EZ-SPOTs: A simple and robust high-throughput liquid handling platform.

Liquid handling is a fundamental capability for many scientific experiments. Previously, we introduced the Surface Patterned Omniphobic Tiles (SPOTs) platform, which enables manipulation of hundreds to thousands of independent experiments without costly equipment or excessive consumable expenses. However, the SPOTs platform requires a custom coating formulation and lacks robustness. To overcome these limitations, we introduce EZ-SPOTs. These devices can be created in an hour with common fabrication tools and just three components - glass, a hydrophobic coating, and acrylic. EZ-SPOTs preserve many of the SPOTs platform's strengths - ease of use, ability to handle a wide range of volumes, and scalability - and adopt a durable and abrasion resistant coating that enables multiple reuses of each device. Here, we describe the fabrication of EZ-SPOTs and showcase how its reusability allows antibiotic susceptibility testing of many isolates using a single device. These results quantitatively match current gold standard assays and the increased throughput provides substantially more information than standard approaches.

ß-Barrel proteins tether the outer membrane in many Gram-negative bacteria.

Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)1. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins2,3. In Escherichia coli, Braun's lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability4; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present5. We used a glycoproteomic analysis of PG to show that ß-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the ß-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.

Peptidoglycan Production by an Insect-Bacterial Mosaic.

Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes.

Noncanonical Inhibition of mTORC1 by Coxiella burnetii Promotes Replication within a Phagolysosome-Like Vacuole.

The Q fever agent Coxiella burnetii is a Gram-negative bacterium that invades macrophages and replicates inside a specialized lysosomal vacuole. The pathogen employs a type 4B secretion system (T4BSS) to deliver effector proteins into the host cell that modify the Coxiella-containing vacuole (CCV) into a replication-permissive niche. Mature CCVs are massive degradative organelles that acquire lysosomal proteins. Inhibition of mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) kinase by nutrient deprivation promotes autophagy and lysosome fusion, as well as activation of the transcription factors TFE3 and TFEB (TFE3/B), which upregulates expression of lysosomal genes. Here, we report that C. burnetii inhibits mTORC1 as evidenced by impaired localization of mTORC1 to endolysosomal membranes and decreased phosphorylation of elF4E-binding protein 1 (4E-BP1) and S6 kinase 1 in infected cells. Infected cells exhibit increased amounts of autophagy-related proteins protein 1A/1B-light chain 3 (LC3) and p62 as well as of activated TFE3. However, C. burnetii did not accelerate autophagy or block autophagic flux triggered by cell starvation. Activation of autophagy or transcription by TFE3/B increased CCV expansion without enhancing bacterial replication. By contrast, knockdown of tuberous sclerosis complex 1 (TSC1) or TSC2, which hyperactivates mTORC1, impaired CCV expansion and bacterial replication. Together, these data demonstrate that specific inhibition of mTORC1 by C. burnetii, but not amplified cell catabolism via autophagy, is required for optimal pathogen replication. These data reveal a complex interplay between lysosomal function and host cell metabolism that regulates C. burnetii intracellular growth.IMPORTANCECoxiella burnetii is an intracellular pathogenic bacterium that replicates within a lysosomal vacuole. Biogenesis of the Coxiella-containing vacuole (CCV) requires effector proteins delivered into the host cell cytosol by the type 4B secretion system (T4BSS). Modifications to lysosomal physiology required for pathogen replication within the CCV are poorly understood. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is a master kinase that regulates lysosome structure and function. Nutrient deprivation inhibits mTORC1, which promotes cell catabolism in the form of accelerated autophagy and increased lysosome biosynthesis. Here, we report that C. burnetii growth is enhanced by T4BSS-dependent inhibition of mTORC1 that does not activate autophagy. Canonical inhibition of mTORC1 by starvation or inhibitor treatment that induces autophagic flux does not benefit C. burnetii growth. Furthermore, hyperactivation of mTORC1 impairs bacterial replication. These findings indicate that C. burnetii inhibition of mTORC1 without accelerated autophagy promotes bacterial growth.

Coxiella burnetii RpoS Regulates Genes Involved in Morphological Differentiation and Intracellular Growth.

