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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Studies on synthetic LuxR solo hybrids.

A sub-group of LuxR family of proteins that plays important roles in quorum sensing, a process of cell-cell communication, is widespread in proteobacteria. These proteins have a typical modular structure consisting of N-ter autoinducer binding and C-ter helix-turn-helix (HTH) DNA binding domains. The autoinducer binding domain recognizes signaling molecules which are most often N-acyl homoserine lactones (AHLs) but could also be other novel and yet unidentified molecules. In this study we carried out a series of specific domain swapping and promoter activation experiments as a first step to engineer synthetic signaling modules, taking advantage of the modularity and the versatile/diverse signal specificities of LuxR proteins. In our experiments the N-ter domains from different LuxR homologs were either interchanged or placed in tandem followed by a C-ter domain. The rational design of the hybrid proteins was supported by a structure-based homology modeling studies of three members of the LuxR family (i.e., LasR, RhlR, and OryR being chosen for their unique ligand binding specificities) and of selected chimeras. Our results reveal that these LuxR homologs were able to activate promoter elements that were not their usual targets; we also show that hybrid LuxR proteins retained the ability to recognize the signal specific for their N- ter autoinducer binding domain. However, the activity of hybrid LuxR proteins containing two AHL binding domains in tandem appears to depend on the organization and nature of the introduced domains. This study represents advances in the understanding of the modularity of LuxR proteins and provides additional possibilities to use hybrid proteins in both basic and applied synthetic biology based research.

A bioinformatic survey of distribution, conservation, and probable functions of LuxR solo regulators in bacteria.

LuxR solo transcriptional regulators contain both an autoinducer binding domain (ABD; N-terminal) and a DNA binding Helix-Turn-Helix domain (HTH; C-terminal), but are not associated with a cognate N-acyl homoserine lactone (AHL) synthase coding gene in the same genome. Although a few LuxR solos have been characterized, their distributions as well as their role in bacterial signal perception and other processes are poorly understood. In this study we have carried out a systematic survey of distribution of all ABD containing LuxR transcriptional regulators (QS domain LuxRs) available in the InterPro database (IPR005143), and identified those lacking a cognate AHL synthase. These LuxR solos were then analyzed regarding their taxonomical distribution, predicted functions of neighboring genes and the presence of complete AHL-QS systems in the genomes that carry them. Our analyses reveal the presence of one or multiple predicted LuxR solos in many proteobacterial genomes carrying QS domain LuxRs, some of them harboring genes for one or more AHL-QS circuits. The presence of LuxR solos in bacteria occupying diverse environments suggests potential ecological functions for these proteins beyond AHL and interkingdom signaling. Based on gene context and the conservation levels of invariant amino acids of ABD, we have classified LuxR solos into functionally meaningful groups or putative orthologs. Surprisingly, putative LuxR solos were also found in a few non-proteobacterial genomes which are not known to carry AHL-QS systems. Multiple predicted LuxR solos in the same genome appeared to have different levels of conservation of invariant amino acid residues of ABD questioning their binding to AHLs. In summary, this study provides a detailed overview of distribution of LuxR solos and their probable roles in bacteria with genome sequence information.

Agrobacterium tumefaciens responses to plant-derived signaling molecules.

As a special phytopathogen, Agrobacterium tumefaciens infects a wide range of plant hosts and causes plant tumors also known as crown galls. The complexity of Agrobacterium-plant interaction has been studied for several decades. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals. Agrobacterium perceives plant-derived signals to activate its virulence genes, which are responsible for transferring and integrating its Transferred DNA (T-DNA) from its Tumor-inducing (Ti) plasmid into the plant nucleus. The expression of T-DNA in plant hosts leads to the production of a large amount of indole-3-acetic acid (IAA), cytokinin (CK), and opines. IAA and CK stimulate plant growth, resulting in tumor formation. Agrobacterium utilizes opines as nutrient sources as well as signals in order to activate its quorum sensing (QS) to further promote virulence and opine metabolism. Intriguingly, Agrobacterium also recognizes plant-derived signals including γ-amino butyric acid and salicylic acid (SA) to activate quorum quenching that reduces the level of QS signals, thereby avoiding the elicitation of plant defense and preserving energy. In addition, Agrobacterium hijacks plant-derived signals including SA, IAA, and ethylene to down-regulate its virulence genes located on the Ti plasmid. Moreover, certain metabolites from corn (Zea mays) also inhibit the expression of Agrobacterium virulence genes. Here we outline the responses of Agrobacterium to major plant-derived signals that impact Agrobacterium-plant interactions.

Bacterial LuxR solos have evolved to respond to different molecules including signals from plants.

A future challenge will be understanding the extensive communication that most likely takes place in bacterial interspecies and interkingdom signaling between plants and bacteria. A major bacterial inter-cellular signaling system in Gram-negative bacteria is LuxI/R quorum sensing (QS) based on the production (via the LuxI-family proteins) and detection (via the LuxR-family proteins) of N-acyl homoserine lactones (AHLs) signaling molecules. LuxR proteins which have the same modular structure of QS LuxRs but are devoid of a cognate LuxI AHL synthase are called solos. LuxR solos have been shown to be responsible to respond to exogenous AHLs produced by neighboring cells as well endogenously produced AHLs. It is now also evident that some LuxR proteins have evolved from the ability to binding AHLs and respond to other molecules/signals. For example, recent research has shown that a sub-family of LuxR solos responds to small molecules produced by plants. This indicates the presence of a uni-directional interkingdom signaling system occurring from plants to bacteria. In addition LuxR solos have now been also implicated to respond to endogenously produced signals which are not AHLs. In this Mini Review article we will discuss current trends and implications of the role of LuxR solos in bacterial responses to other signals using proteins related to AHL QS systems.

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.

Quorum sensing systems influence Burkholderia cenocepacia virulence.

Burkholderia cepacia complex strains communicate using N-acyl homoserine lactones and BDSF-dependent quorum sensing (QS) systems. Burkholderia cenocepacia QS systems include CepIR, CciIR, CepR2 and BDSF. Analysis of CepR, CciIR, CepR2 and RpfF (BDSF synthase) QS regulons revealed that these QS systems both independently regulate and coregulate many target genes, often in an opposing manner. The role of QS and several QS-regulated genes in virulence has been determined using vertebrate, invertebrate and plant infection models. Virulence phenotypes are strain and model dependent, suggesting that different QS-regulated genes are important depending on the strain and type of infection. QS inhibitors in combination with antibiotics can reduce biofilm formation and virulence in infection models.