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Azelaic acid is a dicarboxylic acid that has recently been shown to play a role in plant-bacteria signalling and also occurs naturally in several cereals. Several bacteria have been reported to be able to utilize azelaic acid as a unique source of carbon and energy, including Pseudomonas nitroreducens . In this study, we utilize P. nitroreducens as a model organism to study bacterial degradation of and response to azelaic acid. We report genetic evidence of azelaic acid degradation and the identification of a transcriptional regulator that responds to azelaic acid in P. nitroreducens DSM 9128. Three mutants possessing transposons in genes of an acyl-CoA ligase, an acyl-CoA dehydrogenase and an isocitrate lyase display a deficient ability in growing in azelaic acid. Studies on transcriptional regulation of these genes resulted in the identification of an IclR family repressor that we designated as AzeR, which specifically responds to azelaic acid. A bioinformatics survey reveals that AzeR is confined to a few proteobacterial genera that are likely to be able to degrade and utilize azelaic acid as the sole source of carbon and energy.

Metabolite annotation from imaging mass spectrometry (imaging MS) data is a difficult undertaking that is extremely resource intensive. Here, we adapted METASPACE, cloud software for imaging MS metabolite annotation and data interpretation, to quickly annotate microbial specialized metabolites from high-resolution and high-mass accuracy imaging MS data. Compared with manual ion image and MS1 annotation, METASPACE is faster and, with the appropriate database, more accurate. We applied it to data from microbial colonies grown on agar containing 10 diverse bacterial species and showed that METASPACE was able to annotate 53 ions corresponding to 32 different microbial metabolites. This demonstrates METASPACE to be a useful tool to annotate the chemistry and metabolic exchange factors found in microbial interactions, thereby elucidating the functions of these molecules. ispartof: METABOLITES vol:11 issue:8 ispartof: location:Switzerland status: published

Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling., Endophytes are microorganisms that live inside plants and are often beneficial for the host. Kosakonia is a novel bacterial genus that includes several species that are diazotrophic and plant associated. This study revealed two quorum sensing-related LuxR solos, designated LoxR and PsrR, in the plant endophyte Kosakonia sp. strain KO348. LoxR modeling and biochemical studies demonstrated that LoxR binds N-acyl homoserine lactones (AHLs) in a promiscuous way. PsrR, on the other hand, belongs to the subfamily of plant-associated-bacterium (PAB) LuxR solos that respond to plant compounds. Target promoter studies as well as modeling and phylogenetic comparisons suggest that PAB LuxR solos are likely to respond to different plant compounds. Finally, LoxR is involved in the regulation of T6SS and PsrR plays a role in root endosphere colonization. IMPORTANCE Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling.

Quorum Sensing (QS) signaling, which allows the coordination of the microbial physiology, can be influenced by environmental factors. However, the impact of copper-based fungicides on the QS of rhizosphere microorganisms is currently unknown. The simultaneous presence of other microorganisms may also alter the QS signaling. In this work we report that the fungicide CuSO4 modifies the expression from the promoter of ppuI, ppoR and rsaL, components of Pseudomonas capeferrum WCS358 QS system. ppuI and ppoR promoters showed a biphasic behavior reducing their activities as CuSO4 concentrations increased up to 1 mmol l−1, but with increased values at higher metal concentrations. rsaL promoter attained a minimal activity with 1 mmol l−1, though higher concentrations did not modify its expression. The impact of CuSO4 on biofilm formation by WCS358 was dependent on the bacterial QS activity. Dual biofilms with Papiliotrema laurentii YL2 yeast were also affected by the bacterial QS and the metal. CuSO4 was detrimental for the bacterial colonization, but YL2 exerted a protective effect in dual colonies, an effect that depended on the QS activity. These results suggest the importance of the WCS358 QS systems in the rhizosphere, where copper-based fungicides and concomitant microorganisms conform a complex system.

Pseudomonas fuscovaginae (Pfv) is an emerging plant pathogen causing sheath brown rot in rice, as well as diseases in other gramineae food crops including maize, sorghum and wheat. Pfv possesses two conserved N-acyl homoserine lactone (AHL) quorum sensing (QS) systems called PfvI/R and PfsI/R, which are repressed by RsaL and RsaM, respectively. The two systems are not hierarchically organized and are involved in plant virulence. In this study the AHL QS PfsI/R, PfvI/R and RsaM regulons were determined by transcriptomic analysis. The PfsI/R system regulates 98 genes, whereas 26 genes are regulated by the PfvI/R AHL QS system; only two genes are regulated by both systems. RsaM, on the other hand, regulates over 400 genes: 206 are negatively regulated and 260 are positively regulated. More than half of the genes controlled by the PfsI/R system and 65 % by the PfvI/R system are also part of the RsaM regulon; this is due to RsaM being involved in the regulation of both systems. It is concluded that the two QS systems regulate a unique set of genes and that RsaM is a global regulator mediating the expression of different genes through the two QS systems as well as genes independently of QS.

The phyllosphere is a complex environment where microbes communicate through signalling molecules in a system, generally known as quorum sensing (QS). One of the most common QS systems in Gram-negative proteobacteria is based on the production of N-acyl homoserine lactones (AHLs) by a LuxI synthase and their perception by a LuxR sensor. Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of the bacterial canker of kiwifruit, colonises plant phyllosphere before penetrating via wounds and natural openings. Since Psa genome encodes three LuxR solos without a cognate LuxI, this bacterium may perceive diffusible signals, but it cannot produce AHLs, displaying a non-canonical QS system. The elucidation of the mechanisms underlying the perception of environmental cues in the phyllosphere by this pathogen and their influence on the onset of pathogenesis are of crucial importance for a long-lasting and sustainable management of the bacterial canker of kiwifruit. Here, we report the ability of Psa to sense its own population density and the presence of surrounding bacteria. Moreover, we show that Psa can perceive AHLs, indicating that AHL-producing neighbouring bacteria may regulate Psa virulence in the host. Our results suggest that the ecological environment is important in determining Psa fitness and pathogenic potential. This opens new perspectives in the use of more advanced biochemical and microbiological tools for the control of bacterial canker of kiwifruit.

