Effectiveness of Pseudomonas aeruginosa type VI secretion system relies on toxin potency and type IV pili-dependent interaction.

The type VI secretion system (T6SS) is an antibacterial weapon that is used by numerous Gram-negative bacteria to gain competitive advantage by injecting toxins into adjacent prey cells. Predicting the outcome of a T6SS-dependent competition is not only reliant on presence-absence of the system but instead involves a multiplicity of factors. Pseudomonas aeruginosa possesses 3 distinct T6SSs and a set of more than 20 toxic effectors with diverse functions including disruption of cell wall integrity, degradation of nucleic acids or metabolic impairment. We generated a comprehensive collection of mutants with various degrees of T6SS activity and/or sensitivity to each individual T6SS toxin. By imaging whole mixed bacterial macrocolonies, we then investigated how these P. aeruginosa strains gain a competitive edge in multiple attacker/prey combinations. We observed that the potency of single T6SS toxin varies significantly from one another as measured by monitoring the community structure, with some toxins acting better in synergy or requiring a higher payload. Remarkably the degree of intermixing between preys and attackers is also key to the competition outcome and is driven by the frequency of contact as well as the ability of the prey to move away from the attacker using type IV pili-dependent twitching motility. Finally, we implemented a computational model to better understand how changes in T6SS firing behaviours or cell-cell contacts lead to population level competitive advantages, thus providing conceptual insight applicable to all types of contact-based competition.

Carbon starvation of Pseudomonas aeruginosa biofilms selects for dispersal insensitive mutants.

BACKGROUND: Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation. RESULTS: In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain. CONCLUSIONS: This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation.

Thioether-linked dihydropyrrol-2-one analogues as PqsR antagonists against antibiotic resistant Pseudomonas aeruginosa.

The Pseudomonas quinolone system (pqs) is one of the key quorum sensing systems in antibiotic-resistant P. aeruginosa and is responsible for the production of virulence factors and biofilm formation. Thus, synthetic small molecules that can target the PqsR (MvfR) receptor can be utilized as quorum sensing inhibitors to treat P. aeruginosa infections. In this study, we report the synthesis of novel thioether-linked dihydropyrrol-2-one (DHP) analogues as PqsR antagonists. Compound 7g containing a 2-mercaptopyridyl linkage effectively inhibited the pqs system with an IC50 of 32 µM in P. aeruginosa PAO1. Additionally, these inhibitors significantly reduced bacterial aggregation and biofilm formation without affecting planktonic growth. The molecular docking study suggest that these inhibitors bind with the ligand binding domain of the MvfR as a competitive antagonist.

The afc antifungal activity cluster, which is under tight regulatory control of ShvR, is essential for transition from intracellular persistence of Burkholderia cenocepacia to acute pro-inflammatory infection.

The opportunistic pathogen Burkholderia cenocepacia is particularly life-threatening for cystic fibrosis (CF) patients. Chronic lung infections with these bacteria can rapidly develop into fatal pulmonary necrosis and septicaemia. We have recently shown that macrophages are a critical site for replication of B. cenocepacia K56-2 and the induction of fatal pro-inflammatory responses using a zebrafish infection model. Here, we show that ShvR, a LysR-type transcriptional regulator that is important for biofilm formation, rough colony morphotype and inflammation in a rat lung infection model, is also required for the induction of fatal pro-inflammatory responses in zebrafish larvae. ShvR was not essential, however, for bacterial survival and replication in macrophages. Temporal, rhamnose-induced restoration of shvR expression in the shvR mutant during intramacrophage stages unequivocally demonstrated a key role for ShvR in transition from intracellular persistence to acute fatal pro-inflammatory disease. ShvR has been previously shown to tightly control the expression of the adjacent afc gene cluster, which specifies the synthesis of a lipopeptide with antifungal activity. Mutation of afcE, encoding an acyl-CoA dehydrogenase, has been shown to give similar phenotypes as the shvR mutant. We found that, like shvR, afcE is also critical for the switch from intracellular persistence to fatal infection in zebrafish. The closely related B. cenocepacia H111 has been shown to be less virulent than K56-2 in several infection models, including Galleria mellonella and rats. Interestingly, constitutive expression of shvR in H111 increased virulence in zebrafish larvae to almost K56-2 levels in a manner that absolutely required afc. These data confirm a critical role for afc in acute virulence caused by B. cenocepacia that depends on strain-specific regulatory control by ShvR. We propose that ShvR and AFC are important virulence factors of the more virulent Bcc species, either through pro-inflammatory effects of the lipopeptide AFC, or through AFC-dependent membrane properties.

