AmrZ and FleQ Co-regulate Cellulose Production in Pseudomonas syringae pv. Tomato DC3000.

Pseudomonas syringae pv. tomato DC3000 carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose, whose production is stimulated by increasing the intracellular levels of the second messenger c-di-GMP. This enhances air-liquid biofilm formation and generates a wrinkly colony morphotype in solid media. In the present study we show that cellulose production is a complex process regulated at multiple levels and involving different players in this bacterium. Using different in vitro approaches, including Electrophoretic Mobility Shift Assay (EMSA) and footprint analysis, we demonstrated the interrelated role of two transcriptional regulators, AmrZ and FleQ, over cellulose production in Pto DC3000 and the influence of c-di-GMP in this process. Under physiological c-di-GMP levels, both regulators bind directly to adjacent regions at the wss promoter inhibiting its expression. However, just FleQ responds to c-di-GMP releasing from its wss operator site and converting from a repressor to an activator of cellulose production. The additive effect of the double amrZ/fleQ mutation on the expression of wss, together with the fact that they are not cross-regulated at the transcriptional level, suggest that FleQ and AmrZ behave as independent regulators, unlike what has been described in other Pseudomonas species. Furthermore, this dual co-regulation exerted by AmrZ and FleQ is not limited to cellulose production, but also affects other important phenotypes in Pto DC3000, such as motility and virulence.

AmrZ regulates cellulose production in Pseudomonas syringae pv. tomato DC3000.

In Pseudomonas syringae pv. tomato DC3000, the second messenger c-di-GMP has been previously shown to stimulate pellicle formation and cellulose biosynthesis. A screen for genes involved in cellulose production under high c-di-GMP intracellular levels led to the identification of insertions in two genes, wssB and wssE, belonging to the Pto DC3000 cellulose biosynthesis operon wssABCDEFGHI. Interestingly, beside cellulose-deficient mutants, colonies with a rougher appearance than the wild type also arouse among the transposants. Those mutants carry insertions in amrZ, a gene encoding a transcriptional regulator in different Pseudomonas. Here, we provide evidence that AmrZ is involved in the regulation of bacterial cellulose production at transcriptional level by binding to the promoter region of the wssABCDEFGHI operon and repressing cellulose biosynthesis genes. Mutation of amrZ promotes wrinkly colony morphology, increased cellulose production and loss of motility in Pto DC3000. AmrZ regulon includes putative c-di-GMP metabolising proteins, like AdcA and MorA, which may also impact those phenotypes. Furthermore, an amrZ but not a cellulose-deficient mutant turned out to be impaired in pathogenesis, indicating that AmrZ is a key regulator of Pto DC3000 virulence probably by controlling bacterial processes other than cellulose production.

Programa de saúde bucal domiciliar para crianças e adolescentes com paralisia cerebral / Oral health home program for children and teenagers with cerebral palsy

Objetivo: avaliar um programa de cuidado em Saúde Bucal (SB) domiciliar para Crianças e Adolescentes com Paralisia Cerebral (CAPC) e seus cuidadores. Metodologia: estudo de Intervenção com ensaio clínico não controlado. A amostra compreendeu 12 CAPC entre 2-16 anos e 12 cuidadores. O programa preventivo domiciliar constou de ações individuais semanais. As ações foram planejadas a partir dos dados coletados nos exames clínicos nas CAPC, antes da implantação do programa. O cuidador recebeu noções de educação em saúde e treinamento para Escovação Supervisionada (ES) adaptada com abridores de boca. As CAPC receberam ações de ES, aplicação tópica de flúor, raspagem periodontal supragengival e Tratamento Restaurador Atraumático. O exame clínico registrou, por examinador calibrado (Kappa 0,88), o Índice de Higiene Oral Simplificado (IHOS), Índice de Sangramento Gengival (ISG), Dentes Perdidos, Cariados e Obturados (CPOD) e dentes decíduos cariados, extraídos e obturados (ceo-d) analisados pelo teste Wilcoxon (alfa=0,05). O programa foi avaliado pela comparação entre os dados coletados antes (t0) e após três meses (t1) de implantação do programa. Resultados: Foram realizadas 94 consultas domiciliares, no período de agosto a outubro de 2011. Os resultados do IHOS médio (t0 = 2,26±0,43; t1= 0,62±0,32) e do ISG médio (t0= 19,3±7,68; t1= 4,0±3,39) apresentaram diferença significativa (p0,005). O componente cariado do ceo-d (t0= 1,08±2,27; t1= 0,0±0,0) foi revertido em obturado (t0= 0,34±1,15; t1= 1,42±2,67). Os componentes Cariado (0,0±0,0), Perdido (0,0±0,0) e Obturado (0,5±1,73) do CPO-D e extraído (0,58±2,02) do ceo-d não foram alterados entre t0 e t1. Conclusão: O programa domiciliar avaliado qualificou o cuidado em SB das CAPC e produziu alterações positivas dos índices bucais.

