Expanding the genetic toolkit helps dissect a global stress response in the early-branching species Fusobacterium nucleatum.

Fusobacterium nucleatum, long known as a common oral microbe, has recently garnered attention for its ability to colonize tissues and tumors elsewhere in the human body. Clinical and epidemiological research has now firmly established F. nucleatum as an oncomicrobe associated with several major cancer types. However, with the current research focus on host associations, little is known about gene regulation in F. nucleatum itself, including global stress-response pathways that typically ensure the survival of bacteria outside their primary niche. This is due to the phylogenetic distance of Fusobacteriota to most model bacteria, their limited genetic tractability, and paucity of known gene functions. Here, we characterize a global transcriptional stress-response network governed by the extracytoplasmic function sigma factor, σE. To this aim, we developed several genetic tools for this anaerobic bacterium, including four different fluorescent marker proteins, inducible gene expression, scarless gene deletion, and transcriptional and translational reporter systems. Using these tools, we identified a σE response partly reminiscent of phylogenetically distant Proteobacteria but induced by exposure to oxygen. Although F. nucleatum lacks canonical RNA chaperones, such as Hfq, we uncovered conservation of the noncoding arm of the σE response in form of the noncoding RNA FoxI. This regulatory small RNA acts as an mRNA repressor of several membrane proteins, thereby supporting the function of σE. In addition to the characterization of a global stress response in F. nucleatum, the genetic tools developed here will enable further discoveries and dissection of regulatory networks in this early-branching bacterium.

sRNA-mediated RNA processing regulates bacterial cell division.

Tight control of cell division is essential for survival of most organisms. For prokaryotes, the regulatory mechanisms involved in the control of cell division are mostly unknown. We show that the small non-coding sRNA StsR has an important role in controlling cell division and growth in the alpha-proteobacterium Rhodobacter sphaeroides. StsR is strongly induced by stress conditions and in stationary phase by the alternative sigma factors RpoHI/HII, thereby providing a regulatory link between cell division and environmental cues. Compared to the wild type, a mutant lacking StsR enters stationary phase later and more rapidly resumes growth after stationary phase. A target of StsR is UpsM, the most abundant sRNA in the exponential phase. It is derived from partial transcriptional termination within the 5' untranslated region of the mRNA of the division and cell wall (dcw) gene cluster. StsR binds to UpsM as well as to the 5' UTR of the dcw mRNA and the sRNA-sRNA and sRNA-mRNA interactions lead to a conformational change that triggers cleavage by the ribonuclease RNase E, affecting the level of dcw mRNAs and limiting growth. These findings provide interesting new insights into the role of sRNA-mediated regulation of cell division during the adaptation to environmental changes.

Role of sigma factor RpoS in Cronobacter sakazakii environmental stress tolerance.

Cronobacter sakazakii is a food-borne, conditionally pathogenic bacterium that mainly infects neonates, especially premature infants. Previous studies have indicated that an important route of infection for C. sakazakii is through infant formula, suggesting a high stress resistance of the bacterium. RpoS is a σ-factor that is closely related to the bacterial resistance mechanisms. In this study, a C. sakazakii BAA894 model strain was used. An rpoS-deficient mutant strain Δrpos was constructed using Red homologous recombination, and the differences between the mutant and the wild-type strains were compared. To investigate the functions of the rpoS gene, the membrane formation and cell wall properties of the strains were studied, and the tolerance of each strain to acid, osmotic pressure, desiccation, and drug resistance were compared. The results showed that the membrane formation ability in the mutant strain was increased, auto-aggregation was enhanced, motility, acid resistance and hyperosmotic resistance were alternated to different degrees, and desiccation resistance was stronger than observed in the wild type grown in LB medium but weaker than the wild type cultured in M9 medium. These results showed that rpoS is involved in environmental stress resistance in C. sakazakii BAA894. Finally, transcriptome analysis verified that the deletion of the rpoS gene caused differential expression of resistance-related genes and instigated changes in related metabolic pathways. These messenger RNA results were consistent with the functional experimental results and help explain the phenotypic changes observed in the mutant strain.

Novel self-regulation strategy of a small heat shock protein for prodigious and rapid expression on demand.

In this mini-review, we summarize the known and novel regulation mechanisms of small heat shock proteins (sHsps). sHsps belong to a well-conserved family of ATP-independent oligomeric chaperones that protect denatured proteins from forming irreversible aggregates by co-aggregation. The functions of sHsps as a first line of defense against acute stresses require the high abundance of sHsps on demand. The heat stress-induced expression of IbpA, one of the sHsps in Escherichia coli, is regulated by σ32, an RNA polymerase subunit, and the thermoresponsive mRNA structures in the 5' untranslated region, called RNA thermometers. In addition to the known mechanisms, a recent study has revealed unexpected processes by which the oligomeric IbpA self-represses the ibpA translation via the direct binding of IbpA to its own mRNA, and mediates the mRNA degradation. In summary, the role of IbpA as an aggregation-sensor, combined with other mechanisms, tightly regulates the expression level of IbpA, thus enabling the sHsp to function as a "sequestrase" upon acute aggregation stress, and provides new insights into the mechanisms of other sHsps in both bacteria and eukaryotes.

