Search

Mucoid mucA22 Pseudomonas aeruginosa (PA) is an opportunistic lung pathogen of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients that is highly sensitive to acidified nitrite (A-NO2-). In this study, we first screened PA mutant strains for sensitivity or resistance to 20 mM A-NO2- under anaerobic conditions that represent the chronic stages of the aforementioned diseases. Mutants found to be sensitive to A-NO2- included PA0964 (pmpR, PQS biosynthesis), PA4455 (probable ABC transporter permease), katA (major catalase, KatA) and rhlR (quorum sensing regulator). In contrast, mutants lacking PA0450 (a putative phosphate transporter) and PA1505 (moaA2) were A-NO2- resistant. However, we were puzzled when we discovered that mucA22 mutant bacteria, a frequently isolated mucA allele in CF and to a lesser extent COPD, were more sensitive to A-NO2- than a truncated ΔmucA deletion (Δ157-194) mutant in planktonic and biofilm culture, as well as during a chronic murine lung infection. Subsequent transcriptional profiling of anaerobic, A-NO2--treated bacteria revealed restoration of near wild-type transcript levels of protective NO2- and nitric oxide (NO) reductase (nirS and norCB, respectively) in the ΔmucA mutant in contrast to extremely low levels in the A-NO2--sensitive mucA22 mutant. Proteins that were S-nitrosylated by NO derived from A-NO2- reduction in the sensitive mucA22 strain were those involved in anaerobic respiration (NirQ, NirS), pyruvate fermentation (UspK), global gene regulation (Vfr), the TCA cycle (succinate dehydrogenase, SdhB) and several double mutants were even more sensitive to A-NO2-. Bioinformatic-based data point to future studies designed to elucidate potential cellular binding partners for MucA and MucA22. Given that A-NO2- is a potentially viable treatment strategy to combat PA and other infections, this study offers novel developments as to how clinicians might better treat problematic PA infections in COPD and CF airway diseases.

Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages.

The production of cellulose fibrils is involved in the attachment of Agrobacterium tumefaciens to its plant host. Consistent with previous studies, we reported recently that a putative diguanylate cyclase, celR , is required for synthesis of this polymer in A. tumefaciens . In this study, the effects of celR and other components of the regulatory pathway of cellulose production were explored. Mutational analysis of celR demonstrated that the cyclase requires the catalytic GGEEF motif, as well as the conserved aspartate residue of a CheY-like receiver domain, for stimulating cellulose production. Moreover, a site-directed mutation within the PilZ domain of CelA, the catalytic subunit of the cellulose synthase complex, greatly reduced cellulose production. In addition, deletion of divK , the first gene of the divK-celR operon, also reduced cellulose production. This requirement for divK was alleviated by expression of a constitutively active form of CelR, suggesting that DivK acts upstream of CelR activation. Based on bacterial two-hybrid assays, CelR homodimerizes but does not interact with DivK. The mutation in divK additionally affected cell morphology, and this effect was complementable by a wild-type copy of the gene, but not by the constitutively active allele of celR . These results support the hypothesis that CelR is a bona fide c-di-GMP synthase and that the nucleotide signal produced by this enzyme activates CelA via the PilZ domain. Our studies also suggest that the DivK/CelR signaling pathway in Agrobacterium regulates cellulose production independent of cell cycle checkpoint systems that are controlled by divK .

The opportunistic pathogen Pseudomomas aeruginosa produces multiple pigments during in vitro culture and in vivo during colonization of burn wounds and in the airways of cystic fibrosis (CF) patients. One pigment is a deep 'merlot'-coloured compound known as aeruginosin A (AA). However, the red pigment(s) of P. aeruginosa are often collectively called pyorubrin, of which there is no known chemical composition. Here, we purified and confirmed by MS and assessed the physicochemical properties of AA (2-amino-6-carboxy-10-methylphenazinium betaine) by first focusing on its ability to redox-cycle using cyclic voltammetry and its spectroscopic (as well as fluorescent) properties, experiments that were conducted at physiological pH. AA exhibited reversible electrochemistry at a glassy carbon electrode within a potential range of -500 to -200 mV. Electrochemical anodic and cathodic peak currents were observed at -327 and -360 mV, respectively, with a low formal reduction potential of -343.5 mV versus Ag/AgCl. AA absorbed at 516 nm and fluoresced at 606 nm. Results from the spectro-electrochemistry of pyorubrin revealed that its strongest fluorescence was in its parent or oxidized form. Production of AA by P. aeruginosa was found to be controlled by the rhl component of the intercellular signalling system known as quorum sensing and was produced maximally during the stationary growth phase. However, unlike its downstream blue redox-active toxin, pyocyanin, AA had no adverse effects on methicillin-resistant Staphylococcus aureus USA300, Escherichia coli DH5-α or human keratinocytes. We close with some thoughts on the potential commercial use(s) of AA.

