Domain shuffling in a sensor protein contributed to the evolution of insect pathogenicity in plant-beneficial Pseudomonas protegens.

Pseudomonas protegens is a biocontrol rhizobacterium with a plant-beneficial and an insect pathogenic lifestyle, but it is not understood how the organism switches between the two states. Here, we focus on understanding the function and possible evolution of a molecular sensor that enables P. protegens to detect the insect environment and produce a potent insecticidal toxin specifically during insect infection but not on roots. By using quantitative single cell microscopy and mutant analysis, we provide evidence that the sensor histidine kinase FitF is a key regulator of insecticidal toxin production. Our experimental data and bioinformatic analyses indicate that FitF shares a sensing domain with DctB, a histidine kinase regulating carbon uptake in Proteobacteria. This suggested that FitF has acquired its specificity through domain shuffling from a common ancestor. We constructed a chimeric DctB-FitF protein and showed that it is indeed functional in regulating toxin expression in P. protegens. The shuffling event and subsequent adaptive modifications of the recruited sensor domain were critical for the microorganism to express its potent insect toxin in the observed host-specific manner. Inhibition of the FitF sensor during root colonization could explain the mechanism by which P. protegens differentiates between the plant and insect host. Our study establishes FitF of P. protegens as a prime model for molecular evolution of sensor proteins and bacterial pathogenicity.

Molecular epidemiology of pseudomonas aeruginosae in intensive care units (ICUs) over a 10-year period (1998-2007)

Pseudomonas aeruginosa, une bactérie environnementale ubiquitaire, est un des pathogènes nosocomiaux les plus fréquents aux soins intensifs. La source de ce microorganisme peut être soit endogène, 2,6 à 24 % des patients hospitalisés étant colonisés au niveau digestif, soit exogène. La proportion des cas d'infections à P. aeruginosa d'origine exogène, donc secondaires à une transmission par manuportage ou par l'eau du réseau utilisée pour la toilette ou d'autres soins, reste débattue. Or une meilleure évaluation du taux d'infections exogènes est importante pour la mise en place de mesures de contrôle appropriées. Le but de cette étude était de déterminer sur une période de 10 ans les rôles respectifs des sources exogènes (robinets, autres patients) et endogène dans la colonisation et/ou l'infection par P.aeruginosa chez les patients des Soins Intensifs, ainsi que de documenter les variations épidémiologiques au cours du temps. L'étude a été menée dans les unités de Soins Intensifs du Centre Hospitalier Universitaire Vaudois (CHUV). Les patients colonisés et/ou infectés par P. aeruginosa entre 1998 et 2007ont été identifiés via la base de données du laboratoire de microbiologie. Ils ont été inclus dans l'étude s'ils étaient hospitalisés dans une des unités de Soins Intensifs, Durant cette période, des prélèvements pour recherche de P. aeruginosa ont été effectués sur des robinets des soins intensifs. Un typage moléculaire a été effectué sur toutes les souches cliniques et environnementales isolées en 1998, 2000, 2003, 2004 et 2007. Les patients inclus dans l'étude ont été répartis en quatre catégories (A-D) selon le résultat du typage moléculaire leur souche de P. aeruginosa. La catégorie A inclut les cas pour lesquels le génotype de P. aeruginosa est identique à un des génotypes retrouvé dans l'environnement. La catégorie B comprend les cas pour lesquels le génotype est identique à celui d'au moins un autre patient. La catégorie C comprend les cas avec un génotype unique et la catégorie D comprend les cas pour lesquels la souche était non disponible pour le typage. Les cas des catégories A et B sont considérés comme ayant une origine exogène. Au cours des années de l'étude, le nombre d'admissions aux soins intensifs est resté stable. En moyenne, 86 patients par année ont été identifiés colonisés ou infectés par P. aeruginosa aux Soins Intensifs. Durant la première année d'investigation, un grand nombre de patients colonisés par une souche de P. aeruginosa identique à une de celles retrouvées dans l'environnement a été mis en évidence. Par la suite, possiblement suite à l'augmentation de la température du réseau d'eau chaude, le nombre de cas dans la catégorie A a diminué. Dans la catégorie B, le nombre de cas varie de 1,9 à 20 cas/1000 admissions selon les années. Ce nombre est supérieur à 10 cas/1000 admissions en 1998, 2003 et 2007 et correspond à des situations épidémiques transitoires. Tout au long des 10 ans de l'étude, le nombre de cas dans la catégorie C (source endogène) est demeuré stable et indépendant des variations du nombre de cas dans les catégories A et B. En conclusion, la contribution relative des réservoirs endogène et exogène dans la colonisation et/ou l'infection des patients de soins Intensifs varie au cours du temps. Les facteurs principaux qui contribuent à de telles variations sont probablement le degré de contamination de l'environnement, la compliance des soignants aux mesures de contrôle des infections et la génétique du pathogène lui-même. Etant donné que ce germe est ubiquitaire dans l'environnement aqueux et colonise jusqu'à 15% des patients hospitalisés, la disparition de son réservoir endogène semble difficile. Cependant, cette étude démontre que son contrôle est possible dans l'environnement, notamment dans les robinets en augmentant la température de l'eau. De plus, si une souche multi-résistante est retrouvée de manière répétée dans l'environnement, des efforts doivent être mis en place pour éliminer cette souche. Des efforts doivent être également entrepris afin de limiter la transmission entre les patients, qui est une cause importante et récurrente de contamination exogène. - Pseudomonas aeruginosa is one of the leading nosocomial pathogens in intensive care units (ICUs). The source of this microorganism can be either endogenous or exogenous. The proportion of cases as a result of transmission is still debated, and its elucidation is important for implementing appropriate control measures. To understand the relative importance of exogenous vs. endogenous sources of P. aeru¬ginosa, molecular typing was performed on all available P. aeruginosa isolated from ICU clinical and environmental specimens in 1998, 2000, 2003, 2004 and 2007. Patient samples were classified according to their P. aeruginosa genotypes into three categories: (A) identical to isolate from faucet; (B) identical to at least one other patient sample and not found in faucet; and (C) unique genotype. Cases in cat¬egories A and Β were considered as possibly exogenous, and cases in category C as possibly endogenous. A mean of 34 cases per 1000 admissions per year were found to be colonized or infected by P. aeruginosa. Higher levels of faucet contamination were correlated with a higher number of cases in category A. The number of cases in category Β varied from 1.9 to 20 cases per 1000 admissions. This num¬ber exceeded 10/1000 admissions on three occasions and was correlated with an outbreak on one occasion. The number of cases con¬sidered as endogenous (category C) was stable and independent of the number of cases in categories A and B. The present study shows that repeated molecular typing can help identify variations in the epidemiology of P. aeruginosa in ICU patients and guide infection control measures.

