Draft genome sequence of Stenotrophomonas maltophilia strain M30, isolated from a chronic pressure ulcer in an elderly patient

Stenotrophomonas maltophilia is an emerging opportunistic pathogen with an increasing prevalence of multidrug-resistant strains. Here, we report the draft genome sequence of S. maltophilia strain M30, isolated from a pressure ulcer in an elderly patient

Quorum sensing signaling and quenching in the multidrug-resistant pathogen stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an opportunistic Gram-negative pathogen with increasing incidence in clinical settings. The most critical aspect of S. maltophilia is its frequent resistance to a majority of the antibiotics of clinical use. Quorum Sensing (QS) systems coordinate bacterial populations and act as major regulatory mechanisms of pathogenesis in both pure cultures and poly-microbial communities. Disruption of QS systems, a phenomenon known as Quorum Quenching (QQ), represents a new promising paradigm for the design of novel antimicrobial strategies. In this context, we review the main advances in the field of QS in S. maltophilia by paying special attention to Diffusible Signal Factor (DSF) signaling, Acyl Homoserine Lactone (AHL) responses and the controversial Ax21 system. Advances in the DSF system include regulatory aspects of DSF synthesis and perception by both rpf -1 and rpf -2 variant systems, as well as their reciprocal communication. Interaction via DSF of S. maltophilia with unrelated organisms including bacteria, yeast and plants is also considered. Finally, an overview of the different QQ mechanisms involving S. maltophilia as quencher and as object of quenching is presented, revealing the potential of this species for use in QQ applications. This review provides a comprehensive snapshot of the interconnected QS network that S. maltophilia uses to sense and respond to its surrounding biotic or abiotic environment. Understanding such cooperative and competitive communication mechanisms is essential for the design of effective anti QS strategies

Draft genome sequence of Stenotrophomonas maltophilia strain UV74 reveals extensive variability within its genomic group

We report the draft genome sequence of Stenotrophomonas maltophilia UV74, isolated from a vascular ulcer. This draft genome sequence shall contribute to the understanding of the evolution and pathogenicity of this species, particularly regarding isolates of clinical origin

Decoding the genetic and functional diversity of the DSF Quorum-Sensing system in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia uses the Diffusible Signal Factor (DSF) quorum sensing (QS) system to mediate intra- and inter-specific signaling and regulate virulence-related processes. The components of this system are encoded by the rpf cluster, with genes rpfF and rpfC encoding for the DSF synthase RpfF and sensor RpfC, respectively. Recently, we have shown that there exist two variants of the rpf cluster (rpf-1 and rpf-2), distinguishing two groups of S. maltophilia strains. Surprisingly, only rpf-1 strains produce detectable DSF, correlating with their ability to control biofilm formation, swarming motility and virulence. The evolutive advantage of acquiring two different rpf clusters, the phylogenetic time point and mechanism of this acquisition and the conditions that activate DSF production in rpf-2 strains, are however not known. Examination of this cluster in various species suggests that its variability originated most probably by genetic exchange between rhizosphere bacteria. We propose that rpf-2 variant strains make use of a strategy recently termed as "social cheating." Analysis of cellular and extracellular fatty acids (FAs) of strains E77 (rpf-1) and M30 (rpf-2) suggests that their RpfFs have also a thioesterase activity that facilitates the release of unspecific FAs to the medium in addition to DSF. Production of DSF in rpf-1 strains appears in fact to be modulated by some of these extracellular FAs in addition to other factors such as temperature and nutrients, while in rpf-2 strains DSF biosynthesis is derepressed only upon detection of DSF itself, suggesting that they require cohabitation with DSF-producer bacteria to activate their DSF regulatory machinery. Finally, we show that the mixed rpf-1/rpf-2 population presents synergism in DSF production and virulence capacity in an in vivo infection model. Recovery and quantification of DSF from co-infected animals correlates with the observed mortality rate

Stenotrophomonas maltophilia responds to exogenous AHL signals through the LuxR solo SmoR (Smlt1839)