Here, we constructed stable, constitutively expressed, chromosomal green (GFP) and red fluorescent (RFP) reporters in the genome of the surrogate strain, Francisella tularensis spp. holarctica LVS (herein LVS), and the select agent, F. tularensis Schu S4. A bioinformatic approach was used to identify constitutively expressed genes. Two promoter regions upstream of the FTT1794 and rpsF(FTT1062) genes were selected and fused with GFP and RFP reporter genes in pMP815, respectively. While the LVS strains with chromosomally integrated reporter fusions exhibited fluorescence, we were unable to deliver the same fusions into Schu S4. Neither a temperature-sensitive Francisella replicon nor a pBBR replicon in the modified pMP815 derivatives facilitated integration. However, a mini-Tn7 integration system was successful at integrating the reporter fusions into the Schu S4 genome. Finally, fluorescent F. tularensis LVS and a mutant lacking MglA were assessed for growth in monocyte-derived macrophages (MDMs). As expected, when compared to wild-type bacteria, replication of an mglA mutant was significantly diminished, and the overall level of fluorescence dramatically decreased with infection time. The utility of the fluorescent Schu S4 strain was also examined within infected MDMs treated with clarithromycin and enrofloxacin. Taken together, this study describes the development of an important reagent for F. tularensis research, especially since the likelihood of engineered antibiotic resistant strains will emerge with time. Such strains will be extremely useful in high-throughput screens for novel compounds that could interfere with critical virulence processes in this important bioweapons agent and during infection of alveolar macrophages.

Pseudomonas aeruginosa (PA) is an important airway pathogen of cystic fibrosis and chronic obstructive disease patients. Multiply drug resistant PA is becoming increasing prevalent and new strategies are needed to combat such insidious organisms. We have previously shown that a mucoid, mucA22 mutant PA is exquisitely sensitive to acidified nitrite ([Formula: see text], pH 6.5) at concentrations that are well tolerated in humans. Here, we used a transposon mutagenesis approach to identify PA mutants that are hypersensitive to [Formula: see text]. Among greater than 10,000 mutants screened, we focused on PA4455, in which the transposon was found to disrupt the production of a putative cytoplasmic membrane-spanning ABC transporter permease. The PA4455 mutant was not only highly sensitive to [Formula: see text], but also the membrane perturbing agent, EDTA and the antibiotics doxycycline, tigecycline, colistin, and chloramphenicol, respectively. Treatment of bacteria with [Formula: see text] plus EDTA, however, had the most dramatic and synergistic effect, with virtually all bacteria killed by 10 mM [Formula: see text], and EDTA (1 mM, aerobic, anaerobic). Most importantly, the PA4455 mutant was also sensitive to [Formula: see text] in biofilms. [Formula: see text] sensitivity and an anaerobic growth defect was also noted in two mutants (rmlC and wbpM) that are defective in B-band LPS synthesis, potentially indicating a membrane defect in the PA4455 mutant. Finally, this study describes a gene, PA4455, that when mutated, allows for dramatic sensitivity to the potential therapeutic agent, [Formula: see text] as well as EDTA. Furthermore, the synergy between the two compounds could offer future benefits against antibiotic resistant PA strains.

