The Temperature-Regulation of Pseudomonas aeruginosa cmaX-cfrX-cmpX Operon Reveals an Intriguing Molecular Network Involving the Sigma Factors AlgU and SigX

Pseudomonas aeruginosa is a highly adaptable Gram-negative opportunistic pathogen, notably due to its large number of transcription regulators. The extracytoplasmic sigma factor (ECFσ) AlgU, responsible for alginate biosynthesis, is also involved in responses to cell wall stress and heat shock via the RpoH alternative σ factor. The SigX ECFσ emerged as a major regulator involved in the envelope stress response via membrane remodeling, virulence and biofilm formation. However, their functional interactions to coordinate the envelope homeostasis in response to environmental variations remain to be determined. The regulation of the putative cmaX-cfrX-cmpX operon located directly upstream sigX was investigated by applying sudden temperature shifts from 37°C. We identified a SigX- and an AlgU- dependent promoter region upstream of cfrX and cmaX, respectively. We show that cmaX expression is increased upon heat shock through an AlgU-dependent but RpoH independent mechanism. In addition, the ECFσ SigX is activated in response to valinomycin, an agent altering the membrane structure, and up-regulates cfrX-cmpX transcription in response to cold shock. Altogether, these data provide new insights into the regulation exerted by SigX and networks that are involved in maintaining envelope homeostasis

Mecanismos de comunicación intercelular en Halomonas anticariensis

Halomonas anticariensis FP35T es una gammaproteobacteria perteneciente a la familia Halomonadaceae que fue aislada de suelos salinos de la Laguna de Fuente de Piedra, situada en Antequera (Málaga). En esta bacteria halófila moderada se describió por primera vez en 2004 la producción de moléculas señal N-acil homoserín lactonas (AHLs) típicas de los sistemas quorum sensing (QS). En el presente trabajo se ha caracterizado dicho sistema a nivel genético y está compuesto por genes homólogos a los del tipo luxR/luxI: el gen hanR (regulador transcripcional) y el gen hanI (sintasa de AHLs). Los genes QS hanR/hanI en H. anticariensis FP35T forman parte de un único operón cuya transcripción es dependiente de la densidad celular. El análisis transcripcional de ambos genes indicó que se transcriben a partir de sus propios promotores y de forma independiente de las moléculas señal AHLs lo que sugirió que dicho sistema QS no estaba autorregulado a diferencia del sistema de regulación clásico. La producción de AHLs y la expresión de los genes QS hanR/hanI en H. anticariensis FP35T están regulados de forma positiva por un sistema regulador global de dos componentes (TCS) compuesto por una proteína histidina quinasa híbrida (HK-GacS) y un regulador de respuesta (RR-GacA). Este sistema también controla la producción del exopolisacárido (EPS), la movilidad y la formación del biofilm, aunque el control de estos fenotipos no tiene lugar a través de las moléculas señal AHLs del sistema QS hanR/hanI. Mediante la tecnología del microarray fenotípico se ha puesto de manifiesto que el sistema QS mediado por las AHLs interviene de forma directa o indirecta en el metabolismo de carbohidratos, nitrógeno y fosfato posiblemente a través del control de los sistemas de transporte, y con ello se afecta la resistencia o sensibilidad hacía ciertos compuestos químicos. En definitiva este trabajo representa un primer estudio de los mecanismos de comunicación intercelular en H. anticariensis FP35T y los fenotipos asociados que posiblemente reflejan su papel en la adaptación al medio ambiente. Por otra parte, mediante el análisis de la producción de moléculas señal tipo AHLs en 42 especies de la familia Halomonadaceae se ha demostrado que este tipo de comunicación se encuentra ampliamente distribuida en dicho grupo bacteriano en el que existe una diversidad de AHLs producidas entre los diferentes géneros e incluso entre las diferentes especies.Tesis Univ. Granada. Departamento de MicrobiologíaLa presente tesis doctoral ha sido subvencionada por las ayudas de los siguientes proyectos de investigación, Proyecto de Excelencia P07-CVI-03150 (2007-2011) Junta de Andalucía, y proyecto AGL2009-07656 (2010-2012) CICYT, asimismo, el doctorando ha disfrutado de una beca de Formación de Personal Investigador, concedida por la Junta de Andalucía (2008-2012)

Quorum Sensing in Some Representative Species of Halomonadaceae

Cell-to-cell communication, or quorum-sensing (QS), systems are employed by bacteria for promoting collective behaviour within a population. An analysis to detect QS signal molecules in 43 species of the Halomonadaceae family revealed that they produced N-acyl homoserine lactones (AHLs), which suggests that the QS system is widespread throughout this group of bacteria. Thin-layer chromatography (TLC) analysis of crude AHL extracts, using Agrobacterium tumefaciens NTL4 (pZLR4) as biosensor strain, resulted in different profiles, which were not related to the various habitats of the species in question. To confirm AHL production in the Halomonadaceae species, PCR and DNA sequencing approaches were used to study the distribution of the luxI-type synthase gene. Phylogenetic analysis using sequence data revealed that 29 of the species studied contained a LuxI homolog. Phylogenetic analysis showed that sequences from Halomonadaceae species grouped together and were distinct from other members of the Gammaproteobacteria and also from species belonging to the Alphaproteobacteria and Betaproteobacteria

