Aurélien Carlier

The Ti Plasmid of Agrobacterium tumefaciens Harbors an attM-Paralogous Gene, aiiB, Also Encoding N-Acyl Homoserine Lactonase Activity

ABSTRACT The Agrobacterium tumefaciens C58 genome contains three putative N-acyl homoserine lactone (acyl-HSL) hydrolases, which are closely related to the lactonase AiiA of Bacillus. When expressed in Escherichia coli, two of the putative acyl-HSL hydrolases, AttM and AiiB, conferred the ability to degrade acyl-HSLs on the host. In Erwinia strain 6276, the lactonases reduced the endogenous acyl-HSL level and the bacterial virulence in planta.

The assimilation of gamma-butyrolactone in Agrobacterium tumefaciens C58 interferes with the accumulation of the N-acyl-homoserine lactone signal.

Agrobacterium tumefaciens C58 communicates using N-acyl-homoserine lactones (acyl-HSL) and contains two lactonase-encoding genes, attM and aiiB, the products of which are capable of inactivating the acyl-HSL signal. In A. tumefaciens A6, the expression of the attKLM operon is controlled by the transcriptional repressor encoded by an adjacent gene, attJ. An attJ::Tn5 mutant does not accumulate acyl-HSL because of the constitutive expression of the lactonase AttM, the activity of which inactivates acyl-HSL. In this work, the attKLM operon of A. tumefaciens C58 was shown to be involved in an assimilative pathway of gamma-butyrolactone (GBL), gamma-hydroxybutyrate (GHB), and succinate semialdehyde (SSA), in which AttM and AttL are key enzymes for GBL and GHB assimilation. The expression of the attKLM promoter was activated in the presence of GBL, GHB, and SSA. Under these conditions, A. tumefaciens C58 did not accumulate the acyl-HSL that it naturally synthesizes, and also became able to inactivate exogenous acyl-HSL signals. Therefore, in A. tumefaciens C58, the assimilative pathway of gamma-butyrolactone interferes with the acyl-HSL signaling.

The cell density-dependent expression of stewartan exopolysaccharide in Pantoea stewartii ssp. stewartii is a function of EsaR-mediated repression of the rcsA gene

The LuxR‐type quorum‐sensing transcription factor EsaR functions as a repressor of exopolysaccharide (EPS) synthesis in the phytopathogenic bacterium Pantoea stewartii ssp. stewartii. The cell density‐dependent expression of EPS is critical for Stewarts wilt disease development. Strains deficient in the synthesis of a diffusible acyl‐homoserine lactone inducer remain repressed for EPS synthesis and are consequently avirulent. In contrast, disruption of the esaR gene leads to hypermucoidy and attenuated disease development. Ligand‐free EsaR functions as a negative autoregulator of the esaR gene and responds to exogenous acyl‐homoserine lactone for derepression. The focus of this study was to define the mechanism by which EsaR governs the expression of the cps locus, which encodes functions required for stewartan EPS synthesis and membrane translocation. Genetic and biochemical studies show that EsaR directly represses the transcription of the rcsA gene. RcsA encodes an essential coactivator for RcsA/RcsB‐mediated transcriptional activation of cps genes. In vitro assays identify an EsaR DNA binding site within the rcsA promoter that is reasonably well conserved with the previously described esaR box. We also describe that RcsA positively controls its own expression. Interestingly, promoter proximal genes within the cps cluster are significantly more acyl‐homoserine lactone responsive than genes located towards the middle or 3′ end of the gene cluster. We will discuss a possible role of EsaR‐mediated quorum sensing in the differential expression of the cps operon.

Exposing the third chromosome of Burkholderia cepacia complex strains as a virulence plasmid

The Burkholderia cepacia complex (Bcc) consists of 17 closely related species of opportunistic bacterial pathogens, which are particularly problematic for cystic fibrosis patients and immunocompromised individuals. Bcc genomes consist of multiple replicons, and each strain sequenced to date has three chromosomes. In addition to genes thought to be essential for survival, each chromosome carries at least one rRNA operon. We isolated three mutants during a transposon mutagenesis screen that were non‐pathogenic in a Caenorhabditis elegans infection model. It was demonstrated that these mutants had lost chromosome 3 (c3), and that the observed attenuation of virulence was a consequence of this. We constructed a c3 mini‐replicon and used it to cure c3 from strains of several Bcc species by plasmid incompatibility, resulting in nine c3‐null strains covering seven Bcc species. Phenotypic characterization of c3‐null mutants revealed that they were attenuated in virulence in multiple infection hosts (rat, zebrafish, C. elegans, Galleria mellonella and Drosophila melanogaster), that they exhibited greatly diminished antifungal activity, and that c3 was required for d‐xylose, fatty acid and pyrimidine utilization, as well as for exopolysaccharide production and proteolytic activity in some strains. In conclusion, we show that c3 is not an essential chromosomal element, rather a large plasmid that encodes virulence, secondary metabolism and other accessory functions in Bcc bacteria.

The AHL- and BDSF-dependent quorum sensing systems control specific and overlapping sets of genes in Burkholderia cenocepacia H111.