Many Burkholderia spp. produce in vitro secondary metabolites with relevant biological activities and potential practical applications. Burkholderia gladioli pv. agaricicola (Bga) possess promising biological activities regulated by N-Acyl homoserine lactones (N.AHLs) based quorum sensing (QS) mechanism. In the current study, N.AHLs-deficient (ICMP11096glad-I) and N.AHLs-complemented (ICMP11096glad-IR) mutants were constructed in which the gene coding for AHL synthase was inactivated by allelic exchange in glad I mutant strain. The aims of this research were to (i) assess the antagonistic activity of the wild type (WT) and the glad-I mutant of Bga against Bacillus megaterium (G+ve) and Escherichia coli (G−ve), (ii) screen their hydrolytic enzymes and hemolytic substances, (iii) monitor the pathogenic effect against Agaricus bisporus, and finally (iv) analyze the bioactive secondary metabolites produced by WT and mutant strain using high performance liquid chromatography (HPLC). Results showed that N.AHLs-deficient mutant exhibited high reduction of antagonistic activity against the tested microorganisms and notable reduction of chitinolytic, proteolytic and glucanolytic activities and complete absence of hemolytic activity, and the glad-IR complemented mutant was able to regain the major part of these activities. Furthermore, N.AHLs-deficient mutant strain was unable to degrade flesh cubes pseudo-tissues of A. bisporus. On the other hand, the virulence effect of complemented mutant was like to the parental WT strain. HPLC analysis revealed that some of the single components produced by WT strain were absent in N.AHLs-deficient mutant and others were highly reduced. The out-findings of the current research gave a spot into the regulatory role of N.AHLs and QS phenomenon in the biological activity of Bga bacterium.

In a number of compatible plant‐bacterium interactions, a rise in apoplastic Ca2+ levels is observed, suggesting that Ca2+ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav ) strain DAPP‐PG 722 is mediated by a Na+/Ca2+ exchanger critical for virulence. Using the fluorescent Ca2+ probe Fura 2‐AM, we demonstrate that Ca2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca2+ entry is a Na+/Ca2+ exchanger. In silico analysis of the Psav DAPP‐PG 722 genome revealed the presence of a single gene coding for a Na+/Ca2+ exchanger (cneA ), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL , hrpA and iaaM ) and virulence (ptz ) genes was reduced in this Psav ‐cneA mutant. Complementation of the Psav ‐cneA mutation restored both Ca2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca2+ entry acts as a ‘host signal’ that allows and promotes Psav pathogenicity on olive plants.

Many plant bacterial pathogens monitor their group behaviour and their population density via production of N-acyl homoserine lactone signals which regulate the expression of several genes via the LuxI/R homologs. This regulatory network, termed quorum sensing (QS), is present in the soybean bacterial pathogen Pseudomonas savastanoi pv glycinea (Psg). The sequenced genomes of two strains of Psg, race 4 and B076, contain an N-acyl homoserine lactone (AHL) based LuxI/R QS system named AhlI/R. While studying the QS system of Psg strains race 4 and B076 isolated in USA, LMG5066 in New Zealand and IBSBF355 in Brazil, we found that B076, LMG5066 and IBSBF355 possess a point mutation in the ahlR gene that causes a frameshift resulting in a truncated AhlR protein. Psg race 4 does not possess the mutation in ahlR and the QS system is functional. The same mutation in the ahlR gene was found to be also present in 9 of 19 Psg strains isolated from diseased soybean in Illinois. Phenotypic analysis of strains showed that swarming motility is repressed whereas phosphate solubilisation was activated by QS in Psg. Analysing the secretome, we also found that four proteins were under QS regulation.

The Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. Although yvjB is highly conserved in the genus Lactococcus , the bacteriocin appears to be active only against the subspecies L. lactis subsp. lactis . Comparative analysis of the YvjB proteins of a sensitive strain (YvjB MN ) and a resistant strain (YvjB MG ) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp 25 ) and terminal alanine (Ala 30 ). It was also shown that the distance between Trp 25 and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr 356 ) and alanine (Ala 353 ). In vitro experiments showed that LsbB could interact with both YvjB MN and YvjB MG , but the strength of interaction is significantly less with YvjB MG . In vivo experiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjB MN . IMPORTANCE The antimicrobial activity of LsbB bacteriocin depends on the correct interaction with the corresponding receptor in the bacterial membrane of sensitive cells. Membrane-located bacteriocin receptors have essential primary functions, such as cell wall synthesis or sugar transport, and it seems that interaction with bacteriocins is suicidal for cells. This study showed that the C-terminal part of LsbB is crucial for the bacteriocin activity, most probably through adequate interaction with the third transmembrane domain of the YvjB receptor. The conserved Tyr 356 and Ala 353 residues of YvjB are essential for the function of this Zn-dependent membrane-located protease as a bacteriocin receptor.

Resolution of bacterial infections is often hampered by both resistance to conventional antibiotic therapy and hiding of bacterial cells inside biofilms, warranting the development of innovative therapeutic strategies. Here, we report the efficacy of blue laser light in eradicating Pseudomonas aeruginosa cells, grown in planktonic state, agar plates and mature biofilms, both in vitro and in vivo, with minimal toxicity to mammalian cells and tissues. Results obtained using knock-out mutants point to oxidative stress as a relevant mechanism by which blue laser light exerts its anti-microbial effect. Finally, the therapeutic potential is confirmed in a mouse model of skin wound infection. Collectively, these data set blue laser phototherapy as an innovative approach to inhibit bacterial growth and biofilm formation, and thus as a realistic treatment option for superinfected wounds.

Sponges harbor a remarkable diversity of microbial symbionts in which signal molecules can accumulate and enable cell-cell communication, such as quorum sensing (QS). Bacteria capable of QS were isolated from marine sponges; however, an extremely small fraction of the sponge microbiome is amenable to cultivation. We took advantage of community genome assembly and binning to investigate the uncultured majority of sponge symbionts. We identified a complete N -acyl-homoserine lactone (AHL)-QS system (designated TswIR) and seven partial luxI homologues in the microbiome of Theonella swinhoei . The TswIR system was novel and shown to be associated with an alphaproteobacterium of the order Rhodobacterales , here termed Rhodobacterales bacterium TS309. The tswI gene, when expressed in Escherichia coli , produced three AHLs, two of which were also identified in a T. swinhoei sponge extract. The taxonomic affiliation of the 16S rRNA of Rhodobacterales bacterium TS309 to a sponge-coral specific clade, its enrichment in sponge versus seawater and marine sediment samples, and the presence of sponge-specific features, such as ankyrin-like domains and tetratricopeptide repeats, indicate a likely symbiotic nature of this bacterium.