Coxiella burnetii, the etiological agent of Q fever, undergoes a unique biphasic developmental cycle where bacteria transition from a replicating (exponential-phase) large cell variant (LCV) form to a nonreplicating (stationary-phase) small cell variant (SCV) form. The alternative sigma factor RpoS is an essential regulator of stress responses and stationary-phase physiology in several bacterial species, including Legionella pneumophila, which has a developmental cycle superficially similar to that of C. burnetii Here, we used a C. burnetii ΔrpoS mutant to define the role of RpoS in intracellular growth and SCV development. Growth yields following infection of Vero epithelial cells or THP-1 macrophage-like cells with the rpoS mutant in the SCV form, but not the LCV form, were significantly lower than that of wild-type bacteria. RNA sequencing and whole-cell mass spectrometry of the C. burnetii ΔrpoS mutant revealed that a substantial portion of the C. burnetii genome is regulated by RpoS during SCV development. Regulated genes include those involved in stress responses, arginine transport, peptidoglycan remodeling, and synthesis of the SCV-specific protein ScvA. Genes comprising the dot/icm locus, responsible for production of the Dot/Icm type 4B secretion system, were also dysregulated in the rpoS mutant. These data were corroborated with independent assays demonstrating that the C. burnetii ΔrpoS strain has increased sensitivity to hydrogen peroxide and carbenicillin and a thinner cell wall/outer membrane complex. Collectively, these results demonstrate that RpoS is an important regulator of genes involved in C. burnetii SCV development and intracellular growth.IMPORTANCE The Q fever bacterium Coxiella burnetii has spore-like environmental stability, a characteristic that contributes to its designation as a potential bioweapon. Stability is likely conferred by a highly resistant, small cell variant (SCV) stationary-phase form that arises during a biphasic developmental cycle. Here, we define the role of the alternative sigma factor RpoS in regulating genes associated with SCV development. Genes involved in stress responses, amino acid transport, cell wall remodeling, and type 4B effector secretion were dysregulated in the rpoS mutant. Cellular impairments included defects in intracellular growth, cell wall structure, and resistance to oxidants. These results support RpoS as a central regulator of the Coxiella developmental cycle and identify developmentally regulated genes involved in morphological differentiation.

Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium.

UNLABELLED: Host cell-free (axenic) culture of Coxiella burnetii in acidified citrate cysteine medium-2 (ACCM-2) has provided important opportunities for investigating the biology of this naturally obligate intracellular pathogen and enabled the development of tools for genetic manipulation. However, ACCM-2 has complex nutrient sources that preclude a detailed study of nutritional factors required for C. burnetii growth. Metabolic reconstruction of C. burnetii predicts that the bacterium cannot synthesize all amino acids and therefore must sequester some from the host. To examine C. burnetii amino acid auxotrophies, we developed a nutritionally defined medium with known amino acid concentrations, termed ACCM-D. Compared to ACCM-2, ACCM-D supported longer logarithmic growth, a more gradual transition to stationary phase, and approximately 5- to 10-fold greater overall replication. Small-cell-variant morphological forms generated in ACCM-D also showed increased viability relative to that generated in ACCM-2. Lack of growth in amino acid-deficient formulations of ACCM-D revealed C. burnetii auxotrophy for 11 amino acids, including arginine. Heterologous expression of Legionella pneumophila argGH in C. burnetii permitted growth in ACCM-D missing arginine and supplemented with citrulline, thereby providing a nonantibiotic means of selection of C. burnetii genetic transformants. Consistent with bioinformatic predictions, the elimination of glucose did not impair C. burnetii replication. Together, these results highlight the advantages of a nutritionally defined medium in investigations of C. burnetii metabolism and the development of genetic tools. IMPORTANCE: Host cell-free growth and genetic manipulation of Coxiella burnetii have revolutionized research of this intracellular bacterial pathogen. Nonetheless, undefined components of growth medium have made studies of C. burnetii physiology difficult and have precluded the development of selectable markers for genetic transformation based on nutritional deficiencies. Here, we describe a medium, containing only amino acids as the sole source of carbon and energy, which supports robust growth and improved viability of C. burnetii Growth studies confirmed that C. burnetii cannot replicate in medium lacking arginine. However, genetic transformation of the bacterium with constructs containing the last two genes in the L. pneumophila arginine biosynthesis pathway (argGH) allowed growth on defined medium missing arginine but supplemented with the arginine precursor citrulline. Our results advance the field by facilitating studies of C. burnetii metabolism and allowing non-antibiotic-based selection of C. burnetii genetic transformants, an important achievement considering that selectable makers based on antibiotic resistance are limited.