Hierarchical management of carbon sources is regulated similarly by the CbrA/B systems in Pseudomonas aeruginosa and Pseudomonas putida.

The CbrA/B system in pseudomonads is involved in the utilization of carbon sources and carbon catabolite repression (CCR) through the activation of the small RNAs crcZ in Pseudomonas aeruginosa, and crcZ and crcY in Pseudomonas putida. Interestingly, previous works reported that the CbrA/B system activity in P. aeruginosa PAO1 and P. putida KT2442 responded differently to the presence of different carbon sources, thus raising the question of the exact nature of the signal(s) detected by CbrA. Here, we demonstrated that the CbrA/B/CrcZ(Y) signal transduction pathway is similarly activated in the two Pseudomonas species. We show that the CbrA sensor kinase is fully interchangeable between the two species and, moreover, responds similarly to the presence of different carbon sources. In addition, a metabolomics analysis supported the hypothesis that CCR responds to the internal energy status of the cell, as the internal carbon/nitrogen ratio seems to determine CCR and non-CCR conditions. The strong difference found in the 2-oxoglutarate/glutamine ratio between CCR and non-CCR conditions points to the close relationship between carbon and nitrogen availability, or the relationship between the CbrA/B and NtrB/C systems, suggesting that both regulatory systems sense the same sort or interrelated signal.

Ferripyochelin uptake genes are involved in pyochelin-mediated signalling in Pseudomonas aeruginosa.

In response to iron starvation, Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted to the extracellular environment, pyochelin chelates iron and transports it to the bacterial cytoplasm via its specific outer-membrane receptor FptA and the inner-membrane permease FptX. Exogenously added pyochelin also acts as a signal which induces the expression of the pyochelin biosynthesis and uptake genes by activating PchR, a cytoplasmic regulatory protein of the AraC/XylS family. The importance of ferripyochelin uptake genes in this regulation was evaluated. The fptA and fptX genes were shown to be part of the fptABCX ferripyochelin transport operon, which is conserved in Burkholderia sp. and Rhodospirillum rubrum. The fptB and fptC genes were found to be dispensable for utilization of pyochelin as an iron source, for signalling and for pyochelin production. By contrast, mutations in fptA and fptX not only interfered with pyochelin utilization, but also affected signalling and diminished siderophore production. It is concluded from this that pyochelin-mediated signalling operates to a large extent via the ferripyochelin transport system.

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth and induces encystment in Acanthamoeba castellanii.