Background Pseudomonas syringae pv. actinidiae (PSA) is an emerging kiwifruit bacterial pathogen which since 2008 has caused considerable losses. No quorum sensing (QS) signaling molecule has yet been reported from PSA and the aim of this study was to identify possible intercellular signals produced by PSA. Results A secreted metabolome analysis resulted in the identification of 83 putative compounds, one of them was the nine carbon saturated dicarboxylic acid called azelaic acid. Azelaic acid, which is a nine-carbon (C9) saturated dicarboxylic acid, has been reported in plants as a mobile signal that primes systemic defenses. In addition, its structure,(which is associated with fatty acid biosynthesis) is similar to other known bacterial QS signals like the Diffusible Signal Facor (DSF). For these reason it could be acting as s signal molecule. Analytical and structural studies by NMR spectroscopy confirmed that in PSA spent supernatants azelaic acid was present. Quantification studies further revealed that 20 μg/L of were present and was also found in the spent supernatants of several other P. syringae pathovars. The RNAseq transcriptome study however did not determine whether azelaic acid could behave as a QS molecule. Conclusions This study reports of the possible natural biosynthesis of azelaic acid by bacteria. The production of azelaic acid by P. syringae pathovars can be associated with plant-bacteria signaling. Electronic supplementary material The online version of this article (10.1186/s12866-018-1352-z) contains supplementary material, which is available to authorized users.

Pseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains. Here we show that the mammalian PR-AMP Bac7(1–35) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa . By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1–35) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli . The results evidenced a new mechanism of action for PR-AMPs and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process.

Polyketides and nonribosomal peptides represent two large families of natural products (NPs) with diverse structures and important functions. They are synthesized by polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS), respectively. Lysobacter enzymogenes is emerging as a novel biocontrol agent against pathogens of crop plants and a new source of bioactive NPs, such as antibacterial antibiotic WAP-8294A2 and antifungal antibiotic HSAF. Genome survey of strain OH11, a Chinese L. enzymogenes isolate, detected four novel PKS, NRPS or hybrid gene clusters, designed as cluster A to D. We further individually mutated five genes (PKS or NRPS) located in these four gene clusters, and showed that a PKS gene in cluster A and an NRPS gene in cluster D were involved in the antibacterial activity via a WAP-8294A2 dependent way. The data also showed that none of the five genes was associated with antifungal activity and the regulation of HSAF biosynthesis. Our results reveal the unusual regulatory role of these PKS and NRPS genes that were discovered from genome mining in L. enzymogenes.

Lysobacter enzymogenes is a ubiquitous plant-associated and environmentally friendly bacterium emerging as a novel biological control agent of plant disease. This bacterium produces diverse antifungal factors, such as lytic enzymes and a secondary metabolite (heat-stable antifungal factor [HSAF]) having antifungal activity with a novel structure and mode of action. The regulatory mechanisms for biosynthesis of antifungal factors is largely unknown in L. enzymogenes. The solo LuxR proteins have been shown to be widespread, playing important roles in plant-associated bacteria. Here, we cloned and studied a solo LuxR protein, LesR, from L. enzymogenes strain OH11. Overexpression but not deletion of lesR significantly impaired HSAF biosynthesis levels and antimicrobial activities but did not show visible effect on production of major lytic enzymes. Overexpression of lesR also led to remarkably accelerated cell aggregation and induced production of a melanin-like pigment in L. enzymogenes; these two phenotypes are mediated by the diffusible factor cell-to-cell signaling system of L. enzymogenes. The C-terminus helix-turn-helix domain was shown to be critical for several lesR-controlled functions. Overall, our study provides the first example of the roles and mechanisms of a solo LuxR protein in a plant-associated L. enzymogenes.

Members of the Burkholderia genus of Proteobacteria are capable of living freely in the environment and can also colonize human, animal and plant hosts. Certain members are considered to be clinically important from both medical and veterinary perspectives and furthermore may be important modulators of the rhizosphere. Quorum sensing via N-acyl homoserine lactone signals (AHL QS) is present in almost all Burkholderia species and is thought to play important roles in lifestyle changes such as colonization and niche invasion. Here we present a census of AHL QS genes retrieved from public databases and indicate that the local arrangement (topology) of QS genes, their location within chromosomes and their gene neighborhoods show characteristic patterns that differ between the known Burkholderia clades. In sequence phylogenies, AHL QS genes seem to cluster according to the local gene topology rather than according to the species, which suggests that the basic topology types were present prior to the appearance of current Burkholderia species. The data are available at http://net.icgeb.org/burkholderia/.

Extensive communication is believed to occur between eukaryotes and prokaryotes via signaling molecules; this field of research is now called interkingdom signaling. Recently, it has been discovered that many different plant-associated bacteria possess a protein closely related to the quorum-sensing (QS) LuxR-family protein that binds and responds to plant compounds. This LuxR protein does not have a cognate N -acyl homoserine lactone (AHL) signal synthase and therefore is regarded as a ‘solo' or ‘orphan'. The protein is involved in interkingdom signaling in rhizobia, xanthomonads, and pseudomonads, regulating processes important for plant–bacteria interaction. In this review, we focus on this new interkingdom signaling circuit, which is widespread among pathogenic and beneficial plant-associated bacteria.

The life cycle of bacterial phytopathogens consists of a benign epiphytic phase, during which the bacteria grow in the soil or on the plant surface, and a virulent endophytic phase involving the penetration of host defenses and the colonization of plant tissues. Innovative strategies are urgently required to integrate copper treatments that control the epiphytic phase with complementary tools that control the virulent endophytic phase, thus reducing the quantity of chemicals applied to economically and ecologically acceptable levels. Such strategies include targeted treatments that weaken bacterial pathogens, particularly those inhibiting early infection steps rather than tackling established infections. This chapter describes a reporter gene-based chemical genomic high-throughput screen for the induction of bacterial virulence by plant molecules. Specifically, we describe a chemical genomic screening method to identify agonist and antagonist molecules for the induction of targeted bacterial virulence genes by plant extracts, focusing on the experimental controls required to avoid false positives and thus ensuring the results are reliable and reproducible.