Transcriptional Profiling of Coxiella burnetii Reveals Extensive Cell Wall Remodeling in the Small Cell Variant Developmental Form.

A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3-3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3-3 cross-links as opposed to 16% 3-3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella's environmental resistance.

The broad-spectrum antiviral compound ST-669 restricts chlamydial inclusion development and bacterial growth and localizes to host cell lipid droplets within treated cells.

Novel broad-spectrum antimicrobials are a critical component of a strategy for combating antibiotic-resistant pathogens. In this study, we explored the activity of the broad-spectrum antiviral compound ST-669 for activity against different intracellular bacteria and began a characterization of its mechanism of antimicrobial action. ST-669 inhibits the growth of three different species of chlamydia and the intracellular bacterium Coxiella burnetii in Vero and HeLa cells but not in McCoy (murine) cells. The antichlamydial and anti-C. burnetii activity spectrum was consistent with those observed for tested viruses, suggesting a common mechanism of action. Cycloheximide treatment in the presence of ST-669 abrogated the inhibitory effect, demonstrating that eukaryotic protein synthesis is required for tested activity. Immunofluorescence microscopy demonstrated that different chlamydiae grow atypically in the presence of ST-669, in a manner that suggests the compound affects inclusion formation and organization. Microscopic analysis of cells treated with a fluorescent derivative of ST-669 demonstrated that the compound localized to host cell lipid droplets but not to other organelles or the host cytosol. These results demonstrate that ST-669 affects intracellular growth in a host-cell-dependent manner and interrupts proper development of chlamydial inclusions, possibly through a lipid droplet-dependent process.

Essential role for the response regulator PmrA in Coxiella burnetii type 4B secretion and colonization of mammalian host cells.

Successful host cell colonization by the Q fever pathogen, Coxiella burnetii, requires translocation of effector proteins into the host cytosol by a Dot/Icm type 4B secretion system (T4BSS). In Legionella pneumophila, the two-component system (TCS) PmrAB regulates the Dot/Icm T4BSS and several additional physiological processes associated with pathogenesis. Because PmrA consensus regulatory elements are associated with some dot/icm and substrate genes, a similar role for PmrA in regulation of the C. burnetii T4BSS has been proposed. Here, we constructed a C. burnetii pmrA deletion mutant to directly probe PmrA-mediated gene regulation. Compared to wild-type bacteria, C. burnetii ΔpmrA exhibited severe intracellular growth defects that coincided with failed secretion of effector proteins. Luciferase gene reporter assays demonstrated PmrA-dependent expression of 5 of 7 dot/icm operons and 9 of 11 effector-encoding genes with a predicted upstream PmrA regulatory element. Mutational analysis verified consensus sequence nucleotides required for PmrA-directed transcription. RNA sequencing and whole bacterial cell mass spectrometry of wild-type C. burnetii and the ΔpmrA mutant uncovered new components of the PmrA regulon, including several genes lacking PmrA motifs that encoded Dot/Icm substrates. Collectively, our results indicate that the PmrAB TCS is a critical virulence factor that regulates C. burnetii Dot/Icm secretion. The presence of PmrA-responsive genes lacking PmrA regulatory elements also suggests that the PmrAB TCS controls expression of regulatory systems associated with the production of additional C. burnetii proteins involved in host cell parasitism.

Developmental transitions of Coxiella burnetii grown in axenic media.

Coxiella burnetii undergoes a biphasic developmental cycle within its host cell that generates morphologically and physiologically distinct large cell variants (LCV) and small cell variants (SCV). During the lag phase of the C. burnetii growth cycle, non-replicating SCV differentiate into replicating LCV that in turn differentiate back into SCV during stationary phase. Nearly homogeneous SCV are observed in infected Vero cells after extended incubation (21 to 28days). In the current study, we sought to establish whether C. burnetii developmental transitions in host cells are recapitulated during host cell-free (axenic) growth in first and second generation acidified citrate cysteine media (ACCM-1 and ACCM-2, respectively). We show that ACCM-2 supported developmental transitions and viability. Although ACCM-1 also supported SCV to LCV transition, LCV to SCV transition did not occur after extended incubation (21days). Instead, C. burnetii exhibited a ghost-like appearance with bacteria containing condensed chromatin but otherwise devoid of cytoplasmic content. This phenotype correlated with a near total loss in viability between 14 and 21days of cultivation. Transcriptional profiling of C. burnetii following 14days of incubation revealed elevated expression of oxidative stress genes in ACCM-1 cultivated bacteria. ACCM-2 differs from ACCM-1 by the substitution of methyl-ß-cyclodextrin (Mß-CD) for fetal bovine serum. Addition of Mß-CD to ACCM-1 at 7days post-inoculation rescued C. burnetii viability and lowered expression of oxidative stress genes. Thus, Mß-CD appears to alleviate oxidative stress in ACCM-2 to result in C. burnetii developmental transitions and viability that mimic host cell-cultivated organisms. Axenic cultivation of C. burnetii in ACCM-2 and new methods of genetic manipulation now allow investigation of the molecular basis of C. burnetii biphasic development.