L-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a toxic antimetabolite produced by the opportunistic pathogen Pseudomonas aeruginosa. To evaluate its importance as a potential virulence factor, we tested the host response towards AMB using an Acanthamoeba castellanii cell model. We found that AMB (at concentrations ≥ 0.5 mM) caused amoebal encystment in salt buffer, while inhibiting amoebal growth in rich medium in a dose-dependent manner. However, no difference in amoebal plaque formation was observed on bacterial lawns of wild type and AMB-negative P. aeruginosa strains. We thereby conclude that AMB may eventually act as a virulence factor, but only at relatively high concentrations.

Identification of the biosynthetic gene cluster for the Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid.

L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.

Identification of C(4)-dicarboxylate transport systems in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa utilizes preferentially C(4)-dicarboxylates such as malate, fumarate, and succinate as carbon and energy sources. We have identified and characterized two C(4)-dicarboxylate transport (Dct) systems in P. aeruginosa PAO1. Inactivation of the dctA(PA1183) gene caused a growth defect of the strain in minimal media supplemented with succinate, fumarate or malate, indicating that DctA has a major role in Dct. However, residual growth of the dctA mutant in these media suggested the presence of additional C(4)-dicarboxylate transporter(s). Tn5 insertion mutagenesis of the ΔdctA mutant led to the identification of a second Dct system, i.e., the DctPQM transporter belonging to the tripartite ATP-independent periplasmic (TRAP) family of carriers. The ΔdctA ΔdctPQM double mutant showed no growth on malate and fumarate and residual growth on succinate, suggesting that DctA and DctPQM are the only malate and fumarate transporters, whereas additional transporters for succinate are present. Using lacZ reporter fusions, we showed that the expression of the dctA gene and the dctPQM operon was enhanced in early exponential growth phase and induced by C(4)-dicarboxylates. Competition experiments demonstrated that the DctPQM carrier was more efficient than the DctA carrier for the utilization of succinate at micromolar concentrations, whereas DctA was the major transporter at millimolar concentrations. To conclude, this is the first time that the high- and low-affinity uptake systems for succinate DctA and DctPQM have been reported to function coordinately to transport C(4)-dicarboxylates and that the alternative sigma factor RpoN and a DctB/DctD two-component system regulates simultaneously the dctA gene and the dctPQM operon.

Characterization of cell-to-cell signaling-deficient Pseudomonas aeruginosa strains colonizing intubated patients.

Cell-to-cell signaling involving N-acyl-homoserine lactone compounds termed autoinducers (AIs) is instrumental to virulence factor production and biofilm development by Pseudomonas aeruginosa. In order to determine the importance of cell-to-cell signaling during the colonization of mechanically ventilated patients, we collected 442 P. aeruginosa pulmonary isolates from 13 patients. Phenotypic characterization showed that 81% of these isolates produced the AI-dependent virulence factors elastase, protease, and rhamnolipids. We identified nine genotypically distinct P. aeruginosa strains. Six of these strains produced AIs [N-butanoyl-homoserine lactone or N-(3-oxo-dodecanoyl)-homoserine lactone] and extracellular virulence factors (elastase, total exoprotease, rhamnolipid, hydrogen cyanide, or pyocyanin) in vitro. Three of the nine strains were defective in the production of both AIs and extracellular virulence factors. Two of these strains had mutational defects in both the lasR and rhlR genes, which encode the N-acyl-homoserine lactone-dependent transcriptional regulators LasR and RhlR, respectively. The third of these AI-deficient strains was only mutated in the lasR gene. Our observations suggest that most, but not all, strains colonizing intubated patients are able to produce virulence factors and that mutations affecting the cell-to-cell signaling circuit are preferentially located in the transcriptional regulator genes.

Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa.

In the metabolically versatile bacterium Pseudomonas aeruginosa, the RNA-binding protein Crc is involved in catabolite repression of a range of degradative genes, such as amiE (encoding aliphatic amidase). We found that a CA-rich sequence (termed CA motif) in the amiE translation initiation region was important for Crc binding. The small RNA CrcZ (407 nt) containing 5 CA motifs was able to bind the Crc protein with high affinity and to remove it from amiE mRNA in vitro. Overexpression of crcZ relieved catabolite repression in vivo, whereas a crcZ mutation pleiotropically prevented the utilization of several carbon sources. The sigma factor RpoN and the CbrA/CbrB two-component system, which is known to maintain a healthy carbon-nitrogen balance, were necessary for crcZ expression. During growth on succinate, a preferred carbon source, CrcZ expression was low, resulting in catabolite repression of amiE and other genes under Crc control. By contrast, during growth on mannitol, a poor carbon source, elevated CrcZ levels correlated with relief of catabolite repression. During growth on glucose, an intermediate carbon source, CrcZ levels and amiE expression were intermediate between those observed in succinate and mannitol media. Thus, the CbrA-CbrB-CrcZ-Crc system allows the bacterium to adapt differentially to various carbon sources. This cascade also regulated the expression of the xylS (benR) gene, which encodes a transcriptional regulator involved in benzoate degradation, in an analogous way, confirming this cascade's global role.