Enzymatic degradation of N-acyl homoserine lactone (NAHL) was found to interfere with the quorum sensing (QS) system and related functions in several soil bacteria. In this research, the NAHL lactonase gene aiiA was amplified using aiiA-7F/aiiA7R PCR primers from the quorum sensing inhibitor rhizobacterium Bacillus sp. strain DMS133, and cloned. The plasmid pME7075, carrying the DMS133 aiiA gene under the constitutive lac promoter, was introduced into the plant pathogen Pectobacterium carotovorum EMPCC, creating strain EMPCC/aiiA. Heterologous expression of the DMS133 aiiA gene in EMPCC severely reduced the accumulation of the NAHL throughout growth, and completely prevented pigmentation of the CV026 bioreporter strain. Virulence analysis revealed that the P. carotovorum strain EMPCC/aiiA expressing AiiA lactonase had drastically reduced tissue maceration activity compared with the wild type EMPCC strain. These results provide evidence that AiiA plays an important role in the quorum quenching ability of Bacillus sp. DMS133 whose AHL degradation capacity was investigated previously. In addition, the communication signal-inactivation approach represents a promising strategy for the prevention of diseases in which virulence is regulated by QS signal molecules.

Several soil bacteria were found to degrade N-Acylhomoserine lactones (NAHLs), thereby interfering with the bacterial quorum sensing system. In this research, fifteen strains of NAHL degrading rhizobacteria were isolated from potato rhizosphere. Based on phenotypic characteristics and 16S rDNA sequence analyses, the strains were identified as members of genera Bacillus, Streptomyces, Arthrobacter, Pseudomonas and Mesorhizobium. All tested isolates were capable to degrade both synthetic and natural NAHL produced by Pectobacterium carotovorum subsp. carotovorum (Pcc) strain EMPCC. In quorum quenching experiments selected isolates, especially Mesorhizobium sp., were markedly reduced the pathogenicity of Pcc strain EMPCC in potato tubers and totally suppressed tissue maceration on potato tubers. These led to consider the latter as a useful biocontrol agent against Pectobacterium spp.

Aims: The presented study was aimed to reveal transcriptional regulation of genes involved in SDS degradation (sdsA and sdsB) in Pseudomonas sp. ATCC19151. In addition, the ability of Pseudomonas sp. ATCC19151 to degrade anionic surfactants present in commercial detergent and septic tank drain was analysed. Methods and Results: Strain ATCC19151, at 30°C, degrades all SDS present in the liquid medium (up to 4% w/v of SDS) within 48 h. ATCC19151 grows in the presence up to 15% (v/v) ‘Fairy’ commercial detergent and mineralizes 35% of present anionic surfactants. Analysis of the sdsA (PsdsA) and divergent sdsB (PsdsB) gene promoter activities revealed that SdsB acts as a positive regulator of sdsA and sdsB transcription. PsdsA and PsdsB activities rose significantly in the presence of the SDS, indicating inducibility of sdsA and sdsB transcription. DNA-binding assay indicated that SdsB directly regulates the transcription of sdsA and sdsB genes. Strain ATCC19151 grew in a sterile septic tank drain and on commercial detergent as sole source of carbon. Conclusions: SdsA enables Pseudomonas sp. ATCC19151 to utilize SDS as a sole carbon source. SdsB is positive transcriptional regulator of sdsA and sdsB genes. Significance and Impact of the Study: Ability of ATCC19151 to degrade anionic surfactants makes Pseudomonas sp. ATCC19151 a good candidate for bioremediation.

Pseudomonas fuscovaginae is a Gram-negative fluorescent pseudomonad pathogenic towards several plant species. Despite its importance as a plant pathogen, no molecular studies of virulence have thus far been reported. In this study we show that P. fuscovaginae possesses two conserved N-acyl homoserine lactone (AHL) quorum sensing (QS) systems which we designated PfsI/R and PfvI/R. The PfsI/R system is homologous to the BviI/R system of Burkholderia vietnamiensis and produces and responds to C10-HSL and C12-HSL whereas PfvI/R is homologous to the LasI/R system of Pseudomonas aeruginosa and produces several long-chain 3-oxo-HSLs and responds to 3-oxo-C10-HSL and 3-oxo-C12-HSL and at high AHL concentrations can also respond to structurally different long-chain AHLs. Both systems were found to be negatively regulated by a repressor protein which was encoded by a gene located intergenically between the AHL synthase and LuxR-family response regulator. The pfsI/R system was regulated by a novel repressor designated RsaM while the pfvI/R system was regulated by both the RsaL repressor and by RsaM. The two systems are not transcriptionally hierarchically organized but share a common AHL response and both are required for plant virulence. Pseudomonas fuscovaginae has therefore a unique complex regulatory network composed of at least two different repressors which directly regulate the AHL QS systems and pathogenicity.

The genus Burkholderia includes over 60 species isolated from a wide range of environmental niches and can be tentatively divided into two major species clusters. The first cluster includes pathogens such as Burkholderia glumae , B. pseudomallei , and B. mallei and 17 well-studied species of the Burkholderia cepacia complex. The other recently established cluster comprises at least 29 nonpathogenic species, which in most cases have been found to be associated with plants. It was previously established that Burkholderia kururiensis , a member of the latter cluster, possesses an N -acyl homoserine lactone (AHL) quorum-sensing (QS) system designated “BraI/R,” which is found in all species of the plant-associated cluster. In the present study, two other BraI/R-like systems were characterized in B. xenovorans and B. unamae and were designated the BraI/R XEN and BraI/R UNA systems, respectively. Several phenotypes were analyzed, and it was determined that exopolysaccharide was positively regulated by the BraIR-like system in the species B. kururiensis , B. unamae , and B. xenovorans , highlighting commonality in targets. However, the three BraIR-like systems also revealed differences in targets since biofilm formation and plant colonization were differentially regulated. In addition, a second AHL QS system designated XenI2/R2 and an unpaired LuxR solo protein designated BxeR solo were also identified and characterized in B. xenovorans LB400 T . The two AHL QS systems of B. xenovorans are not transcriptionally regulating each other, whereas BxeR solo negatively regulated xenI2 . The XenI2/R2 and BxeR solo proteins are not widespread in the Burkholderia species cluster. In conclusion, the present study represents an extensive analysis of AHL QS in the Burkholderia plant-associated cluster demonstrating both commonalities and differences, probably reflecting environmental adaptations of the various species.