The cystic fibrosis (CF) airway mucus is an ideal niche in which many bacteria can develop antibiotic- and phagocyte-resistance in unique structures known as "mode II biofilms" where bacteria are embedded within the mucus, yet unattached to airway epithelial cells. Pseudomonas aeruginosa is the dominant CF pathogen, yet herein the authors provide burgeoning evidence that obligate anaerobic bacteria (e.g., Prevotella) actually thrive within the CF mucus, a paradigmatic shift that chronic CF is an "aerobic" disease. Interestingly, CF organisms repress virulence factor production (e.g., P. aeruginosa) while others (e.g., S. aureus) increase them under anaerobic conditions.The authors shed additional light on (i) the anoxic nature of the CF airway mucus, (ii) the relative commonality of anaerobic bacteria isolated from CF sputum, (iii) virulence factor production and cross-talk between obligate anaerobes and P. aeruginosa relative to disease progression/remission, (iv) the role of mucoidy in CF, and (v) the role of nitrosative stress in activation of bacteriophage and pyocins within biofilms.The authors conclude with insight as to how we might treat some CF bacteria during mode II biofilm infections that utilizes a metabolite of bacterial anaerobic respiration and an aerobic oxidation product of airway-generated NO, acidified NO(2)(-).

Conjugative transfer of the Ti plasmids of Agrobacterium tumefaciens is controlled by a quorum-sensing system composed of TraR and its signal N -(3-oxo-octanoyl)- l -homoserine lactone. This system is, in turn, controlled by the conjugative opines produced by crown gall tumors induced on plants by the bacteria. Using nonpolar traI mutants, we examined the kinetics of induction of conjugative transfer in response to exogenous acyl-homoserine lactone. In the absence of the antiactivator TraM, onset of induction of transfer requires about 30 min, 15 to 20 min of which is needed for expression and construction of the conjugative apparatus. TraM delays the onset of conjugation by 30 min. While the rate of development of conjugative competence was not significantly affected by levels of TraR, maximum efficiencies of transfer were correlated with amounts of the activator in the donors. Donors harboring Ti plasmids lacking TraM were fully induced by the quormone at concentrations as low as 100 pM. TraM raised the concentration of signal required for maximum activity to 1 nM. Donors grown in batch culture retained conjugative competence following signal removal, even when in stationary phase. However, donors kept in balanced growth rapidly lost transfer ability following signal removal. Loss of transfer was mirrored by a decrease in levels of active TraR. Decreases in TraR activity and conjugative competence could be accounted for by dilution associated with cell division, suggesting that while induction of Ti plasmid conjugation is an active process, the cells lack a mechanism for disassembling the conjugative apparatus when signals become limiting.

Summary TraM, an 11.2 kDa antiactivator, modulates the acyl-homoserine lactone-mediated autoinduction of Ti plasmid conjugative transfer by interacting directly with TraR, the quorum-sensing transcriptional activator. Most antiactivators and antisigma factors examined to date act in dimer form. However, whether, and if so, how TraM dimerizes is unknown. Analyses based on a genetic assay using fusions of TraM to the λ cI DNA binding domain, and biochemical assays using chemical crosslinking and gel filtration chromatography showed that TraM forms homodimers. Although SDS-PAGE studies suggested that the lone cysteine residue at position 71 was involved in interprotomer disulfide-bridging in TraM, altering Cys-71 to a serine did not significantly affect dimerization or the antiactivator activity of this mutant protein when expressed at wild-type levels in vivo. Analysis of N-terminal, C-terminal, and internal deletion mutants of TraM identified two regions of the protein involved in dimerization; one located within a segment between residues 20 and 50, and the other located to a segment between residues 67 and 96. Both regions are required for formation of fully stable dimers. Analysis of the activity of these deletion mutants in vivo, and their ability to bind TraR and to disrupt TraR-DNA complexes in vitro, suggests that while the internal segment of the protein is required for dimerization, determinants located at the far C-terminus and beginning at between residues 10 and 20 at the N-terminus play a role in TraR binding and antiactivator function. When co-expressed with λ cI′::TraR fusions, wild-type TraM mediated quormone-independent dimerization of the transcriptional activator, suggesting that dimers of TraM can multimerize TraR.