Sec-mediated secretion by Coxiella burnetii.

BACKGROUND: Coxiella burnetii is a Gram-negative intracellular bacterial pathogen that replicates within a phagolysosome-like parasitophorous vacuole (PV) of macrophages. PV formation requires delivery of effector proteins directly into the host cell cytoplasm by a type IVB secretion system. However, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promote pathogen replication. RESULTS: To assess the potential of C. burnetii to secrete proteins into the PV, we analyzed the protein content of modified acidified citrate cysteine medium for the presence of C. burnetii proteins following axenic (host cell-free) growth. Mass spectrometry generated a list of 105 C. burnetii proteins that could be secreted. Based on bioinformatic analysis, 55 proteins were selected for further study by expressing them in C. burnetii with a C-terminal 3xFLAG-tag. Secretion of 27 proteins by C. burnetii transformants was confirmed by immunoblotting culture supernatants. Tagged proteins expressed by C. burnetii transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs ex vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase. CONCLUSIONS: C. burnetii secretes multiple proteins, in vitro and ex vivo, in a Sec-dependent manner. Possible roles for secreted proteins and secretion mechanisms are discussed.

Genomic and phenotypic characterization of in vitro-generated Chlamydia trachomatis recombinants.

BACKGROUND: Pre-genomic and post-genomic studies demonstrate that chlamydiae actively recombine in vitro and in vivo, although the molecular and cellular biology of this process is not well understood. In this study, we determined the genome sequence of twelve Chlamydia trachomatis recombinants that were generated in vitro under antibiotic selection. These strains were used to explore the process of recombination in Chlamydia spp., including analysis of candidate recombination hotspots, and to correlate known C. trachomatis in vitro phenotypes with parental phenotypes and genotypes. RESULTS: Each of the 190 examined recombination events was the product of homologous recombination, and no candidate targeting motifs were identified at recombination sites. There was a single deletion event in one recombinant progeny that resulted in the removal of 17.1 kilobases between two rRNA operons. There was no evidence for preference for any specific region of the chromosome for recombination, and analyses of a total of over 200 individual recombination events do not provide any support for recombination hotspots in vitro. Two measurable phenotypes were analyzed in these studies. First, the efficiency of attachment to host cells in the absence of centrifugation was examined, and this property segregated to regions of the chromosome that carry the polymorphic membrane protein (Pmp) genes. Second, the formation of secondary inclusions within cells varied among recombinant progeny, but this did not cleanly segregate to specific regions of the chromosome. CONCLUSIONS: These experiments examined the process of recombination in C. trachomatis and identified tools that can be used to associate phenotype with genotype in recombinant progeny. There were no data supporting the hypothesis that particular nucleotide sequences are preferentially used for recombination in vitro. Selected phenotypes can be segregated by analysis of recombination, and this technology may be useful in preliminary analysis of the relationship of genetic variation to phenotypic variation in the chlamydiae.

Resistance to a novel antichlamydial compound is mediated through mutations in Chlamydia trachomatis secY.

A novel and quantitative high-throughput screening approach was explored as a tool for the identification of novel compounds that inhibit chlamydial growth in mammalian cells. The assay is based on accumulation of a fluorescent marker by intracellular chlamydiae. Its utility was demonstrated by screening 42,000 chemically defined compounds against Chlamydia caviae GPIC. This analysis led to the identification of 40 primary-hit compounds. Five of these compounds were nontoxic to host cells and had similar activities against both C. caviae GPIC and Chlamydia trachomatis. The inhibitory activity of one of the compounds, (3-methoxyphenyl)-(4,4,7-trimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-C]quinolin-1-ylidene)amine (MDQA), was chlamydia specific and was selected for further study. Selection for resistance to MDQA led to the generation of three independent resistant clones of C. trachomatis. Amino acid changes in SecY, a protein involved in Sec-dependent secretion in Gram-negative bacteria, were associated with the resistance phenotype. The amino acids changed in each of the resistant mutants are located in the predicted central channel of a SecY crystal structure, based on the known structure of Thermus thermophilus SecY. These experiments model a process that can be used for the discovery of antichlamydial, anti-intracellular, or antibacterial compounds and has led to the identification of compounds that may have utility in both antibiotic discovery and furthering our understanding of chlamydial biology.