Objective: To evaluate an oral health (OH) home program for children and teenagers with cerebral palsy (CTCP) and for their respective caregivers. Methods: This was an interventional study with non-controlled clinical trial. The sample consisted of 12 CTCP aged 2 to 16 years and 12 caregivers. The preventive home program consisted of weekly individual interventions. Actions were planned according to data collected in the clinical examinations of the CTCP before implementing the program. Caregivers were instructed as regards health education and training for supervised tooth brushing (STB) adapted with mouth openers. The CTCP underwent STB, fluoride topical application, supragingival scaling and Atraumatic Restorative Treatment. On clinical examination, a calibrated examiner (Kappa 0.88) registered the simplified Oral Hygiene Index (S-OHI); Gingival Bleeding Index (GBI); decayed, missing and filled teeth (DMFT) and decayed, extracted and filled primary teeth (def), which were analyzed by the Wilcoxon test (alpha=0.05). The program was assessed by comparing data collected at baseline (t0) and after three months (t1) upon its implementation. Results: 94 home consultations were performed from August to October 2011. S-OHI mean (t0 = 2.26±0.43; t1= 0.62±0.32) and GBI mean (t0= 19.3±7.68; t1= 4.0±3.39) were found to present significant difference (p0.005). The 'decayed' component of def (t0= 1.08±2.27; t1= 0.0±0.0) turned into 'filled' (t0= 0.34±1.15; t1= 1.42±2.67). The 'decayed' (0.0±0.0), 'missing' (0.0±0.0) and 'filled' (0.5±1.73) components of DMFT and the 'extracted' component of def did not differ significantly between t0 and t1. Conclusion: The assessed home program provided OH care for CTCP and resulted in positive changes of the oral indexes.

Induction of Pseudomonas syringae pv. tomato DC3000 MexAB-OprM multidrug efflux pump by flavonoids is mediated by the repressor PmeR.

In this study, we have analyzed the expression of the Pseudomonas syringae pv. tomato DC3000 mexAB-oprM efflux pump operon and of the regulatory gene pmeR, and we have investigated the role of the PmeR protein on transcription from both promoters. We demonstrate that mexAB-oprM and pmeR are expressed in vivo at a relatively high and moderate basal level, respectively, which, in both cases, increases in the presence of different flavonoids and other compounds, such as butyl and methylparaben. We show that PmeR is the local repressor of the mexAB-oprM promoter and is able to regulate its own expression. The mechanism for this regulation includes binding to a pseudopalindromic operator site which overlaps both mexAB-oprM and pmeR promoters. We have also proven that flavonoids are able to interact with PmeR and induce a conformational change that interferes with the DNA binding ability of PmeR, thereby modulating mexAB-oprM and pmeR expression. Finally, we demonstrate by in vivo experiments that the PmeR/MexAB-OprM system contributes to the colonization of tomato plants. These results provide new insight into a transcriptional regulator and a transport system that play essential roles in the ability of P. syringae pv. tomato DC3000 to resist the action of flavonoids produced by the host.

Complexity in efflux pump control: cross-regulation by the paralogues TtgV and TtgT.

