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1.  Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon 
In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSFts1. The mutations that resulted in a temperature-sensitive phenotype in mSFts1 were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42°C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 hours of growth. In order to facilitate its utilization, the mSFts1 was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSFts1 for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.
doi:10.1016/j.mimet.2009.07.002
PMCID: PMC2782952  PMID: 19615413
stabilizing fragment; conditional replicon; plasmid cure; PCR mutagenesis; DNA sequencing; genetic cassette
2.  Elucidating the Process of Activation of Methyl-Coenzyme M Reductase 
Journal of Bacteriology  2014;196(13):2491-2498.
Methyl-coenzyme M reductase (MCR) catalyzes the reversible reduction of methyl-coenzyme M (CH3-S-CoM) and coenzyme B (HS-CoB) to methane and heterodisulfide CoM-S-S-CoB (HDS). MCR contains the hydroporphinoid nickel complex coenzyme F430 in its active site, and the Ni center has to be in its Ni(I) valence state for the enzyme to be active. Until now, no in vitro method that fully converted the inactive MCRsilent-Ni(II) form to the active MCRred1-Ni(I) form has been described. With the potential use of recombinant MCR in the production of biofuels and the need to better understand this enzyme and its activation process, we studied its activation under nonturnover conditions and achieved full MCR activation in the presence of dithiothreitol and protein components A2, an ATP carrier, and A3a. It was found that the presence of HDS promotes the inactivation of MCRred1, which makes it essential that the activation process is isolated from the methane formation assay, which tends to result in minimal activation rates. Component A3a is a multienzyme complex that includes the mcrC gene product, an Fe-protein homolog, an iron-sulfur flavoprotein, and protein components involved in electron bifurcation. A hypothetical model for the cellular activation process of MCR is presented.
doi:10.1128/JB.01658-14
PMCID: PMC4054172  PMID: 24769699
3.  Isolation and Characterization of a Suppressor Mutation that Restores Myxococcus xanthus Exopolysaccharide Production 
Microbiology (Reading, England)  2009;155(Pt 11):3599-3610.
SUMMARY
Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell-surface associated exopolysaccharide (EPS) is essential for S motility and the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base-pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a difA+ background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wild-type levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production.
doi:10.1099/mic.0.031070-0
PMCID: PMC2879065  PMID: 19684067
4.  Isolation and characterization of a suppressor mutation that restores Myxococcus xanthus exopolysaccharide production 
Microbiology  2009;155(Pt 11):3599-3610.
Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell surface-associated exopolysaccharide (EPS) is essential for S motility and that the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a difA+ background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wild-type levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production.
doi:10.1099/mic.0.031070-0
PMCID: PMC2879065  PMID: 19684067
5.  Pseudomonas aeruginosa Forms Biofilms in Acute Infection Independent of Cell-to-Cell Signaling▿ † 
Infection and Immunity  2007;75(8):3715-3721.
Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.
doi:10.1128/IAI.00586-07
PMCID: PMC1952004  PMID: 17562773
6.  Adaptations of Pseudomonas aeruginosa to the Cystic Fibrosis Lung Environment Can Include Deregulation of zwf, Encoding Glucose-6-Phosphate Dehydrogenase 
Journal of Bacteriology  2005;187(22):7561-7568.
Cystic fibrosis (CF) patients are highly susceptible to chronic pulmonary disease caused by mucoid Pseudomonas aeruginosa strains that overproduce the exopolysaccharide alginate. We showed here that a mutation in zwf, encoding glucose-6-phosphate dehydrogenase (G6PDH), leads to a ∼90% reduction in alginate production in the mucoid, CF isolate, P. aeruginosa FRD1. The main regulator of alginate, sigma-22 encoded by algT (algU), plays a small but demonstrable role in the induction of zwf expression in P. aeruginosa. However, G6PDH activity and zwf expression were higher in FRD1 strains than in PAO1 strains. In PAO1, zwf expression and G6PDH activity are known to be subject to catabolite repression by succinate. In contrast, FRD1 zwf expression and G6PDH activity were shown to be refractory to such catabolite repression. This was apparently not due to a defect in the catabolite repression control (Crc) protein. Such relaxed control of zwf was found to be common among several examined CF isolates but was not seen in other strains of clinical and environmental origin. Two sets of clonal isolates from individual CF patient indicated that the resident P. aeruginosa strain underwent an adaptive change that deregulated zwf expression. We hypothesized that high-level, unregulated G6PDH activity provided a survival advantage to P. aeruginosa within the lung environment. Interestingly, zwf expression in P. aeruginosa was shown to be required for its resistance to human sputum. This study illustrates that adaptation to the CF pulmonary environment by P. aeruginosa can include altered regulation of basic metabolic activities, including carbon catabolism.
doi:10.1128/JB.187.22.7561-7568.2005
PMCID: PMC1280303  PMID: 16267280
8.  The eutT Gene of Salmonella enterica Encodes an Oxygen-Labile, Metal-Containing ATP:Corrinoid Adenosyltransferase Enzyme 
Journal of Bacteriology  2004;186(17):5708-5714.
The eutT gene of Salmonella enterica was cloned and overexpressed, and the function of its product was established in vivo and in vitro. The EutT protein has an oxygen-labile, metal-containing ATP:co(I)rrinoid adenosyltransferase activity associated with it. Functional redundancy between EutT and the housekeeping ATP:co(I)rrinoid adenosyltransferase CobA enzyme was demonstrated through phenotypic analyses of mutant strains. Lack of CobA and EutT blocked ethanolamine utilization. EutT was necessary and sufficient for growth of an S. enterica cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source. A eutT+ gene provided in trans corrected the adenosylcobalamin-dependent transcription of a eut-lacZ operon fusion in a cobA strain. Cell extracts enriched for EutT protein contained strong, readily detectable ATP:co(I)rrinoid adenosyltransferase activity. The activity was only detected in extracts maintained under anoxic conditions, with complete loss of activity upon exposure to air or treatment with the Fe2+ ion chelator bathophenanthroline. While the involvement of another metal ion cannot be ruled out, the observed sensitivity to air and bathophenanthroline suggests involvement of Fe2+. We propose that the EutT protein is a unique metal-containing ATP:co(I)rrinoid adenosyltransferase. It is unclear whether the metal ion plays a structural or catalytic role.
doi:10.1128/JB.186.17.5708-5714.2004
PMCID: PMC516830  PMID: 15317775
9.  The Pseudomonas fluorescens AlgG Protein, but Not Its Mannuronan C-5-Epimerase Activity, Is Needed for Alginate Polymer Formation 
Journal of Bacteriology  2003;185(12):3515-3523.
Bacterial alginates are produced as 1-4-linked β-d-mannuronan, followed by epimerization of some of the mannuronic acid residues to α-l-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-l-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.
doi:10.1128/JB.185.12.3515-3523.2003
PMCID: PMC156231  PMID: 12775688
10.  Effect of rpoS Mutation on the Stress Response and Expression of Virulence Factors in Pseudomonas aeruginosa 
Journal of Bacteriology  1999;181(13):3890-3897.
The sigma factor RpoS (ςS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS in Pseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa, including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1 rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50°C, increased osmolarity, and prolonged exposure to high concentrations of H2O2. In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa. The rpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoS mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium. On a solid medium, the FRD1 rpoS mutant produced approximately 70% less alginate than did the wild-type strain. Thus, our data indicate that although some of the functions of RpoS in P. aeruginosa physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.
PMCID: PMC93876  PMID: 10383954

Results 1-10 (10)