Quorum sensing in Burkholderia cenocepacia H111 involves two signalling systems that depend on different signal molecules, namely N-acyl homoserine lactones (AHLs) and the diffusible signal factor cis-2-dodecenoic acid (BDSF). Previous studies have shown that AHLs and BDSF control similar phenotypic traits, including biofilm formation, proteolytic activity and pathogenicity. In this study we mapped the BDSF stimulon by RNA-Seq and shotgun proteomics analysis. We demonstrate that a set of the identified BDSF-regulated genes or proteins are also controlled by AHLs, suggesting that the two regulons partially overlap. The detailed analysis of two mutually regulated operons, one encoding three lectins and the other one encoding the large surface protein BapA and its type I secretion machinery, revealed that both AHLs and BDSF are required for full expression, suggesting that the two signalling systems operate in parallel. In accordance with this, we show that both AHLs and BDSF are required for biofilm formation and protease production.

Molecular mechanisms underlying the close association between soil Burkholderia and fungi

Bacterial species belonging to the genus Burkholderia have been repeatedly reported to be associated with fungi but the extent and specificity of these associations in soils remain undetermined. To assess whether associations between Burkholderia and fungi are widespread in soils, we performed a co-occurrence analysis in an intercontinental soil sample collection. This revealed that Burkholderia significantly co-occurred with a wide range of fungi. To analyse the molecular basis of the interaction, we selected two model fungi frequently co-occurring with Burkholderia, Alternaria alternata and Fusarium solani, and analysed the proteome changes caused by cultivation with either fungus in the widespread soil inhabitant B. glathei, whose genome we sequenced. Co-cultivation with both fungi led to very similar changes in the B. glathei proteome. Our results indicate that B. glathei significantly benefits from the interaction, which is exemplified by a lower abundance of several starvation factors that were highly expressed in pure culture. However, co-cultivation also gave rise to stress factors, as indicated by the increased expression of multidrug efflux pumps and proteins involved in oxidative stress response. Our data suggest that the ability of Burkholderia to establish a close association with fungi mainly lies in the capacities to utilize fungal-secreted metabolites and to overcome fungal defense mechanisms. This work indicates that beneficial interactions with fungi might contribute to the survival strategy of Burkholderia species in environments with sub-optimal conditions, including acidic soils.

Identification and characterization of three novel EsaI/EsaR quorum-sensing controlled stewartan exopolysaccharide biosynthetic genes in Pantoea stewartii ssp. stewartii

Pantoea stewartii ssp. stewartii (P. stewartii ), formerly Erwinia stewartii, is a Gram-negative bacterium that causes Stewart’s wilt disease in susceptible maize cultivars by colonizing the xylem as cell wall-adherent biofilms (Braun, 1982; Koutsoudis et al., 2006). This mode of growth requires the production of large amounts of stewartan exopolysaccharide (EPS), which impedes the flow of xylem sap, leading to plant wilt. Stewartan EPS is a high-molecular-weight heteropolysaccharide and represents the primary virulence factor in P. stewartii (Bradshaw-Rouse et al., 1981; Jumel et al., 1997). Mutants unable to secrete EPS adhere strongly to surfaces, tend to generate compact, amorphous biofilms and fail to spread beyond the site of infection in the xylem vessels (Koutsoudis et al., 2006). Stewartan EPS is an anionic polymer composed largely of heptasaccharide repeat units that contain galactose, glucose and glucuronic acid in a 3:3:1 ratio (Fig. 1) (Nimtz et al., 1996a; Yang et al., 1996). Its chemical structure is related to that of amylovoran, a polysaccharide and virulence determinant in Erwinia amylovora (Nimtz et al., 1996b). E. amylovora is also a xylem-dwelling pathogen and causes Fireblight disease in rosaceous plants (Geider, 2000). Sequence homology and partial genetic and biochemical verification indicate that both polymer repeat units are assembled on a polyisoprenoid lipid carrier and translocated via the Wzx membraneassociated polysaccharide specific transport (PST) protein across the inner membrane. A predicted inner membrane-localized Wzy polymerase is thought to facilitate oligomerization of the repeat units, suggesting that the mechanism of stewartan and amylovoran synthesis is related to colanic acid synthesis in Escherichia coli (Coplin et al., 1996; Reeves et al., 1996; Geider, 2000; Whitfield, 2006). All three biosynthetic pathways are under the control of the Rcs phosphorelay signal transduction system (Torres-Cabassa et al., 1987). In P. stewartii, the biosynthesis of stewartan EPS is cell density-dependent governed by the EsaI/EsaR cell–cell signalling or quorum-sensing (QS) system (von Bodman and Farrand, 1995; von Bodman et al., 1998). The EsaR regulator dimerizes and binds target DNA in the absence of inducing levels of the acyl-homoserine lactone (AHL) signal, and loses DNA binding affinity in its presence (Minogue et al., 2002). EsaR inhibits EPS synthesis by repressing the expression of rcsA at low cell density (Carlier and von Bodman, 2006). The rcsA gene encodes an important regulatory component of the Rcs environmental signal sensing phosphorelay system (Majdalani and Gottesman, 2005). At high cell density, inducing levels of AHL, neutralize EsaR DNA binding at the rcsA promoter allowing expression of RcsA to levels required for formation of the RcsA/RcsB activation complex. This complex is necessary for the stimulated expression of the stewartan biosynthetic cps gene cluster. We have adopted the gene designation wce in place of cps following the suggested nomenclature for various bacterial polysaccharide biosynthetic genes (Reeves et al., 1996). The primary gene cluster for stewartan EPS synthesis, now termed wce-I, is structurally and functionally related to the ams gene cluster of E. amylovora (Coplin et al., 1996; Geider, 2000). For example, it is possible to complement specific wce-I mutants with corresponding genes of the ams gene cluster and vice versa (Bernhard et al., 1996). In our attempt to more fully understand the regulated functions involved in stewartan EPS synthesis, we realized that additional functions, not contained within the primary wce-I gene system, must exist elsewhere in the P. stewartii genome. First, it seemed unlikely that biosynthesis of the hexasaccharidic subunits of amylovoran and Accepted 26 September, 2009. *For correspondence. E-mail susanne.vonbodman@uconn.edu; Tel. (+1) 860 486 4408; Fax (+1) 860 486 0534. Molecular Microbiology (2009) 74(4), 903–913 doi:10.1111/j.1365-2958.2009.06906.x First published online 23 October 2009