Phenotypic switching in Pseudomonas brassicacearum involves GacS- and GacA-dependent Rsm small RNAs.

The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as noncoding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small RNA (sRNA) genes rsmX, rsmY, and rsmZ. Naturally occurring mutations in the gacS-gacA system accounted for phenotypic switching, which was characterized by downregulation of antifungal secondary metabolites (2,4-diacetylphloroglucinol and cyanide), indoleacetate, exoenzymes (lipase and protease), and three different N-acyl-homoserine lactone molecules. Moreover, in addition to abrogating these biocontrol traits, gacS and gacA mutations resulted in reduced expression of the type VI secretion machinery, alginate biosynthesis, and biofilm formation. In a gacA mutant, the expression of rsmX was completely abolished, unlike that of rsmY and rsmZ. Overexpression of any of the three sRNAs in the gacA mutant overruled the pleiotropic changes and restored the wild-type phenotypes, suggesting functional redundancy of these sRNAs. In conclusion, our data show that phenotypic switching in P. brassicacearum results from mutations in the gacS-gacA system.

Impact of quorum sensing on fitness of Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, cell-cell communication based on N-acyl-homoserine lactone (AHL) signal molecules (termed quorum sensing) is known to control the production of extracellular virulence factors. Hence, in pathogenic interactions with host organisms, the quorum-sensing (QS) machinery can confer a selective advantage on P. aeruginosa. However, as shown by transcriptomic and proteomic studies, many intracellular metabolic functions are also regulated by quorum sensing. Some of these serve to regenerate the AHL precursors methionine and S-adenosyl-methionine and to degrade adenosine via inosine and hypoxanthine. The fact that a significant percentage of clinical and environmental isolates of P. aeruginosa is defective for QS because of mutation in the major QS regulatory gene lasR, raises the question of whether the QS machinery can have a negative impact on the organism's fitness. In vitro, lasR mutants have a higher probability to escape lytic death in stationary phase under alkaline conditions than has the QS-proficient wild type. Similar selective forces might also operate in natural environments.

Construction of Pseudomonas strains with improved biocontrol activity

Selected strains of fluorescent pseudomonads suppress various plant diseases caused by soil-borne pathogenic fungi, by a blend of several mechanisms including aggressive root colonization, antibiosis, competition for nutrients, induction of resistance in the plant, and enzymatic attack of the pathogen. These traits are amenable to genetic analysis and, therefore, to modification by genetic engineering. Biocontrol activities of Pseudomonas spp. have been enhanced in two ways: (i) by overexpression of traits known to involved in diseaese suppression, and (ii) by introduction of additional beneficial traits into strains having basal biocontrol activities. Under experimental conditions in microcosms, a number of genetically modified Pseudomonas strains have given promising results. It remains to be seen whether such strains will be superior to the best naturally occurring strains, applied singly or in combination, under greenhouse and field conditions.

Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.

Emergence of secretion-defective sublines of Pseudomonas aeruginosa PAO1 resulting from spontaneous mutations in the vfr global regulatory gene.

Pseudomonas aeruginosa undergoes spontaneous mutation that impairs secretion of several extracellular enzymes during extended cultivation in vitro in rich media, as well as during long-term colonization of the cystic fibrosis lung. A frequent type of strong secretion deficiency is caused by inactivation of the quorum-sensing regulatory gene lasR. Here we analyzed a spontaneously emerging subline of strain PAO1 that exhibited moderate secretion deficiency and partial loss of quorum-sensing control. Using generalized transduction, we mapped the secretion defect to the vfr gene, which is known to control positively the expression of the lasR gene and type II secretion of several proteases. We confirmed this secretion defect by sequencing and complementation of the vfr mutation. In a reconstruction experiment conducted with a 1:1 mixture of wild-type strain PAO1 and a vfr mutant of PAO1, we observed that the vfr mutant had a selective advantage over the wild type after growth in static culture for 4 days. Under these conditions, spontaneous vfr emerged in a strain PAO1 population after four growth cycles, and these mutants accounted for more than 40% of the population after seven cycles. These results suggest that partial or complete loss of quorum sensing and secretion can be beneficial to P. aeruginosa under certain environmental conditions.