Quorum Sensing (QS) mediated by Acyl Homoserine Lactone (AHL) molecules are probably the most widespread and studied among Gram-negative bacteria. Canonical AHL systems are composed by a synthase (LuxI family) and a regulator element (LuxR family), whose genes are usually adjacent in the genome. However, incomplete AHL-QS machinery lacking the synthase LuxI is frequently observed in Proteobacteria, and the regulator element is then referred as LuxR solo. It has been shown that certain LuxR solos participate in interspecific communication by detecting signals produced by different organisms. In the case of Stenotrophomonas maltophilia, a preliminary genome sequence analysis revealed numerous putative luxR genes, none of them associated to a luxI gene. From these, the hypothetical LuxR solo Smlt1839, here designated SmoR, presents a conserved AHL binding domain and a helix-turn-helix DNA binding motif. Its genomic organization-adjacent to hchA gene-indicate that SmoR belongs to the new family "LuxR regulator chaperone HchA-associated." AHL-binding assays revealed that SmoR binds to AHLs in-vitro, at least to oxo-C8-homoserine lactone, and it regulates operon transcription, likely by recognizing a conserved palindromic regulatory box in the hchA upstream region. Supplementation with concentrated supernatants from Pseudomonas aeruginosa, which contain significant amounts of AHLs, promoted swarming motility in S. maltophilia. Contrarily, no swarming stimulation was observed when the P. aeruginosa supernatant was treated with the lactonase AiiA from Bacillus subtilis, confirming that AHL contributes to enhance the swarming ability of S. maltophilia. Finally, mutation of smoR resulted in a swarming alteration and an apparent insensitivity to the exogenous AHLs provided by P. aeruginosa. In conclusion, our results demonstrate that S. maltophilia senses AHLs produced by neighboring bacteria through the LuxR solo SmoR, regulating population behaviors such as swarming motility

Improved mini-Tn 7 Delivery Plasmids for Fluorescent Labeling of

Fluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such as . Using a Tn 7 -based genomic integration system, we report the construction of improved mini-Tn 7 delivery plasmids for labeling of with sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn 7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3'-end of the conserved glmS gene of different wild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence in . It is also shown that the mini-Tn 7 elements remained stably integrated in the genome of over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn 7 delivery plasmids are valuable tools for generating fluorescently labeled strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium is an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence of infections worldwide. One important virulence-related trait of is the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn 7 -based labeling system for to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions

Fatty acid-mediated quorum sensing systems in stenotrophomonas maltophilia

Els sistemes de comunicació bacteriana -coneguts com quorum sensing (QS)- a través de molècules senyalitzadores del tipus àcid gras han despertat molt d’interès en els darrers anys ja que s’ha vist que molts bacteris patògens els utilitzen per regular funcions relacionades amb la virulència. Es coneix que Stenotrophomonas maltophilia presenta el sistema de QS DSF (Diffusible Signal Factor) el qual és controlat pels gens que conformen el clúster rpf (Regulation of Pathogenecity Factors). No obstant, no està clar els mecanismes pels quals S. maltophilia sintetitza i sensa les molècules senyal així com quines funcions estan regulades per aquest sistema. En aquest treball hem demostrat que existeixen dues poblacions de S. maltophilia les quals es diferencien en base al clúster rpf (rpf-1 o rpf-2) que presenten. Cada variant difereix bàsicament en els gens que codifiquen per la sintasa RpfF i el sensor RpfC. A més, hem observat que existeix una associació entre ambdós components, generant-se la parella RpfF-1/RpfC-1 per les soques rpf-1 i RpfF-2/RpfC-2 per les soques rpf-2. Addicionalment, hem demostrat que només aquelles soques que presenten la variant rpf-1 produeixen nivells detectables de DSF i aquest regula motilitat bacteriana, formació de biofilm i virulència. Per altra banda, les soques de la variant rpf-2 necessiten més còpies de la sintasa RpfF-2 o l’absència del repressor RpfC-2 per produir DSF. En aquest cas, el sistema de QS DSF sembla només regular pocs fenotips relacionats amb virulència en situacions molt específiques. També hem mostrat que existeix un feedback positiu en la síntesi de DSF i que ambdós grups de soques actuen de manera sinèrgica en la producció de DSF i la virulència de tota la població. Addicionalment, hem observat que, mentre la variant RpfC-1 és un sensor promiscu el qual permet l’alliberació de la sintasa RpfF-1 tant punt detecta no només DSF sinó també àcids grassos de cadena mitja, el sensor RpfC-2 és molt més específic, alliberant RpfF-2 només quan detecta DSF. A més a més, aquí també mostrem com el sistema de QS cis-DA (cis-decenoic) descrit recentment a Pseudomonas aeruginosa és també present a S. maltophilia i regula un alt nombre de factors de virulència. En aquesta línia, hem sigut capaços de caracteritzar preliminarment dos components importants en la biosíntesi de l’àcid gras cis-DA: les enoil coA hidratases (ECH) Smlt0266 i Smlt0267. Hem observat que, mentre la mutació de la hipotètica sintasa Smlt0266 només condueix a l’increment de la formació de biofilm, la mutació en el gen que codifica per la ECH alternativa Smlt0267 implica una reducció dràstica en la formació de biofilm, la motilitat bacteriana, la producció d’exopolisacàrids, la resistència a antibiòtics i la virulència. Resultats similars s’han obtingut per els mutants dels gens ortòlegs a P. aeruginosa, la qual cosa recolza la importància d’aquestes dues ECHs, a més a més del sistema DSF, en la regulació de la virulència i aporta noves dianes interessants pel desenvolupament de teràpies antimicrobianes contra aquest potencial patogen humàFatty-acid mediated Quorum Sensing (QS) systems have aroused considerably interest in the last years since it has been reported that many important bacterial pathogens use these communication systems to regulate virulence-related functions. It is known that Stenotrophomonas maltophilia presents the DSF (Diffusible Signal Factor) QS system, which is controlled by components that are encoded in the rpf cluster (Regulation of Pathogenicity Factors). However, the mechanisms by which S. maltophilia synthesize and sense as well as the biological functions that are under control of DSF-QS remain unclear. Here, we have first demonstrated that two populations of S. maltophilia can be distinguished depending on the rpf cluster (rpf-1 or rpf-2) they harbour. Each variant cluster differs basically in the genes that encode for the synthase RpfF and the sensor RpfC. Moreover, we have observed that there exist a full association between both components, existing the pair RpfF-1/RpfC-1 for the rpf-1 variant and RpfF-2/RpfC-2 for the rpf-2 variant. In addition, we have demonstrated that only strains harbouring the rpf-1 variant produce detectable levels of DSF and it seems to regulate bacterial motility, biofilm development and virulence. On the other hand, strains harbouring the rpf-2 variant need extra copies of rpfF-2 or the absence of rpfC-2 to achieve detectable levels of DSF. In this case, DSF-QS seems to control only some virulence-related phenotypes in very specific environments (e.g., zebrafish infection). We also have shown that DSF is produced in a positive feedback-manner in S. maltophilia, and also, that both rpf-variant groups act synergistically in the DSF production and virulence ability of the whole population. In addition, we have observed that while RpfC-1 is a promiscuous sensor that liberates free active-RpfF-1 -with the subsequent DSF synthesis- upon detection not only DSF, but also saturated medium-length fatty acids, the sensor RpfC-2 only allows activation of RpfF-2 upon detection of DSF-itself, indicating that this sensor component is much more specific. Here, we further report that the cis-DA (cis-decenoic acid) QS system recently described in Pseudomonas aeruginosa is also present in S. maltophilia, and it regulates various virulence factors. In this line, we have preliminary characterized two important components in the biosynthesis of cis-DA, the enoyl-CoA hydratases (ECH) Smlt0266 and Smlt0267. We have observed that while the mutation in the putative synthase smlt0266 lead to alteration basically in biofilm formation, the mutation of the alternative ECH smlt0267 results in a drastic effect in many virulence-related behaviours such as biofilm formation, bacterial motility, exopolysaccharide production, antibiotic resistance and virulence. Similar results have been obtained for the mutants in the orthologous P. aeruginosa genes ∆dspI and ∆dspII. These results further support the significance of these two ECH, in addition to DSF-QS system, in virulence regulation of S. maltophilia and provide new interesting targets for developing new antimicrobial therapies against this potential human pathogen