Pseudomonas putida DOT-T1E, known for its high tolerance to solvents, possesses three Resistance-Nodulation-Cell Division-type (RND) efflux pumps, namely TtgABC, TtgDEF and TtgGHI, which are involved in the active extrusion of solvents. Expression of the ttgABC and ttgGHI operons was previously shown to be regulated by the adjacently encoded repressors, TtgR and TtgV, respectively. Upstream of the third RND operon, ttgDEF, is located a putative regulator gene, ttgT. In this study, TtgT is shown to bind to the promoter region of the ttgDEF operon, and to be released from DNA in the presence of organic solvents. In vitro studies revealed that TtgV and TtgT bind the same operator sites in both the ttgDEF and the ttgGHI promoters. However, the affinity of TtgV for the ttgDEF operator was higher than that of TtgT, which, together with the fact that the ttgV promoter seems to be almost twice stronger than the ttgT promoter, explains why TtgV takes over in the regulation of the two efflux pump operons. The functional replacement of the cognate, chromosomally encoded TtgT by the plasmid-encoded paralogue TtgV illustrates a new mode of efflux pump regulation of which the physiological relevance is discussed.

The multidrug efflux regulator TtgV recognizes a wide range of structurally different effectors in solution and complexed with target DNA: evidence from isothermal titration calorimetry.

TtgV modulates the expression of the ttgGHI operon, which encodes an efflux pump that extrudes a wide variety of chemicals including mono- and binuclear aromatic hydrocarbons, aliphatic alcohols, and antibiotics of dissimilar chemical structure. Using a 'lacZ fusion to the ttgG promoter, we show that the most efficient in vivo inducers were 1-naphthol, 2,3-dihydroxynaphthalene, 4-nitrotoluene, benzonitrile, and indole. The thermodynamic parameters for the binding of different effector molecules to purified TtgV were determined by isothermal titration calorimetry. For the majority of effectors, the interaction was enthalpy-driven and counterbalance by unfavorable entropy changes. The TtgV-effector dissociation constants were found to vary between 2 and 890 mum. There was a relationship between TtgV affinity for the different effectors and their potential to induce gene expression in vivo, indicating that the effector binding constant is a major determinant for efficient efflux pump gene expression. Equilibrium dialysis and isothermal titration calorimetry studies indicated that a TtgV dimer binds one effector molecule. No evidence for the simultaneous binding of multiple effectors to TtgV was obtained. The binding of TtgV to a 63-bp DNA fragment containing its cognate operator was tight and entropy-driven (K(D) = 2.4 +/- 0.35 nm, DeltaH = 5.5 +/- 0.04 kcal/mol). The TtgV-DNA complex was shown to bind 1-napthol with an affinity comparable with the free soluble TtgV protein, K(D) = 4.8 +/- 0.19 and 3.0 +/- 0.15 mum, respectively. The biological relevance of this finding is discussed.

Antibiotic-dependent induction of Pseudomonas putida DOT-T1E TtgABC efflux pump is mediated by the drug binding repressor TtgR.

Pseudomonas putida is well known for its metabolic capabilities, but recently, it has been shown to exhibit resistance to a wide range of antibiotics. In P. putida DOT-T1E, the TtgABC efflux pump, which has a broad substrate specificity, extrudes antibiotics such as ampicillin, carbenicillin, tetracycline, nalidixic acid, and chloramphenicol. We have analyzed the expression of the ttgABC efflux pump operon and its regulatory gene, ttgR, in response to several structurally unrelated antibiotics at the transcriptional level and investigated the role of the TtgR protein in this process. ttgABC and ttgR are expressed in vivo at a moderate basal level, which increases in the presence of hydrophobic antibiotics like chloramphenicol and tetracycline. In vitro experiments show that, in the absence of inducers, TtgR binds to a palindromic operator site which overlaps both ttgABC and ttgR promoters and dissociates from it in the presence of chloramphenicol and tetracycline. These results suggest that the TtgR repressor is able to bind to structurally different antibiotics, which allows induction of TtgABC multidrug efflux pump expression in response to these antimicrobial agents. This is the first case in which the expression of a drug transporter of the resistance-nodulation-division family has been shown to be regulated directly by antibiotics.