Design, Synthesis and Biological Evaluation of Novel Anthraniloyl-AMP Mimics as PQS Biosynthesis Inhibitors Against Pseudomonas aeruginosa Resistance.

The Pseudomonas quinolone system (PQS) is one of the three major interconnected quorum sensing signaling systems in Pseudomonas aeruginosa. The virulence factors PQS and HHQ activate the transcription regulator PqsR (MvfR), which controls several activities in bacteria, including biofilm formation and upregulation of PQS biosynthesis. The enzyme anthraniloyl-CoA synthetase (PqsA) catalyzes the first and critical step in the biosynthesis of quinolones; therefore, it is an attractive target for the development of anti-virulence therapeutics against Pseudomonas resistance. Herein, we report the design and synthesis of novel triazole nucleoside-based anthraniloyl- adenosine monophosphate (AMP) mimics. These analogues had a major impact on the morphology of bacterial biofilms and caused significant reduction in bacterial aggregation and population density. However, the compounds showed only limited inhibition of PQS and did not exhibit any effect on pyocyanin production.

Cinnamaldehyde disrupts biofilm formation and swarming motility of Pseudomonas aeruginosa.

Bacterial biofilms can cause serious health care complications associated with increased morbidity and mortality. There is an urge to discover and develop new biofilm inhibitors from natural products or by modifying natural compounds or understanding the modes of action of existing compounds. Cinnamaldehyde (CAD), one of the major components of cinnamon oil, has been demonstrated to act as an antimicrobial agent against a number of Gram-negative and Gram-positive pathogens, including Pseudomonas aeruginosa, Helicobacter pylori and Listeria monocytogenes. Despite the mechanism of action of CAD against the model organism P. aeruginosa being undefined, based on its antimicrobial properties, we hypothesized that it may disrupt preformed biofilms of P. aeruginosa. The minimum inhibitory concentration (MIC) of CAD for planktonic P. aeruginosa was determined to be 11.8 mM. Membrane depolarization assays demonstrated disruption of the transmembrane potential of P. aeruginosa. CAD at 5.9 mM (0.5 MIC) disrupted preformed biofilms by 75.6 % and 3 mM CAD (0.25 MIC) reduced the intracellular concentrations of the secondary messenger, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), which controls P. aeruginosa biofilm formation. The swarming motility of P. aeruginosa was also reduced by CAD in a concentration-dependent manner. Collectively, these findings show that sub-MICs of CAD can disrupt biofilms and other surface colonization phenotypes through the modulation of intracellular signalling processes.

Identification of Loci of Pseudomonas syringae pv. actinidiae Involved in Lipolytic Activity and Their Role in Colonization of Kiwifruit Leaves.

Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae, an emerging pathogen of kiwifruit plants, has recently brought about major economic losses worldwide. Genetic studies on virulence functions of P. syringae pv. actinidiae have not yet been reported and there is little experimental data regarding bacterial genes involved in pathogenesis. In this study, we performed a genetic screen in order to identify transposon mutants altered in the lipolytic activity because it is known that mechanisms of regulation, production, and secretion of enzymes often play crucial roles in virulence of plant pathogens. We aimed to identify the set of secretion and global regulatory loci that control lipolytic activity and also play important roles in in planta fitness. Our screen for altered lipolytic activity phenotype identified a total of 58 Tn5 transposon mutants. Mapping all these Tn5 mutants revealed that the transposons were inserted in genes that play roles in cell division, chemotaxis, metabolism, movement, recombination, regulation, signal transduction, and transport as well as a few unknown functions. Several of these identified P. syringae pv. actinidiae Tn5 mutants, notably the functions affected in phosphomannomutase AlgC, lipid A biosynthesis acyltransferase, glutamate-cysteine ligase, and the type IV pilus protein PilI, were also found affected in in planta survival and/or growth in kiwifruit plants. The results of the genetic screen and identification of novel loci involved in in planta fitness of P. syringae pv. actinidiae are presented and discussed.

Functionality of chimeric TssA proteins in the type VI secretion system reveals sheath docking specificity within their N-terminal domains.

