Ferrous iron sensing and responding in Pseudomonas aeruginosa

Controlling iron distribution is important for all organisms, and is key in bacterial pathogenesis. It has long been understood that cystic fibrosis (CF) patient sputum contains elevated iron concentrations. However, anaerobic bacteria have been isolated from CF sputum and hypoxic zones in sputum have been measured. Because ferrous iron [Fe(II)] is stable in reducing, acidic conditions, it could exist in the CF lung. I show that a two-component system, BqsRS, specifically responds to Fe(II) in the CF pathogen, Pseudomonas aeruginosa. Concurrently, a clinical study found that Fe(II) is present in CF sputum at all stages of lung function decline. Fe(II), not Fe(III) correlates with patients in the most severe disease state. Furthermore, transcripts of the newly identified BqsRS were detected in sputum. Two component systems are the main method bacteria interact with their extracellular environment. A typical two-component system contains a sensor histidine kinase, which upon activation phosphorylates a response regulator that then acts as a transcription factor to elicit a cellular response to stimuli. To explore the mechanism of BqsRS, I describe the Fe(II)-sensing RExxE motif in the sensor BqsS and determine the consensus DNA sequence BqsR binds. With the BqsR binding sequence, I identify novel regulon members through bioinformatic and molecular biology techniques. From the predicted function of new BqsR regulon members, I find that Fe(II) elicits a response that globally protects the cells against cationic stressors, including clinically relevant antibiotics. Subsequently, I use BqsR as a case study to determine if promoter outputs can accurately be predicted based only on a deep understanding of a transcriptional activator’s operator or if a broader regulatory context is required for accurate predictions at all genomic loci. This work highlights the importance of Fe(II) as a (micro)environmental factor, even in conditions typically thought of as aerobic. Since the presence of Fe(II) can alter P. aeruginosa’s antibiotic susceptibility, combining the current strategy of targeting Fe(III) with a new approach targeting Fe(II) may help eradicate infections in the CF lung in the future.

Bioorthogonal Noncanonical Amino Acid Tagging for Selective Analysis of the Pseudomonas aeruginosa Proteome

In natural environments, bacterial physiology is frequently characterized by slow metabolic rates and complex cellular heterogeneities. The opportunistic pathogen Pseudomonas aeruginosa provides one such example; P. aeruginosa forms untreatable chronic biofilm infections of the cystic fibrosis lung, where oxygen limitation can lead to states of metabolic dormancy. To better understand the biology of these states, in vitro experiments must be adapted to better recapitulate natural settings. However, low rates of protein turnover and cellular or phenotypic complexity make these systems difficult to study using established methods. Here we adapt the bioorthogonal noncanonical amino acid tagging (BONCAT) method for time- and cell-selective proteomic analysis to the study of P. aeruginosa. Analysis of proteins synthesized in an anoxic dormancy state led to the discovery of a new type of transcriptional regulator which we designated SutA. We performed detailed analyses of SutA’s role in transcription under slow growth states and we elucidated the structural basis for its regulatory behavior. Additionally, we used cell-selective targeting of BONCAT labeling to measure the dynamic proteomic response of an antibiotic-tolerant biofilm subpopulation. Overall this work shows the utility of selective proteomics as applied to bacterial physiology and describes the broad biological insight obtained from that application.

Avaliação da segurança de uso de pseudomonas como biorremediador em áreas degradadas de pastagens.

A integração agricultura pecuária consiste em uma alternativa promissora de produção, pelo aumento da eficiência de utilização de recursos naturais e a preservação do meio ambiente, além de cooperar com a segurança alimentar. Porém, para se obter sucesso em seu uso, a pastagem não deve estar em estádio avançado de degradação. Em áreas já degradadas, pode-se utilizar as Pseudomonas que são rizobactérias promotoras de crescimento. Neste trabalho, foram avaliados os efeitos decorrentes da exposição a P. putida em mamíferos e um invertebrado aquático como sistema teste para avaliação de risco da introdução desses agentes microbiológicos nessas áreas. Não foram encontrados sinais de patogenicidade e infectividade desses agentes microbianos utilizados como biorremediadores. Apesar disso, seria interessante a realização de testes adicionais a fim de garantir a inocuidade dos agentes bem como a sua segurança. Os protocolos empregados fornecem subsídios técnicos para gerenciar os possíveis riscos envolvidos na liberação e/ou uso do produto. Os resultados obtidos, além de sua aplicação na identificação de efeitos prejudiciais à saúde ambiental, poderão subsidiar e orientar avaliações P. putida por agências reguladoras, quanto ao seu uso comercial para fins de biorremediação.

Impacto da utilização de ácido gálhico, ácido ferúlico e eugenol em modo contínuo na suscetibilidade de Pseudomonas aeruginosa

Dissertação de Mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Influence of Pseudomonas putida AF7 inoculation on soil enzymes.