The hanR/hanI quorum-sensing system of Halomonas anticariensis, a moderately halophilic bacterium

International audienceQuorum sensing is a cell density-dependent gene expression mechanism found in many Gram- negative bacteria which involves the production of signal molecules such as N-acylhomoserine lactones (AHLs). One significant group of micro-organisms in which quorum sensing has not been previously studied, however, are the moderate halophiles. We describe here the results of our studies of the quorum-sensing system in Halomonas anticariensis FP35T, which is composed of luxR/luxI homologues: hanR (the putative transcriptional regulator gene) and hanI (the autoinducer synthase gene). To understand how the hanR/hanI system is organized and regulated we conducted RT-PCR and quantitative real-time PCR assays. Transcriptional analysis indicated that the hanR and hanI genes are on the same transcript and that their transcription is growth phase-dependent. HanI seems to be the only autoinducer synthase responsible for the synthesis of AHLs by the bacterium, since the inactivation of hanI resulted in the complete loss of its AHLs. We also found that the hanI gene appears to be transcribed from its own promoter and that its expression does not depend upon HanR. This finding was supported by the fact that the FP35hanR mutant showed AHL-producing activity and hanI expression similar to that of the wild- type strain, the latter being measured by RT-PCR. Moreover, hanR is expressed from its own promoter and appears to be independent of the AHL signalling molecules produced by HanI

Draft Genome Sequence of the Moderately Halophilic Gammaproteobacterium Halomonas anticariensis FP35T

International audienceHalomonas anticariensis strain FP35(T) is a moderately halophilic bacterium isolated from a soil sample taken from Fuente de Piedra, a saline wetland in the province of Málaga (Spain), which produces an exopolysaccharide and quorum-sensing signaling molecules of the type N-acylhomoserine lactone. We report here the draft genome sequence of this gammaproteobacterium

Genetic and phenotypic analysis of the GacS/GacA system in the moderate halophile Halomonas anticariensis

International audienceA multisensory, hybrid histidine kinase (HK) and a response regulator (RR), which together may well constitute a two-component regulatory system (TCS), have been located in Halomonas anticariensis FP35(T) by transposon mutagenesis. This TCS is homologous to the GacS/GacA system described for many Gram-negative bacteria. An analysis of crude N-acylhomoserine lactone (AHL) extracts from cultures of FP35gacS and FP35gacA mutants showed that they produced lower quantities of AHLs than the wild-type strain. In addition, RT-PCR analysis revealed a considerable decrease in the expression of the quorum-sensing (QS) genes hanR and hanI compared with the wild-type strain. This result indicates that the GacS/GacA TCS exerts a positive effect upon the QS HanR/HanI system and suggests its integral involvement in the intercellular communication strategies of this bacterium. We have also demonstrated the influence of GacS and GacA upon exopolysaccharide production and biofilm formation, in which this regulatory machinery appears to play a key role in an overall system that co-ordinates gene expression and behaviour in H. anticariensis FP35(T) in response to environmental conditions

Quorum Sensing in Some Representative Species of Halomonadaceae

International audienceCell-to-cell communication, or quorum-sensing (QS), systems are employed by bacteria for promoting collective behaviour within a population. An analysis to detect QS signal molecules in 43 species of the Halomonadaceae family revealed that they produced N-acyl homoserine lactones (AHLs), which suggests that the QS system is widespread throughout this group of bacteria. Thin-layer chromatography (TLC) analysis of crude AHL extracts, using Agrobacterium tumefaciens NTL4 (pZLR4) as biosensor strain, resulted in different profiles, which were not related to the various habitats of the species in question. To confirm AHL production in the Halomonadaceae species, PCR and DNA sequencing approaches were used to study the distribution of the luxI-type synthase gene. Phylogenetic analysis using sequence data revealed that 29 of the species studied contained a LuxI homolog. Phylogenetic analysis showed that sequences from Halomonadaceae species grouped together and were distinct from other members of the Gammaproteobacteria and also from species belonging to the Alphaproteobacteria and Betaproteobacteria

N -Acylsulfonamide: a valuable moiety to design new sulfa drug analogues

International audienceWe report here the synthesis and antibacterial/antibiofilm activities of 18 sulfa drug analogues with an N -acylsulfonamide moiety obtained efficiently by sulfo-click reactions between readily available thioacid and sulfonyl azide synthons

Expanding the gene regulatory network in response to osmotic stress in the halophilic Chromohalobacter salexigens

International audienceBackground: Chromohalobacter salexigens is a broad-growing halophilic bacterium considered as a biological model to study osmoadaptation and a cell factory for the production of ectoines, biostabilizing compatible solutes which have many biotechnological applications. To design new metabolic engineering strategies to obtain improved ectoines production strains of C. salexigens, a preliminary transcriptional regulatory network (TRN) core was reconstructed by using an integrative approach that combined RNAseq data from wild type and different in silico analyses. This core constituted by 598 genes and 1192 regulatory interactions was used to analyse molecular regulation events involved in metabolic osmotic stress response and ectoines metabolism