAA production by Ech138 was reduced in M9 minimal medium supplemented with l-tryptophan. Compared with wild-type Ech3937, Ech138 exhibited reduced ability to produce local maceration, but its multiplication in Saintpaulia ionantha was unaffected. The pectate lyase production of Ech138 was diminished. Compared with wild-type Ech3937, the expression levels of an oligogalacturonate lyase gene, ogl, and three endopectate lyase genes, pelD, pelI, and pelL, were reduced in Ech138 as determined by a green fluorescent protein-based fluorescence-activated cell sorting promoter activity assay. In addition, the transcription of type III secretion system (T3SS) genes, dspE (a putative T3SS effector) and hrpN (T3SS harpin), was found to be diminished in the iaaM mutant Ech138. Compared with Ech3937, reduced expression of hrpL (a T3SS alternative sigma factor) and gacA but increased expression of rsmA in Ech138 was also observed, suggesting that the regulation of T3SS and pectate lyase genes by IAA biosynthesis might be partially due to the posttranscriptional regulation of the Gac-Rsm regulatory pathway.

Genome-wide identification of plant-upregulated genes of Erwinia chrysanthemi 3937 using a GFP-based IVET leaf array

A green fluorescent protein-based in vivo expression technology leaf array was used to identify genes in Erwinia chrysanthemi 3937 that were specifically upregulated in plants compared with growth in a laboratory culture medium. Of 10,000 E. chrysanthemi 3937 clones, 61 were confirmed as plant upregulated. On the basis of sequence similarity, these were recognized with probable functions in metabolism (20%), information transfer (15%), regulation (11%), transport (11%), cell processes (11%), and transposases (2%); the function for the remainder (30%) is unknown. Upregulated genes included transcriptional regulators, iron uptake systems, chemotaxis components, transporters, stress response genes, and several already known or new putative virulence factors. Ten independent mutants were constructed by insertions in these plant-upregulated genes and flanking genes. Two different virulence assays, local leaf maceration and systemic invasion in African violet, were used to evaluate these mutants. Among these, mutants of a purM homolog from Escherichia coli (purM::Tn5), and hrpB, hrcJ, and a hrpD homologs from the Erwinia carotovorum hrpA operon (hrpB::Tn5, hrcJ::Tn5, and hrpD::Tn5) exhibited reduced abilities to produce local and systemic maceration of the plant host. Mutants of rhiT from E. chrysanthemi (rhiT::Tn5), and an eutR homolog from Salmonella typhimurium (eutR::TnS) showed decreased ability to cause systemic inva sion on African violet. However, compared with the wild-type E. chrysanthemi 3937, these mutants exhibited no significant differences in local leaf maceration. The pheno type of hrpB::Tn5, hrcC::Tn5, and hrpD::Tn5 mutants further confirmed our previous findings that hrp genes are crucial virulence determinants in E. chrysanthemi 3937.

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 by 5′ to the translational start site of copA, and the transcriptional initiation site of PcopH 88 by 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. syringae or E. coli. The results presented delineate important regions on two copper-inducible promoters from P. syringae.

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

ABSTRACT 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′ andlacZ. 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 byP. putida 06909.

Phylogenetic Relationships of Xylella fastidiosa Strains Isolated from Landscape Ornamentals in Southern California

ABSTRACT Xylella fastidiosa is an insect-borne, xylem-limited pathogenic bacterium that has been associated with a rise in incidence of diseased landscape ornamentals in southern California. The objective of this study was to genetically characterize strains isolated from ornamental hosts to understand their distribution and identity. Strains of X. fastidiosa isolated from ornamentals were characterized using a multiprimer polymerase chain reaction (PCR) system, random amplified polymorphic DNA (RAPD)-PCR, and sequence analysis of the 16S-23S rDNA intergenic spacer region (ISR). Based on RAPD-PCR and 16S-23S rDNA ISR, strains isolated from daylily, jacaranda, and magnolia clustered with members of X. fastidiosa subsp. sandyi and caused oleander leaf scorch but not Pierces disease symptoms in glasshouse assays on oleander and grape, respectively. This demonstrated both that our groupings based on genetic characterization were valid and that strains of X. fastidiosa subsp. sandyi are present in hosts other than oleander. Strains isolated from Spanish broom, cherry, and one strain isolated from western redbud clustered with X. fastidiosa subsp. fastidiosa members. Strains isolated from purple-leafed plum, olive, peach, plum, sweetgum, maidenhair tree, crape myrtle, and another western redbud strain clustered with members of X. fastidiosa subsp. multiplex. All strains isolated from mulberry and one from heavenly bamboo formed a separate cluster that has not yet been defined as a subspecies.

Regulation of resistance to copper in Xanthomonas axonopodis pv. vesicatoria

ABSTRACT Copper-resistant strains of Xanthomonas axonopodis pv. vesicatoria were previously shown to carry plasmid-borne copper resistance genes related to the cop and pco operons of Pseudomonas syringae and Escherichia coli, respectively. However, instead of the two-component (copRS and pcoRS) systems determining copper-inducible expression of the operons in P. syringae and E. coli, a novel open reading frame, copL, was found to be required for copper-inducible expression of the downstream multicopper oxidase copA in X. axonopodis. copL encodes a predicted protein product of 122 amino acids that is rich in histidine and cysteine residues, suggesting a possible direct interaction with copper. Deletions or frameshift mutations within copL, as well as an amino acid substitution generated at the putative start codon of copL, caused a loss of copper-inducible transcriptional activation of copA. A nonpolar insertion of a kanamycin resistance gene in copL resulted in copper sensitivity in the wild-type strain. However, repeated attempts to complement copL mutations in trans failed. Analysis of the genomic sequence databases shows that there are copL homologs upstream of copAB genes in X. axonopodis pv. citri, X. campestris pv. campestris, and Xylella fastidiosa. The cloned promoter area upstream of copA in X. axonopodis pv. vesicatoria did not function in Pseudomonas syringae or in E. coli, nor did the P. syringae cop promoter function in Xanthomonas. However, a transcriptional fusion of the Xanthomonas cop promoter with the Pseudomonas copABCDRS was able to confer resistance to copper in Xanthomonas, showing divergence in the mechanisms of regulation of the resistance to copper in phytopathogenic bacteria.

Characterization of Regulatory Pathways in Xylella fastidiosa: Genes and Phenotypes Controlled by algU

ABSTRACT Many virulence genes in plant bacterial pathogens are coordinately regulated by “global” regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierces disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.

The exopolysaccharide of Xylella fastidiosa is essential for biofilm formation, plant virulence, and vector transmission.

Exopolysaccharides (EPS) synthesized by plant-pathogenic bacteria are generally essential for virulence. The role of EPS produced by the vector-transmitted bacterium Xylella fastidiosa was investigated by knocking out two genes implicated in the EPS biosynthesis, gumD and gumH. Mutant strains were affected in growth characteristics in vitro, including adhesion to surfaces and biofilm formation. In addition, different assays were used to demonstrate that the mutant strains produced significantly less EPS compared with the wild type. Furthermore, gas chromatography-mass spectrometry showed that both mutant strains did not produce oligosaccharides. Biologically, the mutants were deficient in movement within plants, resulting in an avirulent phenotype. Additionally, mutant strains were affected in transmission by insects: they were very poorly transmitted by and retained within vectors. The gene expression profile indicated upregulation of genes implicated in cell-to-cell signaling and adhesins while downregulation in genes was required for within-plant movement in EPS-deficient strains. These results suggest an essential role for EPS in X. fastidiosa interactions with both plants and insects.