There has been some concern about the environmental impact of microbial agents. Pseudomonas may be used as bioremediator and as biopesticide. In this study, we report the use of soil enzyme assays as biological indicator of possible negative effects in soil functioning after the P. putida AF7 inoculation. For that, P. putida AF7 was originally isolated from the rizosphere of rice and was inoculated on three soil types: Rhodic Hapludox (RH), Typic Hapludox (TH); and Arenic Hapludult (AH). The acid phosphatase, b-glucosidase and protease enzymes activities were measured for three period of evaluation (7, 14 and 21 days). In general, the enzymatic activities pre- sented variation among the tested soils. The highest activities of b-glucosidase and acid phosphatase were observed in the RH and AH soils, while the protease activity was higher in the TH soil. Also, the soil charac- teristics were measured for each plot. The activity of enzymes from the carbon cycle was positively correlated with the N and the P and the enzyme from the nitrogen cycle was negatively correlated with N and C.org. The presented data indicate that soil biochemical properties can be an useful tool for use as an indicator of soil perturba- tions by microbial inoculation in a risk assessment.

Assessing the safety of Pseudomonas putida introduction in the environment: an overview of ecotoxicological tests.

Risk assessment guidelines for the environmental release of microbial agents are performed in a tiered sequence which includes evaluation of exposure effects on non target organisms. However, it becomes important to verify whether environmental risk assessment from temperate studies is applicable to tropical countries, as Brazil. Pseudomonas putida is a bacteria showing potential to be used for environmental applications as bioremediation and plant disease control. This study investigates the effects of this bacteria exposure on rodents and aquatic organisms (Daphnia similes) that are recommended to be used as non-target organism in environmental risk assessments. Also, the microbial activity in three different soils under P. putida exposure was evaluated. Rats did not show clinical alterations, although the agent was recovered 16 h after the exposure in lung homogenates. The bacteria did not reduce significantly the reproduction and survival of D. similis. The soil enzymatic activities presented fluctuating values after inoculation with bacteria. The measurement of perturbations in soil biochemical characteristics is presented as an alternative way of monitoring the overall effects of the microbial agent to be introduced even in first stage (Tier I) of the risk assessment in tropical ecosystems.

Efeito de Pseudomonas chlororaphis sobre a podridão radicular (Pythium aphanidermatum)e o crescimento vegetal em pimentão hidropônico.

A podridão radicular causada por Pythium spp. é uma das principais doenças em cultivos hidropônicos. O objetivo do trabalho foi avaliar o efeito de Pseudomonas chlororaphis (63-28) sobre a podridão radicular e o crescimento vegetal em pimentão hidropônico. Plantas de pimentão cv. 35-206 RZ foram cultivadas em unidades hidropônicas, e infestadas com P. chlororaphis sete dias antes da inoculação com o patógeno. Os tratamentos foram: testemunha, testemunha inoculada com o patógeno, P. chlororaphis, P. chlororaphis com a adição do patógeno. As variáveis avaliadas foram: severidade da doença, expansão foliar, clorofila foliar e incidência do patógeno. A severidade da doença decresceu em 51% nas plantas inoculadas com a bactéria aos 12 dias após a inoculação, em comparação com a testemunha inoculada. O conteúdo de clorofila decresceu em 12% nas plantas inoculadas com Pythium, em comparação com a testemunha. Plantas tratadas com P. chlororaphis sem a presença do patógeno tiveram maior taxa de expansão foliar. Conclui-se que P. chlororaphis é um promissor agente de controle biológico da podridão radicular e promotor de crescimento de pimentão hidropônico.

Formulação e vida de prateleira de Pseudomonas chlororaphis 63- 28 em fibra de coco, talco e turfa.

2009

Pseudomonas Aeruginosa AmpR Transcriptional Regulatory Network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Characterization of the poxAB operon encoding a class D carbapenemase in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a dreaded opportunistic pathogen that causes severe and often intractable infections in immunocompromised and critically ill patients. This bacterium is also the primary cause of fatal lung infections in patients with cystic fibrosis and a leading nosocomial pathogen responsible for nearly 10% of all hospital-acquired infections. P. aeruginosa is intrinsically recalcitrant to most classes of antibiotics and has the ability to acquire additional resistance during treatment. In particular, resistance to the widely used β-lactam antibiotics is frequently mediated by the expression of AmpC, a chromosomally encoded β-lactamase that is ubiquitously found in P. aeruginosa strains. This dissertation delved into the role of a recently reported chromosomal β-lactamase in P. aeruginosa called PoxB. To date, no detailed studies have addressed the regulation of poxB expression and its contribution to β-lactam resistance in P. aeruginosa. In an effort to better understand the role of this β-lactamase, poxB was deleted from the chromosome and expressed in trans from an IPTG-inducible promoter. The loss of poxB did not affect susceptibility. However, expression in trans in the absence of ampC rendered strains more resistant to the carbapenem β-lactams. The carbapenem-hydrolyzing phenotype was enhanced, reaching intermediate and resistant clinical breakpoints, in the absence of the carbapenem-specific outer membrane porin OprD. As observed for most class D β-lactamases, PoxB was only weakly inhibited by the currently available β-lactamase inhibitors. Moreover, poxB was shown to form an operon with the upstream located poxA, whose expression in trans decreased pox promoter (Ppox) activity suggesting autoregulation. The transcriptional regulator AmpR negatively controlled Ppox activity, however no direct interaction could be demonstrated. A mariner transposon library identified genes involved in the transport of polyamines as potential regulators of pox expression. Unexpectedly, polyamines themselves were able induce resistance to carbapenems. In summary, P. aeruginosa carries a chromosomal-encoded β-lactamase PoxB that can provide resistance against the clinically relevant carbapenems despite its narrow spectrum of hydrolysis and whose activity in vivo may be regulated by polyamines.^