Advances in genetic manipulation of obligate intracellular bacterial pathogens.

Infections by obligate intracellular bacterial pathogens result in significant morbidity and mortality worldwide. These bacteria include Chlamydia spp., which causes millions of cases of sexually transmitted disease and blinding trachoma annually, and members of the α-proteobacterial genera Anaplasma, Ehrlichia, Orientia, and Rickettsia, agents of serious human illnesses including epidemic typhus. Coxiella burnetii, the agent of human Q fever, has also been considered a prototypical obligate intracellular bacterium, but recent host cell-free (axenic) growth has rescued it from obligatism. The historic genetic intractability of obligate intracellular bacteria has severely limited molecular dissection of their unique lifestyles and virulence factors involved in pathogenesis. Host cell restricted growth is a significant barrier to genetic transformation that can make simple procedures for free-living bacteria, such as cloning, exceedingly difficult. Low transformation efficiency requiring long-term culture in host cells to expand small transformant populations is another obstacle. Despite numerous technical limitations, the last decade has witnessed significant gains in genetic manipulation of obligate intracellular bacteria including allelic exchange. Continued development of genetic tools should soon enable routine mutation and complementation strategies for virulence factor discovery and stimulate renewed interest in these refractory pathogens. In this review, we discuss the technical challenges associated with genetic transformation of obligate intracellular bacteria and highlight advances made with individual genera.

Antibiotic resistance in Chlamydiae.

There are few documented reports of antibiotic resistance in Chlamydia and no examples of natural and stable antibiotic resistance in strains collected from humans. While there are several reports of clinical isolates exhibiting resistance to antibiotics, these strains either lost their resistance phenotype in vitro, or lost viability altogether. Differences in procedures for chlamydial culture in the laboratory, low recovery rates of clinical isolates and the unknown significance of heterotypic resistance observed in culture may interfere with the recognition and interpretation of antibiotic resistance. Although antibiotic resistance has not emerged in chlamydiae pathogenic to humans, several lines of evidence suggest they are capable of expressing significant resistant phenotypes. The adept ability of chlamydiae to evolve to antibiotic resistance in vitro is demonstrated by contemporary examples of mutagenesis, recombination and genetic transformation. The isolation of tetracycline-resistant Chlamydia suis strains from pigs also emphasizes their adaptive ability to acquire antibiotic resistance genes when exposed to significant selective pressure.

Social cheating in Pseudomonas aeruginosa quorum sensing.

In a process termed quorum sensing, bacteria use diffusible chemical signals to coordinate cell density-dependent gene expression. In the human pathogen Pseudomonas aeruginosa, quorum sensing controls hundreds of genes, many of which encode extracellular virulence factors. Quorum sensing is required for P. aeruginosa virulence in animal models. Curiously, quorum sensing-deficient variants, most of which carry a mutation in the gene encoding the central quorum sensing regulator lasR, are frequently isolated from acute and chronic infections. The mechanism for their emergence is not known. Here we provide experimental evidence suggesting that these lasR mutants are social cheaters that cease production of quorum-controlled factors and take advantage of their production by the group. We detected an emerging subpopulation of lasR mutants after approximately 100 generations of in vitro evolution of the P. aeruginosa wild-type strain under culture conditions that require quorum sensing for growth. Under such conditions, quorum sensing appears to impose a metabolic burden on the proliferating bacterial cell, because quorum-controlled genes not normally induced until cessation of growth were highly expressed early in growth, and a defined lasR mutant showed a growth advantage when cocultured with the parent strain. The emergence of quorum-sensing-deficient variants in certain environments is therefore an indicator of high quorum sensing activity of the bacterial population as a whole. It does not necessarily indicate that quorum sensing is insignificant, as has previously been suggested. Thus, novel antivirulence strategies aimed at disrupting bacterial communication may be particularly effective in such clinical settings.