Chorismate mutase (CM) is a key enzyme in the shikimate pathway which is responsible for the synthesis of aromatic amino acids. There are two classes of CMs, AroQ and AroH, and several pathogenic bacteria have been reported to possess a subgroup of CMs designated AroQγ. These CMs are usually exported to the periplasm or outside the cell; in a few cases, they have been reported to be involved in virulence and their precise role is currently unknown. Here, we report that the important rice pathogen Xanthomonas oryzae pv. oryzae XKK.12 produces an AroQγ CM which we have purified and characterized from spent supernatants. This enzyme is synthesized in planta and X. oryzae pv. oryzae knock-out mutants are hypervirulent to rice. The role of this enzyme in X. oryzae pv. oryzae rice virulence is discussed.

Burkholderia glumae is an emerging seed-borne rice pathogen in many areas around the world. Previous studies have demonstrated that B. glumae produces two major virulence factors: the phytotoxin toxoflavin and a secreted lipase. This synthesis of both of these factors is regulated by an N-acyl homoserine lactone (AHL)-dependent, cell-density-dependent quorum-sensing regulation system. This study reports the production and secretion of two highly similar endo-polygalacturonases (designated PehA and PehB) by B. glumae. The two enzymes were purified to homogeneity and the corresponding genetic determinants were identified and characterized. When either polygalacturonase gene was inactivated, B. glumae retained rice virulence comparable to that of the wild-type parent strain. Furthermore, the role of AHL-dependent quorum sensing and of plant cell wall degradation compounds in their regulation was investigated.

The sigmas subunit of RNA polymerase is a central regulator which governs the expression of a host of stationary phase-induced and osmotically regulated genes in Gram-negative bacteria. The Pseudomonas putida rpoS gene is transcribed as a monocistronic rpoS mRNA with a 368 nucleotide-long 5' untranslated region (5' UTR). In this study, we investigate the posttranscriptional control of RpoS synthesis using rpoS-lacZ transcriptional and translational fusions consisting of the native promoter and deletions of 5' UTR or insertion into UTR. The differing activity of constructed translational fusions strongly indicated that the 5' UTR is involved in the translational regulation of RpoS expression in the stationary phase. The results obtained herein demonstrated that the structure of UTR performs an important function in the translational regulation of the rpoS gene.

Summary The quorum sensing (QS) signalling system of Pseudomonas aeruginosa controls many important functions, including virulence. Although the production of the QS signal molecule N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) is positively autoregulated, its concentration reaches a steady level long before stationary phase. The RsaL protein represses transcription of the lasI signal synthase gene, and thus reduces QS signal production. We show that RsaL binds simultaneously with LasR to the rsaL-lasI bidirectional promoter thereby preventing the LasR-dependent activation of both genes. In an rsaL mutant, 3OC12-HSL production continues to increase throughout growth. Thus RsaL provides homeostasis by functioning in opposition to LasR and limiting 3OC12-HSL production to a physiological concentration. Furthermore, transcription profiling revealed that RsaL regulates 130 genes independent of its effect on QS signal molecule production, including genes involved in virulence. We show that RsaL can repress pyocyanin and hydrogen cyanide virulence genes in two ways: directly, by binding to their promoters, and indirectly, by decreasing levels of the signals for their QS signal-dependent transcription. These investigations highlight the importance of RsaL as a global regulator of P. aeruginosa physiology that provides a counterbalance to 3OC12-HSL-dependent gene activation via multiple mechanisms.

Aims: Isolation and characterization of chitinases from a halotolerant Bacillus pumilus. Methods and Results: Bacillus pumilus strain SG2 was isolated from saline conditions. It is able to produce chitinase activity at high salt concentration. SDS-PAGE analysis of the B. pumilus SG2 culture supernatant showed two major bands that were induced by chitin. The amino acid sequence of the two proteins, designated ChiS and ChiL, showed a high homology with the chitinase of B. subtilis CHU26, and chitinase A of B. licheniformis, respectively. N-terminal signal peptide of both proteins was also determined. The molecular weight and isoelectric point of the chitinases were determined to be 63 and 74 kDa, and 4·5 and 5·1, for ChiS and ChiL respectively. The genes encoding for both chitinases were isolated and their sequence determined. The regulation of the chitinase genes is under the control of the catabolite repression system. Conclusions: Secreted chitinase genes and their flanking region on the genome of B. pumilus SG2 have been identified and sequenced. Significance and Impact of the Study: This is the first report of a multiple chitinases-producing B. pumilus halotolerant strain. We have identified two chitinases by using a reverse genetics approach. The chitinases show resistance to salt.

Pseudomonas corrugata is a phytopathogenic bacterium, causal agent of tomato pith necrosis, yet it is an ubiquitous bacterium that is part of the microbial community in the soil and in the rhizosphere of different plant species. Although it is a very heterogeneous species, all the strains tested were able to produce short chain acyl homoserine lactone (AHL) quorum sensing signal molecules. The main AHL produced was N-hexanoyl-l-homoserine lactone (C6-AHL). An AHL quorum sensing system, designated PcoI/PcoR, was identified and characterized. The role of the quorum sensing system in the expression of a variety of traits was evaluated. Inactivation of pcoI abolished the production of AHLs. The pcoR mutant, but not the pcoI mutant, was impaired in swarming, unable to cause a hypersensitivity response on tobacco and resulted in a reduced tomato pith necrosis phenotype. The pcoI mutant showed a reduced antimicrobial activity against various fungi and bacteria when assayed on King's B medium. These results demonstrate that the AHL quorum sensing in Ps. corrugata regulates traits that contribute to virulence, antimicrobial activity and fitness. This is the first report of genes of Ps. corrugata involved in the disease development and biological control activity.