The human pathogen Pseudomonas aeruginosa is capable of causing both acute and chronic infections. Differences in virulence are attributable to the mode of growth: bacteria growing planktonically cause acute infections, while bacteria growing in matrix-enclosed aggregates known as biofilms are associated with chronic, persistent infections. While the contribution of the planktonic and biofilm modes of growth to virulence is now widely accepted, little is known about the role of dispersion in virulence, the active process by which biofilm bacteria switch back to the planktonic mode of growth. Here, we demonstrate that P. aeruginosa dispersed cells display a virulence phenotype distinct from those of planktonic and biofilm cells. While the highest activity of cytotoxic and degradative enzymes capable of breaking down polymeric matrix components was detected in supernatants of planktonic cells, the enzymatic activity of dispersed cell supernatants was similar to that of biofilm supernatants. Supernatants of non-dispersing ΔbdlA biofilms were characterized by a lack of many of the degradative activities. Expression of genes contributing to the virulence of P. aeruginosa was nearly 30-fold reduced in biofilm cells relative to planktonic cells. Gene expression analysis indicated dispersed cells, while dispersing from a biofilm and returning to the single cell lifestyle, to be distinct from both biofilm and planktonic cells, with virulence transcript levels being reduced up to 150-fold compared to planktonic cells. In contrast, virulence gene transcript levels were significantly increased in non-dispersing ΔbdlA and ΔdipA biofilms compared to wild-type planktonic cells. Despite this, bdlA and dipA inactivation, resulting in an inability to disperse in vitro, correlated with reduced pathogenicity and competitiveness in cross-phylum acute virulence models. In contrast, bdlA inactivation rendered P. aeruginosa more persistent upon chronic colonization of the murine lung, overall indicating that dispersion may contribute to both acute and chronic infections., Author Summary Pathogenic bacteria, including the human pathogen Pseudomonas aeruginosa, can cause acute and chronic infections. The difference in these infection modes can be explained by how bacteria grow. Acute infections occur when individual bacteria rapidly replicate, produce high levels of virulence factors, and disseminate from the nidus of infection. Chronic infections occur when bacteria adhere to tissue or implanted medical devices and form multi-cellular, matrix-encased aggregates known as biofilms. The acute-to-chronic infection switch occurs when bacteria transition from planktonic to biofilm growth. However, the contribution of dispersion, the process by which bacteria leave a biofilm to return to planktonic growth, remains unclear. Here, we demonstrate that, while having left a biofilm, dispersed cells are distinct from planktonic cells with respect to gene expression, release of matrix-degrading enzymes, and pathogenicity. We found that a mutant impaired in nutrient-induced dispersion, while enhancing chronic infections, is impaired in mounting acute infections in both plant and mouse hosts. Overall, this work establishes that dispersed cells have a unique virulence phenotype, with nutrient-induced dispersion not only serving as an integral part of both acute and chronic infections but also as a potential mechanism of infection control.

Pseudomonas aeruginosa is especially adept at colonizing the airways of individuals afflicted with the autosomal recessive disease cystic fibrosis (CF). CF patients suffer from chronic airway inflammation, which contributes to lung deterioration. Once established in the airways, P. aeruginosa continuously adapts to the changing environment, in part through acquisition of beneficial mutations via a process termed pathoadaptation. MutS and DinB are proposed to play opposing roles in P. aeruginosa pathoadaptation: MutS acts in replication-coupled mismatch repair, which acts to limit spontaneous mutations; in contrast, DinB (DNA polymerase IV) catalyzes error-prone bypass of DNA lesions, contributing to mutations. As part of an ongoing effort to understand mechanisms underlying P. aeruginosa pathoadaptation, we characterized hydrogen peroxide (H(2)O(2))-induced phenotypes of isogenic P. aeruginosa strains bearing different combinations of mutS and dinB alleles. Our results demonstrate an unexpected epistatic relationship between mutS and dinB with respect to H(2)O(2)-induced cell killing involving error-prone repair and/or tolerance of oxidized DNA lesions. In striking contrast to these error-prone roles, both MutS and DinB played largely accurate roles in coping with DNA lesions induced by ultraviolet light, mitomycin C, or 4-nitroquinilone 1-oxide. Models discussing roles for MutS and DinB functionality in DNA damage-induced mutagenesis, particularly during CF airway colonization and subsequent P. aeruginosa pathoadaptation are discussed.