The genome of Pseudomonas aeruginosa encodes three type VI secretion systems, each comprising a dozen distinct proteins, which deliver toxins upon T6SS sheath contraction. The least conserved T6SS component, TssA, has variations in size which influence domain organisation and structure. Here we show that the TssA Nt1 domain interacts directly with the sheath in a specific manner, while the C-terminus is essential for oligomerisation. We built chimeric TssA proteins by swapping C-termini and showed that these can be functional even when made of domains from different TssA sub-groups. Functional specificity requires the Nt1 domain, while the origin of the C-terminal domain is more permissive for T6SS function. We identify two regions in short TssA proteins, loop and hairpin, that contribute to sheath binding. We propose a docking mechanism of TssA proteins with the sheath, and a model for how sheath assembly is coordinated by TssA proteins from this position.

Role of Burkholderia cenocepacia afcE and afcF genes in determining lipid-metabolism-associated phenotypes.

Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis patients. Previously we have reported that mutations in shvR, a LysR-type transcriptional regulator, and ShvR-regulated genes BCAS0208 and BCAS0201 (designated afcE and afcF, respectively) affect colony morphotype, biofilm and pellicle formation and virulence in B. cenocepacia. In this study we investigated the role of afcE and afcF in influencing lipid-metabolism-associated phenotypes. As previously reported for K56-2ΔshvR, the Δ2afcE and afcF : : lux mutants had no antifungal activity against Fusarium and Rhizoctonia solani, suggesting that these genes are involved in synthesis of a membrane-associated antifungal lipopeptide. Strains Δ2afcE and afcF : : lux had reduced swarming motility and altered cell membrane morphology, both of which were restored to wild-type levels upon providing these genes in trans. Both K56-2ΔshvR and Δ2afcE showed increased uptake of the hydrophobic fluorescent probe N-phenylnaphthylamine (NPN), indicating altered outer membrane properties. Total lipid profiles determined by TLC revealed distinct differences in cellular lipid compositions of K56-2ΔshvR, Δ2afcE and afcF : : lux compared with K56-2. Taken together, these results indicate that afcE and afcF are involved in metabolic pathway(s) influencing lipid profiles and affect both cell surface and antifungal properties of B. cenocepacia.

Burkholderia cenocepacia ShvR-regulated genes that influence colony morphology, biofilm formation, and virulence.

Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis (CF) patients. Previously, we reported that ShvR, a LysR regulator, influences colony morphology, virulence, and biofilm formation and regulates the expression of an adjacent 24-kb genomic region encoding 24 genes. In this study, we report the functional characterization of selected genes in this region. A Tn5 mutant with shiny colony morphology was identified with a polar mutation in BCAS0208, predicted to encode an acyl-coenzyme A dehydrogenase. Mutagenesis of BCAS0208 and complementation analyses revealed that BCAS0208 is required for rough colony morphology, biofilm formation, and virulence on alfalfa seedlings. It was not possible to complement with BCAS0208 containing a mutation in the catalytic site. BCAS0201, encoding a putative flavin adenine dinucleotide (FAD)-dependent oxidoreductase, and BCAS0207, encoding a putative citrate synthase, do not influence colony morphology but are required for optimum levels of biofilm formation and virulence. Both BCAS0208 and BCAS0201 contribute to pellicle formation, although individual mutations in each of these genes had no appreciable effect on pellicle formation. A mutant with a polar insertion in BCAS0208 was significantly less virulent in a rat model of chronic lung infection as well as in the alfalfa model. Genes in this region were shown to influence utilization of branched-chain fatty acids, tricarboxylic acid cycle substrates, l-arabinose, and branched-chain amino acids. Together, our data show that the ShvR-regulated genes BCAS0208 to BCAS0201 are required for the rough colony morphotype, biofilm and pellicle formation, and virulence in B. cenocepacia.

Bacterial subfamily of LuxR regulators that respond to plant compounds.

Pseudomonas fluorescens are rhizobacteria known for their biocontrol properties. Several antimicrobial functions are crucial for this process, and the experiments described here investigate the modulation of their expression during the plant-bacterium interaction. The role of a LuxR family regulator in interkingdom signaling has been investigated using genome-scale transcriptome analysis, gene promoter studies in vivo and in vitro, biocontrol assays, and response to plant compounds. PsoR, a LuxR solo or orphan regulator of P. fluorescens, was identified. PsoR is solubilized and activates a lux-box-containing promoter only in the presence of macerated plants, suggesting the presence of a plant molecule(s) that most likely binds to PsoR. Gene expression profiles revealed that genes involved in the inhibition of plant pathogens were affected by PsoR, including a chitinase gene, iron metabolism genes, and biosynthetic genes of antifungal compounds. 2,4-Diacetylphloroglucinol production is PsoR dependent both in vitro and in vivo. psoR mutants were significantly reduced for their ability to protect wheat plants from root rot, and damping-off caused by Pythium ultimum infection. PsoR most likely senses a molecule(s) in the plant and modulates expression of genes that have a role in biocontrol. PsoR and related proteins form a subfamily of LuxR family regulators in plant-associated bacteria.