Fatty acid-mediated quorum sensing systems in stenotrophomonas maltophilia

Els sistemes de comunicació bacteriana -coneguts com quorum sensing (QS)- a través de molècules senyalitzadores del tipus àcid gras han despertat molt d'interès en els darrers anys ja que s'ha vist que molts bacteris patògens els utilitzen per regular funcions relacionades amb la virulència. Es coneix que Stenotrophomonas maltophilia presenta el sistema de QS DSF (Diffusible Signal Factor) el qual és controlat pels gens que conformen el clúster rpf (Regulation of Pathogenecity Factors). No obstant, no està clar els mecanismes pels quals S. maltophilia sintetitza i sensa les molècules senyal així com quines funcions estan regulades per aquest sistema. En aquest treball hem demostrat que existeixen dues poblacions de S. maltophilia les quals es diferencien en base al clúster rpf (rpf-1 o rpf-2) que presenten. Cada variant difereix bàsicament en els gens que codifiquen per la sintasa RpfF i el sensor RpfC. A més, hem observat que existeix una associació entre ambdós components, generant-se la parella RpfF-1/RpfC-1 per les soques rpf-1 i RpfF-2/RpfC-2 per les soques rpf-2. Addicionalment, hem demostrat que només aquelles soques que presenten la variant rpf-1 produeixen nivells detectables de DSF i aquest regula motilitat bacteriana, formació de biofilm i virulència. Per altra banda, les soques de la variant rpf-2 necessiten més còpies de la sintasa RpfF-2 o l'absència del repressor RpfC-2 per produir DSF. En aquest cas, el sistema de QS DSF sembla només regular pocs fenotips relacionats amb virulència en situacions molt específiques. També hem mostrat que existeix un feedback positiu en la síntesi de DSF i que ambdós grups de soques actuen de manera sinèrgica en la producció de DSF i la virulència de tota la població. Addicionalment, hem observat que, mentre la variant RpfC-1 és un sensor promiscu el qual permet l'alliberació de la sintasa RpfF-1 tant punt detecta no només DSF sinó també àcids grassos de cadena mitja, el sensor RpfC-2 és molt més específic, alliberant RpfF-2 només quan detecta DSF. A més a més, aquí també mostrem com el sistema de QS cis-DA (cis-decenoic) descrit recentment a Pseudomonas aeruginosa és també present a S. maltophilia i regula un alt nombre de factors de virulència. En aquesta línia, hem sigut capaços de caracteritzar preliminarment dos components importants en la biosíntesi de l'àcid gras cis-DA: les enoil coA hidratases (ECH) Smlt0266 i Smlt0267. Hem observat que, mentre la mutació de la hipotètica sintasa Smlt0266 només condueix a l'increment de la formació de biofilm, la mutació en el gen que codifica per la ECH alternativa Smlt0267 implica una reducció dràstica en la formació de biofilm, la motilitat bacteriana, la producció d'exopolisacàrids, la resistència a antibiòtics i la virulència. Resultats similars s'han obtingut per els mutants dels gens ortòlegs a P. aeruginosa, la qual cosa recolza la importància d'aquestes dues ECHs, a més a més del sistema DSF, en la regulació de la virulència i aporta noves dianes interessants pel desenvolupament de teràpies antimicrobianes contra aquest potencial patogen humàFatty-acid mediated Quorum Sensing (QS) systems have aroused considerably interest in the last years since it has been reported that many important bacterial pathogens use these communication systems to regulate virulence-related functions. It is known that Stenotrophomonas maltophilia presents the DSF (Diffusible Signal Factor) QS system, which is controlled by components that are encoded in the rpf cluster (Regulation of Pathogenicity Factors). However, the mechanisms by which S. maltophilia synthesize and sense as well as the biological functions that are under control of DSF-QS remain unclear. Here, we have first demonstrated that two populations of S. maltophilia can be distinguished depending on the rpf cluster (rpf-1 or rpf-2) they harbour. Each variant cluster differs basically in the genes that encode for the synthase RpfF and the sensor RpfC. Moreover, we have observed that there exist a full association between both components, existing the pair RpfF-1/RpfC-1 for the rpf-1 variant and RpfF-2/RpfC-2 for the rpf-2 variant. In addition, we have demonstrated that only strains harbouring the rpf-1 variant produce detectable levels of DSF and it seems to regulate bacterial motility, biofilm development and virulence. On the other hand, strains harbouring the rpf-2 variant need extra copies of rpfF-2 or the absence of rpfC-2 to achieve detectable levels of DSF. In this case, DSF-QS seems to control only some virulence-related phenotypes in very specific environments (e.g., zebrafish infection). We also have shown that DSF is produced in a positive feedback-manner in S. maltophilia, and also, that both rpf-variant groups act synergistically in the DSF production and virulence ability of the whole population. In addition, we have observed that while RpfC-1 is a promiscuous sensor that liberates free active-RpfF-1 -with the subsequent DSF synthesis- upon detection not only DSF, but also saturated medium-length fatty acids, the sensor RpfC-2 only allows activation of RpfF-2 upon detection of DSF-itself, indicating that this sensor component is much more specific. Here, we further report that the cis-DA (cis-decenoic acid) QS system recently described in Pseudomonas aeruginosa is also present in S. maltophilia, and it regulates various virulence factors. In this line, we have preliminary characterized two important components in the biosynthesis of cis-DA, the enoyl-CoA hydratases (ECH) Smlt0266 and Smlt0267. We have observed that while the mutation in the putative synthase smlt0266 lead to alteration basically in biofilm formation, the mutation of the alternative ECH smlt0267 results in a drastic effect in many virulence-related behaviours such as biofilm formation, bacterial motility, exopolysaccharide production, antibiotic resistance and virulence. Similar results have been obtained for the mutants in the orthologous P. aeruginosa genes ∆dspI and ∆dspII. These results further support the significance of these two ECH, in addition to DSF-QS system, in virulence regulation of S. maltophilia and provide new interesting targets for developing new antimicrobial therapies against this potential human pathogen