Burkholderia glumae is an emerging rice pathogen in several areas around the world. Closely related Burkholderia species are important opportunistic human pathogens for specific groups of patients, such as patients with cystic fibrosis and patients with chronic granulomatous disease. Here we report that the first clinical isolate of B. glumae , strain AU6208, has retained its capability to be very pathogenic to rice. As previously reported for rice isolate B. glumae BGR1 (and also for the clinical isolate AU6208), TofI or TofR acyl homoserine lactone (AHL) quorum sensing played a pivotal role in rice virulence. We report that AHL quorum sensing in B. glumae AU6208 regulates secreted LipA lipase and toxoflavin, the phytotoxin produced by B. glumae. B. glumae AU6208 lipA mutants were no longer pathogenic to rice, indicating that the lipase is an important virulence factor. It was also established that type strain B. glumae ATCC 33617 did not produce toxoflavin and lipase and was nonpathogenic to rice. It was determined that in strain ATCC 33617 the LuxR family quorum-sensing sensor/regulator TofR was inactive. Introducing the tofR gene of B. glumae AU6208 in strain ATCC 33617 restored its ability to produce toxoflavin and the LipA lipase. This study extends the role of AHL quorum sensing in rice pathogenicity through the regulation of a lipase which was demonstrated to be a virulence factor. It is the first report of a clinical B. glumae isolate retaining strong rice pathogenicity and finally determined that B. glumae can undergo phenotypic conversion through a spontaneous mutation in the tofR regulator.

Endo-polygalacturonases (endoPGs) belong to the glycoside hydrolase family 28 and hydrolyze the alpha-1,4 glycosidic bond present in the smooth regions of pectins. Pectic substances are among the principal macromolecular components of the primary plant cell walls and are subjected to enzymatic degradation not only in the course of important physiological processes such as plant senescence and ripening, but also during infection events by plant pathogens. Here we report, for the first time, the isolation and the purification of an endoPG (PehA) from the supernatant of the plant pathogen Burkholderia cepacia strain ATCC 25416. In order to obtain adequate amounts of protein required for structural and functional studies, the gene coding for pehA was PCR-amplified and cloned in Escherichia coli cells. The recombinant protein was purified to homogeneity and characterized. PehA exhibited a pI value of 8.0 and an optimal activity at pH 3.5. Far-UV circular dichroism (CD) measurements show that PehA assumes a beta-helix fold super-secondary structural motif.

Many Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum-sensing (QS) signal molecules. AHL QS has been the subject of extensive investigation in the last decade and has become a paradigm for bacterial intercellular signaling. Research in AHL QS has been considerably aided by simple methods devised to detect AHLs using bacterial biosensors that phenotypically respond when exposed to exogenous AHLs. This article reviews and discusses the currently available bacterial biosensors which can be used in detecting and studying the different AHLs.

Burkholderia plantarii is a plant pathogen responsible for causing rice seedling blight. The molecular mechanisms responsible for this pathogenicity are currently unknown. In this study, we report the identification and characterization of N-acyl homoserine lactone quorum sensing and the stationary phase RpoS sigma factor of B. plantarii. Both global regulatory systems are involved in causing rice seedling blight. This is the first report of gene regulators of B. plantarii implicated in the disease.

We purified to homogeneity an intracellular esterase from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The enzyme hydrolyzes p-nitrophenyl acetate and other acetylated substrates. The N-terminal amino acid sequence was analyzed and 11 residues, SEPLILDAPNA, were determined. The corresponding gene PA3859 was identified in the P. aeruginosa PAO1 genome as the only gene encoding for a protein with this N-terminus. The encoding gene was cloned in Escherichia coli, and the recombinant protein expressed and purified to homogeneity. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and analytical gel filtration chromatography, the esterase was found to be a monomer of approximately 24 kDa. The experimentally determined isoelectric point was 5.2 and the optimal enzyme activity was at 55 degrees C and at pH 9.0. The esterase preferentially hydrolyzed short-chain fatty acids. It is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by ethylendiaminotetraacetic acid (EDTA). Native enzyme preparations typically showed a Michaelis constant (K(m)) and V(max) of 0.43 mM and 12,500 U mg(-1), respectively, using p-nitrophenyl acetate as substrate. Homology-based database searches clearly revealed the presence of the consensus GXSXG signature motif that is present in the serine-dependent acylhydrolase protein family.

We report for the first time the production of acyl homoserine lactones (AHLs) by Erwina amylovora, an important quarantine bacterial pathogen that causes fire blight in plants. E. amylovora produces one N-acyl homoserine lactone [a N-(3-oxo-hexanoyl)-homoserine lactone or a N-(3-hydroxy-hexanoyl)-homoserine lactone] quorum sensing signal molecule both in vitro and in planta (pear plant). Given the involvement of AHLs in plant pathogenesis, we speculate that AHL-dependent quorum sensing could play an important role in the regulation of E. amylovora virulence.

The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa , to cause infection. Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking relA exhibited reduced virulence in a D. melanogaster feeding assay. In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N -[3-oxododecanoyl]- l -homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of relA also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P. aeruginosa adaptation in pathogenic relationships.