N-Acyl Homoserine Lactone-Mediated Quorum Sensing Regulates Species Interactions in Multispecies Biofilm Communities.

Bacterial biofilms are important medically, environmentally and industrially and there is a need to understand the processes that govern functional synergy and dynamics of species within biofilm communities. Here, we have used a model, mixed-species biofilm community comprised of Pseudomonas aeruginosa PAO1, Pseudomonas protegens Pf-5 and Klebsiella pneumoniae KP1. This biofilm community displays higher biomass and increased resilience to antimicrobial stress conditions such as sodium dodecyl sulfate and tobramycin, compared to monospecies biofilm populations. P. aeruginosa is present at low proportions in the community and yet, it plays a critical role in community function, suggesting it acts as a keystone species in this community. To determine the factors that regulate community composition, we focused on P. aeruginosa because of its pronounced impact on community structure and function. Specifically, we evaluated the role of the N-acyl homoserine lactone (AHL) dependent quorum sensing (QS) system of P. aeruginosa PAO1, which regulates group behaviors including biofilm formation and the production of effector molecules. We found that mixed species biofilms containing P. aeruginosa QS mutants had significantly altered proportions of K. pneumoniae and P. protegens populations compared to mixed species biofilms with the wild type P. aeruginosa. Similarly, inactivation of QS effector genes, e.g. rhlA and pvdR, also governed the relative species proportions. While the absence of QS did not alter the proportions of the two species in dual species biofilms of P. aeruginosa and K. pneumoniae, it resulted in significantly lower proportions of P. aeruginosa in dual species biofilms with P. protegens. These observations suggest that QS plays an important role in modulating community biofilm structure and physiology and affects interspecific interactions.

Functional metagenomic analysis of quorum sensing signaling in a nitrifying community.

Quorum sensing (QS) can function to shape the microbial community interactions, composition, and function. In wastewater treatment systems, acylated homoserine lactone (AHL)-based QS has been correlated with the conversion of floccular biomass into microbial granules, as well as EPS production and the nitrogen removal process. However, the role of QS in such complex communities is still not fully understood, including the QS-proficient taxa and the functional QS genes involved. To address these questions, we performed a metagenomic screen for AHL genes in an activated sludge microbial community from the Ulu Pandan wastewater treatment plant (WWTP) in Singapore followed by functional validation of luxI activity using AHL biosensors and LC-MSMS profiling. We identified 13 luxI and 30 luxR homologs from the activated sludge metagenome. Of those genes, two represented a cognate pair of luxIR genes belonging to a Nitrospira spp. and those genes were demonstrated to be functionally active. The LuxI homolog synthesized AHLs that were consistent with the dominant AHLs in the activated sludge system. Furthermore, the LuxR homolog was shown to bind to and induce expression of the luxI promoter, suggesting this represents an autoinduction feedback system, characteristic of QS circuits. Additionally, a second, active promoter was upstream of a gene encoding a protein with a GGDEF/EAL domain, commonly associated with modulating the intracellular concentration of the secondary messenger, c-di-GMP. Thus, the metagenomic approach used here was demonstrated to effectively identify functional QS genes and suggests that Nitrospira spp. maybe QS is active in the activated sludge community.

The biofilm matrix scaffold of Pseudomonas aeruginosa contains G-quadruplex extracellular DNA structures.

Extracellular DNA, or eDNA, is recognised as a critical biofilm component; however, it is not understood how it forms networked matrix structures. Here, we isolate eDNA from static-culture Pseudomonas aeruginosa biofilms using ionic liquids to preserve its biophysical signatures of fluid viscoelasticity and the temperature dependency of DNA transitions. We describe a loss of eDNA network structure as resulting from a change in nucleic acid conformation, and propose that its ability to form viscoelastic structures is key to its role in building biofilm matrices. Solid-state analysis of isolated eDNA, as a proxy for eDNA structure in biofilms, reveals non-canonical Hoogsteen base pairs, triads or tetrads involving thymine or uracil, and guanine, suggesting that the eDNA forms G-quadruplex structures. These are less abundant in chromosomal DNA and disappear when eDNA undergoes conformation transition. We verify the occurrence of G-quadruplex structures in the extracellular matrix of intact static and flow-cell biofilms of P. aeruginosa, as displayed by the matrix to G-quadruplex-specific antibody binding, and validate the loss of G-quadruplex structures in vivo to occur coincident with the disappearance of eDNA fibres. Given their stability, understanding how extracellular G-quadruplex structures form will elucidate how P. aeruginosa eDNA builds viscoelastic networks, which are a foundational biofilm property.