Structure/Function Analysis of the Pantoea stewartii Quorum-Sensing Regulator EsaR as an Activator of Transcription

In Pantoea stewartii subsp. stewartii, two regulatory proteins are key to the process of cell-cell communication known as quorum sensing: the LuxI and LuxR homologues EsaI and EsaR. Most LuxR homologues function as activators of transcription in the presence of their cognate acylated homoserine lactone (AHL) signal. However, EsaR was initially found to function as a repressor in the absence of AHL. Previous studies demonstrated that, in the absence of AHL, EsaR retains the ability to function as a weak activator of the lux operon in recombinant Escherichia coli. Here it is shown that both the N-terminal and the C-terminal domains of EsaR are necessary for positive regulation. A site-directed mutagenesis study, guided by homology modeling to LuxR and TraR, has revealed three critical residues in EsaR that are involved in activation of RNA polymerase. In addition, a native EsaR-activated promoter has been identified, which controls expression of a putative regulatory sRNA in P. stewartii.

Identification of Burkholderia cenocepacia Strain H111 Virulence Factors Using Nonmammalian Infection Hosts

ABSTRACT Burkholderia cenocepacia H111, a strain isolated from a cystic fibrosis patient, has been shown to effectively kill the nematode Caenorhabditis elegans. We used the C. elegans model of infection to screen a mini-Tn5 mutant library of B. cenocepacia H111 for attenuated virulence. Of the approximately 5,500 B. cenocepacia H111 random mini-Tn5 insertion mutants that were screened, 22 showed attenuated virulence in C. elegans. Except for the quorum-sensing regulator cepR, none of the mutated genes coded for the biosynthesis of classical virulence factors such as extracellular proteases or siderophores. Instead, the mutants contained insertions in metabolic and regulatory genes. Mutants attenuated in virulence in the C. elegans infection model were also tested in the Drosophila melanogaster pricking model, and those also attenuated in this model were further tested in Galleria mellonella. Six of the 22 mutants were attenuated in D. melanogaster, and five of these were less pathogenic in the G. mellonella model. We show that genes encoding enzymes of the purine, pyrimidine, and shikimate biosynthesis pathways are critical for virulence in multiple host models of infection.

The Third Replicon of Members of the Burkholderia cepacia Complex, Plasmid pC3, Plays a Role in Stress Tolerance

ABSTRACT The metabolically versatile Burkholderia cepacia complex (Bcc) occupies a variety of niches, including the plant rhizosphere and the cystic fibrosis lung (where it is often fatal to the patient). Bcc members have multipartite genomes, of which the third replicon, pC3 (previously chromosome 3), has been shown to be a nonessential megaplasmid which confers virulence and both antifungal and proteolytic activity on several strains. In this study, pC3 curing was extended to cover strains of 16 of the 17 members of the Bcc, and the phenotypes conferred by pC3 were determined. B. cenocepacia strains H111, MCO-3, and HI2424 were previously cured of pC3; however, this had not proved possible in the epidemic strain K56-2. Here, we investigated the mechanism of this unexpected stability and found that efficient toxin-antitoxin systems are responsible for maintaining pC3 of strain K56-2. Identification of these systems allowed neutralization of the toxins and the subsequent deletion of K56-2pC3. The cured strain was found to exhibit reduced antifungal activity and was attenuated in both the zebrafish and the Caenorhabditis elegans model of infection. We used a PCR screening method to examine the prevalence of pC3 within 110 Bcc isolates and found that this replicon was absent in only four cases, suggesting evolutionary fixation. It is shown that plasmid pC3 increases the resistance of B. cenocepacia H111 to various stresses (oxidative, osmotic, high-temperature, and chlorhexidine-induced stresses), explaining the prevalence of this replicon within the Bcc.