Quorum sensing is a cell population-density dependent regulatory system which in gram-negative bacteria often involves the production and detection of N -acyl homoserine lactones (AHLs). Some Pseudomonas putida strains have been reported to produce AHLs, and one quorum-sensing locus has been identified. However, it appears that the majority of strains do not produce AHLs. In this study we report the identification and regulation of the AHL-dependent system of rhizosphere P. putida WCS358. This system is identical to the recently identified system of P. putida strain IsoF and very similar to the las system of Pseudomonas aeruginosa . It is composed of three genes, the luxI family member ppuI , the putative repressor rsaL , and the luxR family member ppuR . A genomic ppuR ::Tn 5 mutant of strain WCS358 was identified by its inability to produce AHLs when it was cross-streaked in close proximity to an AHL biosensor, whereas an rsaL ::Tn 5 genomic mutant was identified by its ability to overproduce AHL molecules. Using transcriptional promoter fusions, we studied expression profiles of the rsaL , ppuI , and ppuR promoters in various genetic backgrounds. At the onset of the stationary phase, the autoinducer synthase ppuI gene expression is under positive regulation by PpuR-AHL and under negative regulation by RsaL, indicating that the molecules could be in competition for binding at the ppuI promoter. In genomic rsaL ::Tn 5 mutants ppuI expression and production of AHL levels increased dramatically; however, both processes were still under growth phase regulation, indicating that RsaL is not involved in repressing AHL production at low cell densities. The roles of the global response regulator GacA and the stationary-phase sigma factor RpoS in the regulation of the AHL system at the onset of the stationary phase were also investigated. The P. putida WCS358 gacA gene was cloned and inactivated in the genome. It was determined that the three global regulatory systems are closely linked, with quorum sensing and RpoS regulating each other and GacA positively regulating ppuI expression. Studies of the regulation of AHL quorum-sensing systems have lagged behind other studies and are important for understanding how these systems are integrated into the overall growth phase and metabolic status of the cells.

In Escherichia coli, the stationary phase alternative sigma factor sigmas controls the expression of genes involved cell survival in response to cessation of growth (stationary phase) and provides cross-protection to various stresses. Levels of sigmas increase dramatically at the onset of stationary phase and are regulated at the transcriptional, post-transcriptional and post-translational level, making this one of the most complex regulatory systems in bacteria. The basic mechanisms for the control of translation and sigmas proteolysis have been understood. However, studies on the transcriptional control in E. coli lag behind and are controversial. The cAMP-CRP complex and the two component BarA/UvrY system have been implicated and, ppGpp and polyphosphate appear to have a signalling role. sigmas has also been reported to be a general stress regulator in the fluorescent pseudomonads (Pseudomonas aeruginosa, P. fluorescens and P. putida) and recent studies on sigmas regulation highlight that transcriptional regulation in these bacteria apparently plays a major role. Global regulatory systems, the GacA/GacS two component system and quorum sensing all affect rpoS expression, as does the TetR family PsrA regulator that directly binds to- and activates the rpoS promoter in stationary phase. This striking difference in regulation between E. coli and Pseudomonas can be partly attributed to the differences in the functional role of sigmas in the two bacterial species. This report will review mainly recent studies on rpoS transcriptional regulation and will try to rationalize the current knowledge into a working model.

Bacterial strains belonging to Burkholderia cepacia can be human opportunistic pathogens, plant pathogens, and plant growth promoting and have remarkable catabolic activity. B. cepacia consists of several genomovars comprising what is now known as the B. cepacia complex. Here we report the quorum-sensing system of a genomovar I onion rot type strain ATCC 25416. Quorum sensing is a cell-density-dependent regulatory response which involves the production of N -acyl homoserine lactone (HSL) signal molecules. The cep locus has been inactivated in the chromosome, and it has been shown that CepI is responsible for the biosynthesis of an N -hexanoyl HSL (C 6 -HSL) and an N -octanoyl HSL (C 8 -HSL) and that the cep locus regulates protease production as well as onion pathogenicity via the expression of a secreted polygalacturonase. A cep - lacZ -based sensor plasmid has been constructed and used to demonstrate that CepR responded to C 6 -HSL with only 15% of the molar efficiency of C 8 -HSL, that a cepR knockout mutant synthesized 70% less HSLs, and that CepR responded best towards long-chain HSLs. In addition, we also report the cloning and characterization of the stationary-phase sigma factor gene rpoS of B. cepacia ATCC 25416. It was established that quorum sensing in B. cepacia has a negative effect on rpoS expression as determined by using an rpoS - lacZ transcriptional fusion; on the other hand, rpoS -null mutants displayed no difference in the accumulation of HSL signal molecules.

Bacillus pumilus PS213 secretes an alpha-L-arabinofuranosidase (AF) when grown in the presence of arabinogalactan or oat meal. The enzyme has been purified to homogeneity and characterised. Its molecular mass, as determined by gel filtration, is 220 kDa, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band of approximately 60 kDa. According to the result of the mass spectrometry analysis showing a molecular mass of 56 kDa, the enzyme should be a homotetramer. The isoelectric point was found to be 5.2, the enzyme activity was optimal at 55 degrees C and pH 7.0. The enzyme retained 80% of its activity after 2 h at 65 degrees C and lost 50% of activity at 75 degrees C after 135 min. The Michaelis constant K(m) and V(max) for p-nitrophenylarabinofuranoside at 37 degrees C were 1.7 mM and 52.9 U mg(-1), respectively. N-terminal sequence analysis and internal peptide fragments showed homology with glycosyl hydrolases of family 51.

Background Small-molecule hormones are well known to play key roles in the plant immune signaling network that is activated upon pathogen perception. In contrast, little is known about whether phytohormones also directly influence microbial virulence, similar to what has been reported in animal systems. Results In this paper, we tested the hypothesis that hormones fulfill dual roles in plant-microbe interactions by orchestrating host immune responses, on the one hand, and modulating microbial virulence traits, on the other. Employing the rice-Xanthomonas oryzae pv. oryzae (Xoo) interaction as a model system, we show that Xoo uses the classic immune hormone salicylic acid (SA) as a trigger to activate its virulence-associated quorum sensing (QS) machinery. Despite repressing swimming motility, sodium salicylate (NaSA) induced production of the Diffusible Signal Factor (DSF) and Diffusible Factor (DF) QS signals, with resultant accumulation of xanthomonadin and extracellular polysaccharides. In contrast, abscisic acid (ABA), which favors infection by Xoo, had little impact on DF- and DSF-mediated QS, but promoted bacterial swimming via the LuxR solo protein OryR. Moreover, we found both DF and DSF to influence SA- and ABA-responsive gene expression in planta. Conclusions Together our findings indicate that the rice SA and ABA signaling pathways cross-communicate with the Xoo DF and DSF QS systems and underscore the importance of bidirectional interkingdom signaling in molding plant-microbe interactions. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0411-3) contains supplementary material, which is available to authorized users.