Francisella tularensisrequires iron (Fe) for growth, but the biologic sources of Fe for this organism are largely unknown. We found thatFrancisellasp. growing in broth culture or within human macrophages can acquire Fe from the two major host Fe-binding proteins, lactoferrin (Lf) and transferrin (Tf). Fe acquisition is a potential target for novel therapies. Gallium (Ga) is a transition metal that interferes with cellular Fe metabolism by competing with Fe for uptake/utilization. Growth of eitherF. tularensislive vaccine strain (LVS) orFrancisella novicidawas inhibited by ≥2 μM Ga chelated to Tf or Lf, with GaLf being somewhat more potent.Francisellaspp. express two Fe-containing antioxidant enzymes, catalase (KatG) and Fe cofactored superoxide dismutase (FeSOD). Growth of LVS with 10 μM GaTf or GaLf led to a dramatic decrease in bacterial catalase activity and in FeSOD activity that was associated with an increased susceptibility to H2O2. Ga also protected mice from intranasal challenge withF. novicida. Whereas 100% of theF. novicida-infected mice died by day 9, 75% of the mice receiving Ga continued to survive to at least day 15. Thus, a single intranasal dose of Ga followed by daily intraperitoneal Ga at a dose tolerated by the animals resulted in prolonged survival. These data support the potential utility of Ga as a therapy forF. tularensisinfection of the lung.

Agrobacterium radiobacter K84 is a commercial agent used worldwide to control crown gall disease caused by pathogenic isolates of A. tumefaciens. More than 2,000 transposon insertion derivatives of strain K84 were screened by a standardized greenhouse bioassay to identify mutants defective in biocontrol. Three mutants affected in biocontrol properties were identified. All three mutants displayed normal levels of attachment to tomato seed and root colonization. One of these mutants, M19-164, exhibited partial biocontrol and did not produce detectable levels of agrocin 84. In this mutant, the transposon is located in the agn locus of pAgK84, which codes for agrocin 84 biosynthesis. The second mutant, M19-158, also exhibited partial biocontrol and produced reduced amounts of agrocin 84 as a result of a mutation in a chromosomal gene of unknown function. The third mutant, M9-22, failed to biocontrol, was impaired in both growth in minimal medium and siderophore production, and failed to produce detectable levels of agrocin 84. The chromosomal gene ahcY, which encodes S-adenosyl-l-homocysteine hydrolase, was disrupted in this mutant. Expression of a functional copy of ahcY in M9-22 restored all of the altered phenotypes. The fact that all identified biocontrol mutants exhibited a partial or total defect in production of agrocin 84 indicates that this antibiotic is required for optimum biocontrol. This study also identified two chromosomally encoded genes required for agrocin 84 production. That a mutation in ahcY abolishes biocontrol suggests that the intracellular ratio of S-adenosyl-l-methionine to S-adenosyl-l-homocysteine is an important factor for agrocin 84 biosynthesis. Finally, we demonstrate that the ahcY gene in strain K84 is also required for optimal growth as well as for antibiotic production and biocontrol of crown gall disease.

The ATP-dependent Lon (La) protease is ubiquitous in nature and regulates a diverse set of physiological responses in bacteria. In this paper alonmutant of theα-proteobacteriumAgrobacterium tumefaciensC58 has been characterized. Unlikelonmutants ofEscherichia coli, thelonmutant ofA. tumefaciensgrows very slowly, is not filamentous and exhibits normal resistance to UV irradiation. The mutant retains motility and chemotaxis, produces apparently normal amounts of exopolysacchride, but displays severe defects in cell morphology, with 80 % of the mutant cells appearing Y-shaped. Lon protease ofA. tumefaciensshares high homology with its counterparts inE. coliand inSinorhizobium meliloti, and functionally complements anE. coli lonmutant for defects in morphology and RcsA-mediated regulation of capsular polysaccharide production. Mutations at sites of LonAtcorresponding to the ATP-binding site and the active site serine of theE. coliLon protease abolish complementation of phenotypes of theA. tumefaciensandE. coli lonmutants. The nucleotide sequence upstream ofA. tumefaciens loncontains an element similar to the consensusσ32heat-shock promoter ofE. coli. Northern and Western blot analyses indicated that expression oflonis induced by elevated temperature, albeit to a much lower level than that ofgroEL. Thelonmutant is highly attenuated for virulence, suggesting that Lon may be required for the proper expression, assembly or function of the VirB/D4-mediated T-DNA transfer system.

Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ∼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (K d ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.