A non-classical LysR-type transcriptional regulator PA2206 is required for an effective oxidative stress response in Pseudomonas aeruginosa

LysR-type transcriptional regulators (LTTRs) are emerging as key circuit components in regulating microbial stress responses and are implicated in modulating oxidative stress in the human opportunistic pathogen Pseudomonas aeruginosa. The oxidative stress response encapsulates several strategies to overcome the deleterious effects of reactive oxygen species. However, many of the regulatory components and associated molecular mechanisms underpinning this key adaptive response remain to be characterised. Comparative analysis of publically available transcriptomic datasets led to the identification of a novel LTTR, PA2206, whose expression was altered in response to a range of host signals in addition to oxidative stress. PA2206 was found to be required for tolerance to H2O2 in vitro and lethality in vivo in the Zebrafish embryo model of infection. Transcriptomic analysis in the presence of H2O2 showed that PA2206 altered the expression of 58 genes, including a large repertoire of oxidative stress and iron responsive genes, independent of the master regulator of oxidative stress, OxyR. Contrary to the classic mechanism of LysR regulation, PA2206 did not autoregulate its own expression and did not influence expression of adjacent or divergently transcribed genes. The PA2214-15 operon was identified as a direct target of PA2206 with truncated promoter fragments revealing binding to the 5'-ATTGCCTGGGGTTAT-3' LysR box adjacent to the predicted -35 region. PA2206 also interacted with the pvdS promoter suggesting a global dimension to the PA2206 regulon, and suggests PA2206 is an important regulatory component of P. aeruginosa adaptation during oxidative stress.

Resistência de representantes dos gêneros pseudomonas sp e bacillus sp mediante exposição à radiação ultravioleta c.

As mudanças climáticas causadas pela degradação da camada de ozônio e pelo aumento da emissão de gases causadores do efeito estufa, podem ocasionar alterações não somente aos seres humanos, mamíferos, répteis ou aves, mas também aos microrganismos. Dentro deste contexto, o aumento nos níveis de radiação solar incidente na superfície da Terra pode afetar as interações que ocorrem entre bactérias e plantas, alterando tanto a ocorrência de doenças como as interações benéficas entre estes organismos. Muitos são os gêneros de bactérias que interagem com as plantas, colonizando os mais diferentes nichos presentes neste hospedeiro. Dentre os grupos bacterianos destacam-se os gêneros Bacillus sp e Pseudomonas sp, que possuem efeitos benéficos como a promoção de crescimento vegetal e a proteção de plantas contra doenças e pragas. Dessa forma, o atual trabalho tem a princípio, como enfoque, constatar a resistência de isolados dos dois gêneros mencionados, à radiação ultravioleta C (200 a 250 nm). Os resultados obtidos demonstram maior resistência da linhagem de Bacillus sp à radiação ultravioleta C em comparação à linhagem de Pseudomonas sp. fato que possivelmente está relacionado com a capacidade de produzir esporos, característica do gênero Bacillus. Este trabalho fornece dados iniciais para futuras avaliações do comportamento destas bactérias quando em interação com plantas e sob maiores níveis de radiação ultravioleta no ambiente.

Influence of Pseudomonas putida AF7 inoculation on soil enzymes.

Pseudomonas may use as bioremediator and as biopesticide. The use of soil enzymatic assays as biological indicator of possible negative effects in soil functioning was evaluated after P.putida AF7 inoculation. For that, AF7 was originally isolated from the rizosphere of rice and was inoculated on three soils: Rhodic Hapludox (RH), Typic Hapludox (TH); and Arenic Hapludult (AH). Soil characteristics were measured in each plot. Acid phosphatase, ?-glucosidase and protease activities were measured at 7, 14 and 21 days. The enzyme activity waved during the experimental period but there is a significant reduction of ?-glucosidase activity in RH soil on day 14. Corg was positively correlated to the activities of ?-glucosidase and protease. The presented data indicate that soil biochemical properties may be useful as indicator of soil perturbations.

Influence of pseudomonas putida af7 inoculation on soil enzymes activities.

2008

Influence of Pseudomonas putida AF7 inoculation on soil enzymes activities.

2008