Quorum sensing signaling and quenching in the multidrug-resistant pathogen stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an opportunistic Gram-negative pathogen with increasing incidence in clinical settings. The most critical aspect of S. maltophilia is its frequent resistance to a majority of the antibiotics of clinical use. Quorum Sensing (QS) systems coordinate bacterial populations and act as major regulatory mechanisms of pathogenesis in both pure cultures and poly-microbial communities. Disruption of QS systems, a phenomenon known as Quorum Quenching (QQ), represents a new promising paradigm for the design of novel antimicrobial strategies. In this context, we review the main advances in the field of QS in S. maltophilia by paying special attention to Diffusible Signal Factor (DSF) signaling, Acyl Homoserine Lactone (AHL) responses and the controversial Ax21 system. Advances in the DSF system include regulatory aspects of DSF synthesis and perception by both rpf -1 and rpf -2 variant systems, as well as their reciprocal communication. Interaction via DSF of S. maltophilia with unrelated organisms including bacteria, yeast and plants is also considered. Finally, an overview of the different QQ mechanisms involving S. maltophilia as quencher and as object of quenching is presented, revealing the potential of this species for use in QQ applications. This review provides a comprehensive snapshot of the interconnected QS network that S. maltophilia uses to sense and respond to its surrounding biotic or abiotic environment. Understanding such cooperative and competitive communication mechanisms is essential for the design of effective anti QS strategies

Gene duplications in the E. coli genome : common themes among pathotypes

Background: gGene duplication underlies a significant proportion of gene functional diversity and genome complexity in both eukaryotes and prokaryotes. Although several reports in the literature described the duplication of specific genes in E. coli, a detailed analysis of the extent of gene duplications in this microorganism is needed. - Results: the genomes of the E. coli enteroaggregative strain 042 and other pathogenic strains contain duplications of the gene that codes for the global regulator Hha. To determine whether the presence of additional copies of the hha gene correlates with the presence of other genes, we performed a comparative genomic analysis between E. coli strains with and without hha duplications. The results showed that strains harboring additional copies of the hha gene also encode the yeeR irmA (aec69) gene cluster, which, in turn, is also duplicated in strain 042 and several other strains. The identification of these duplications prompted us to obtain a global map of gene duplications, first in strain 042 and later in other E. coli genomes. Duplications in the genomes of the enteroaggregative strain 042, the uropathogenic strain CFT073 and the enterohemorrhagic strain O145:H28 have been identified by a BLASTp protein similarity search. This algorithm was also used to evaluate the distribution of the identified duplicates among the genomes of a set of 28 representative E. coli strains. Despite the high genomic diversity of E. coli strains, we identified several duplicates in the genomes of almost all studied pathogenic strains. Most duplicated genes have no known function. Transcriptomic analysis also showed that most of these duplications are regulated by the H-NS/Hha proteins. - Conclusions: several duplicated genes are widely distributed among pathogenic E. coli strains. In addition, some duplicated genes are present only in specific pathotypes, and others are strain specific. This gene duplication analysis shows novel relationships between E. coli pathotypes and suggests that newly identified genes that are duplicated in a high percentage of pathogenic E. coli isolates may play a role in virulence. Our study also shows a relationship between the duplication of genes encoding regulators and genes encoding their target