The rpoS gene which encodes a stationary phase sigma factor has been identified and characterised from the rhizosphere-colonising plant growth-promoting Pseudomonas putida strain WCS358. The predicted protein sequence has extensive homologies with the RpoS proteins form other bacteria, in particular with the RpoS sigma factors of the fluorescent pseudomonads. A genomic transposon insertion in the rpoS gene was constructed, these mutants were analysed for their ability to produce siderophore (iron-transport agent) and the autoinducer quorum-sensing molecules called homoserine lactones (AHL). It was determined that RpoS was not involved in the regulation of siderophore and AHL production, synthesis of these molecules is important for gene expression at stationary phase. P. putida WCS358 produces at least three different AHL molecules.

Eight luxI-family gene promoters (luxI, cviI, ahlI, rhlI, cepI, phzI, traI and ppuI) were cloned in tandem, upstream a promoterless lacZ gene in a promoter probe vector yielding pMULTIAHLPROM. This unique construct is useful in determining whether a bacterial strain not producing N-acyl homoserine lactone signal molecules (AHLs) possesses orphan LuxR type proteins able to respond to AHLs and activate transcription from quorum sensing target genes. Using pMULTIAHLPROM, it was demonstrated that Enterobacter aerogenes possibly contains a LuxR-family orphan able to activate luxI-family promoters independently from AHLs.

Recombinants Pseudomonas putida BO14 and Escherichia coli QEFCA8 capable of ferulic acid biotransformation to vanillin were constructed using homologous recombination and a PCR based cloning strategy, respectively. In the liquid culture of P. putida BO14, 26.81+/-2.30 microg vanillin ml(-1) of culture filtrate was detected. In the case of recombinant E. coli QEFCA8, 19.37+/-1.95 microg vanillin ml(-1) of culture filtrate was detected. Results indicate that the strains could be useful for the biotechnological production of vanillin, a very important flavoring substance.

Bacillus pumilusPS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (Km) andVmaxfor α-naphthyl acetate were 1.54 mM and 360 μmol min−1mg of protein−1, respectively.

Pseudomonas syringae pv. actinidiae (Psa) is an emerging phytopathogen causing bacterial canker disease in kiwifruit plants worldwide. Quorum sensing (QS) gene regulation plays important roles in many different bacterial plant pathogens. In this study we analyzed the presence and possible role of N-acyl homoserine lactone (AHL) quorum sensing in Psa. It was established that Psa does not produce AHLs and that a typical complete LuxI/R QS system is absent in Psa strains. Psa however possesses three putative luxR solos designated here as PsaR1, PsaR2 and PsaR3. PsaR2 belongs to the sub-family of LuxR solos present in many plant associated bacteria (PAB) that binds and responds to yet unknown plant signal molecules. PsaR1 and PsaR3 are highly similar to LuxRs which bind AHLs and are part of the canonical LuxI/R AHL QS systems. Mutation in all the three luxR solos of Psa showed reduction of in planta survival and also showed additive effect if more than one solo was inactivated in double mutants. Gene promoter analysis revealed that the three solos are not auto-regulated and investigated their possible role in several bacterial phenotypes.

Lysobacter enzymogenes is a ubiquitous environmental bacterium that is emerging as a potentially novel biological control agent and a new source of bioactive secondary metabolites, such as the heat-stable antifungal factor (HSAF) and photoprotective polyene pigments. Thus far, the regulatory mechanism(s) for biosynthesis of these bioactive secondary metabolites remains largely unknown in L. enzymogenes . In the present study, the diffusible signal factor (DSF) and diffusible factor (DF)-mediated cell-cell signaling systems were identified for the first time from L. enzymogenes . The results show that both Rpf/DSF and DF signaling systems played critical roles in modulating HSAF biosynthesis in L. enzymogenes . Rpf/DSF signaling and DF signaling played negative and positive effects in polyene pigment production, respectively, with DF playing a more important role in regulating this phenotype. Interestingly, only Rpf/DSF, but not the DF signaling system, regulated colony morphology of L. enzymgenes . Both Rpf/DSF and DF signaling systems were involved in the modulation of expression of genes with diverse functions in L. enzymogenes , and their own regulons exhibited only a few loci that were regulated by both systems. These findings unveil for the first time new roles of the Rpf/DSF and DF signaling systems in secondary metabolite biosynthesis of L. enzymogenes .

Marine natural products with antibiotic activity have been a rich source of drug discovery; however, the emergence of antibiotic-resistant bacterial strains has turned attention towards the discovery of alternative innovative strategies to combat pathogens. In many pathogenic bacteria, the expression of virulence factors is under the regulation of quorum sensing (QS). QS inhibitors (QSIs) present a promising alternative or potential synergistic treatment since they disrupt the signaling pathway used for intra- and interspecies coordination of expression of virulence factors. This review covers the set of molecules showing QSI activity that were isolated from marine organisms, including plants (algae), animals (sponges, cnidarians, and bryozoans), and microorganisms (bacteria, fungi, and cyanobacteria). The compounds found and the methods used for their isolation are the emphasis of this review.

Recent studies have identified a novel interkingdom signaling circuit, via plant signaling molecules, and a bacterial sub-family of LuxR proteins, bridging eukaryotes and prokaryotes. Indeed pivotal plant-bacteria interactions are regulated by the so called Plant Associated Bacteria (PAB) LuxR solo regulators that, although closely related to the quorum sensing (QS) LuxR family, do not bind or respond to canonical quorum sensing N-acyl homoserine lactones (AHLs), but only to specific host plant signal molecules. The large body of structural data available for several members of the QS LuxR family complexed with different classes of ligands (AHLs and other compounds), has been exploited to dissect the cartography of their regulatory domains through structure-based multiple sequence alignments, structural superimposition and a comparative analysis of the contact residues involved in ligand binding. In the absence of experimentally determined structures of members of the PAB LuxR solos subfamily, an homology model of its prototype OryR is presented, aiming to elucidate the architecture of its ligand-binding site. The obtained model, in combination with the cartography of the regulatory domains of the homologous QS LuxRs, provides novel insights into the 3D structure of its ligand-binding site and unveils the probable molecular determinants responsible for differences in selectivity towards specific host plant signal molecules, rather than to canonical QS compounds.