PpoR is a conserved unpaired LuxR solo of Pseudomonas putida which binds N-acyl homoserine lactones.

BACKGROUND: Only a small number of Pseudomonas putida strains possess the typical N-acyl homoserine lactone quorum sensing system (AHL QS) that consists of a modular LuxR family protein and its cognate LuxI homolog that produces the AHL signal. Moreover, AHL QS systems in P. putida strains are diverse in the type of AHLs they produce and the phenotypes that they regulate. RESULTS: We identified an unpaired LuxR solo (QS luxR homolog that occurs without the corresponding luxI homolog), which is highly conserved in both the AHL producing and non-AHL producing P. putida strains that we analyzed. In this study we report the cloning and functional characterization of this unpaired LuxR homolog designated PpoR. An AHL binding assay showed that PpoR protein binds to 3-oxo-C6-HSL. Studies using a ppoR promoter-lacZ reporter fusion revealed that it exhibits stringent growth phase dependent expression. Functional interaction of PpoR with the endogenous complete AHL QS systems of P. putida WCS358 (PpuI/R system) and PpoR was also investigated. Microarray analysis of P. putida WCS358 wild type and a PpoR over-expressing strain revealed several putative target genes that may be directly or indirectly regulated by PpoR. CONCLUSION: Our results indicate that PpoR in P. putida strains may have a conserved role in detecting an AHL signal, either self or foreign, and regulating specific target genes.

A versatile plasmid biosensor useful to identify quorum sensing LuxR-family orphans in bacterial strains.

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.

Methods to Study Solo/Orphan Quorum-Sensing Receptors.

LuxR solos/orphans are very widespread among Proteobacteria; however they are surprisingly understudied given that they are likely to play a major role in cell-cell communication in bacteria. Here we describe three simple methodologies/approaches that can be used in order to begin to study this subgroup of quorum sensing-related LuxR receptors.

Negative Regulation of Violacein Biosynthesis in Chromobacterium violaceum.

In Chromobacteium violaceum, the purple pigment violacein is under positive regulation by the N-acylhomoserine lactone CviI/R quorum sensing system and negative regulation by an uncharacterized putative repressor. In this study we report that the biosynthesis of violacein is negatively controlled by a novel repressor protein, VioS. The violacein operon is regulated negatively by VioS and positively by the CviI/R system in both C. violaceum and in a heterologous Escherichia coli genetic background. VioS does not regulate the CviI/R system and apart from violacein, VioS, and quorum sensing regulate other phenotypes antagonistically. Quorum sensing regulated phenotypes in C. violaceum are therefore further regulated providing an additional level of control.

Rice bacterial endophytes: isolation of a collection, identification of beneficial strains and microbiome analysis.

Endophytes are harmless or beneficial microorganisms that live inside plants between cells. The relationship they develop with the plant as well as their potential role in plant health is at large unexplored and it is believed that the opportunity to find new and interesting endophytes among the large variety of plants is great. Here, we present the isolation and analysis of a large collection of endophytes from one cultivar of rice grown in Italy. A total 1318 putative endophytes were isolated from roots, leaves and stems from rice grown in submerged and dry conditions and a working collection of 229 isolates was created. Among these, several isolates were confirmed to be endophytes and a few displayed the trait of plant growth promotion. A cultivation independent analysis via 16S rDNA amplicons of the bacterial community of the endosphere was also performed providing information on bacterial diversity in the rice endopshere.

Growth deficiency of a Xanthomonas oryzae pv. oryzae fur mutant in rice leaves is rescued by ascorbic acid supplementation.

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. A mutation was isolated in the ferric uptake regulator (fur) gene of X. oryzae pv. oryzae and it was shown to result in the production of siderophores in a constitutive manner. The fur mutant is hypersensitive to the metallo-antibiotic streptonigrin, a phenotype that is indicative of intracellular free-iron overload, and also exhibits a slow growth phenotype on rich medium. The fur mutant is virulence deficient, hypersensitive to hydrogen peroxide, and exhibits reduced catalase activity. Exogenous supplementation with ascorbic acid (an antioxidant) rescues the growth deficiency of the fur mutant in rice leaves. The virulence deficiency of the X. oryzae pv. oryzae fur mutant is proposed to be due, at least in part, to an impaired ability to cope with the oxidative stress conditions that are encountered during infection.