Tightly controlled response to oxidative stress; an important factor in the tolerance of Bacteroides fragilis.

The exposure of Bacteroides fragilis to highly oxygenated tissues induces an oxidative stress due to a shift from the reduced condition of the gastrointestinal tract to an aerobic environment of host tissues. The potent and effective responses to reactive oxygen species (ROS) make the B. fragilis tolerant to atmospheric oxygen for several days. The response to oxidative stress in B. fragilis is a complicated event that is induced and regulated by different agents. In this review, we will focus on the B. fragilis response to oxidative stress and present an overview of the regulators of responses to oxidative stress in this bacterium.

The novel ECF56 SigG1-RsfG system modulates morphological differentiation and metal-ion homeostasis in Streptomyces tsukubaensis.

Extracytoplasmic function (ECF) sigma factors are key transcriptional regulators that prokaryotes have evolved to respond to environmental challenges. Streptomyces tsukubaensis harbours 42 ECFs to reprogram stress-responsive gene expression. Among them, SigG1 features a minimal conserved ECF σ2-σ4 architecture and an additional C-terminal extension that encodes a SnoaL_2 domain, which is characteristic for ECF σ factors of group ECF56. Although proteins with such domain organisation are widely found among Actinobacteria, the functional role of ECFs with a fused SnoaL_2 domain remains unknown. Our results show that in addition to predicted self-regulatory intramolecular amino acid interactions between the SnoaL_2 domain and the ECF core, SigG1 activity is controlled by the cognate anti-sigma protein RsfG, encoded by a co-transcribed sigG1-neighbouring gene. Characterisation of ∆sigG1 and ∆rsfG strains combined with RNA-seq and ChIP-seq experiments, suggests the involvement of SigG1 in the morphological differentiation programme of S. tsukubaensis. SigG1 regulates the expression of alanine dehydrogenase, ald and the WhiB-like regulator, wblC required for differentiation, in addition to iron and copper trafficking systems. Overall, our work establishes a model in which the activity of a σ factor of group ECF56, regulates morphogenesis and metal-ions homeostasis during development to ensure the timely progression of multicellular differentiation.

Complementary Tendencies in the Use of Regulatory Elements (Transcription Factors, Sigma Factors, and Riboswitches) in Bacteria and Archaea.

In prokaryotes, the key players in transcription initiation are sigma factors and transcription factors that bind to DNA to modulate the process, while premature transcription termination at the 5' end of the genes is regulated by attenuation and, in particular, by attenuation associated with riboswitches. In this study, we describe the distribution of these regulators across phylogenetic groups of bacteria and archaea and find that their abundance not only depends on the genome size, as previously described, but also varies according to the phylogeny of the organism. Furthermore, we observed a tendency for organisms to compensate for the low frequencies of a particular type of regulatory element (i.e., transcription factors) with a high frequency of other types of regulatory elements (i.e., sigma factors). This study provides a comprehensive description of the more abundant COG, KEGG, and Rfam families of transcriptional regulators present in prokaryotic genomes.IMPORTANCE In this study, we analyzed the relationship between the relative frequencies of the primary regulatory elements in bacteria and archaea, namely, transcription factors, sigma factors, and riboswitches. In bacteria, we reveal a compensatory behavior for transcription factors and sigma factors, meaning that in phylogenetic groups in which the relative number of transcription factors was low, we found a tendency for the number of sigma factors to be high and vice versa. For most of the phylogenetic groups analyzed here, except for Firmicutes and Tenericutes, a clear relationship with other mechanisms was not detected for transcriptional riboswitches, suggesting that their low frequency in most genomes does not constitute a significant impact on the global variety of transcriptional regulatory elements in prokaryotic organisms.

The Alternative Sigma Factor SigB Is Required for the Pathogenicity of Bacillus thuringiensis.

To adapt to changing and potentially hostile environments, bacteria can activate the transcription of genes under the control of alternative sigma factors, such as SigB, a master regulator of the general stress response in several Gram-positive species. Bacillus thuringiensis is a Gram-positive spore-forming invertebrate pathogen whose life cycle includes a variety of environments, including plants and the insect hemocoel or gut. Here, we assessed the role of SigB during the infectious cycle of B. thuringiensis in a Galleria mellonella insect model. We used a fluorescent reporter coupled to flow cytometry and showed that SigB was activated in vivo We also showed that the pathogenicity of the ΔsigB mutant was severely affected when inoculated via the oral route, suggesting that SigB is critical for B. thuringiensis adaptation to the gut environment of the insect. We could not detect an effect of the sigB deletion on the survival of the bacteria or on their sporulation efficiency in the cadavers. However, the gene encoding the pleiotropic regulator Spo0A was upregulated in the ΔsigB mutant cells during the infectious process.IMPORTANCE Pathogenic bacteria often need to transition between different ecosystems, and their ability to cope with such variations is critical for their survival. Several Gram-positive species have developed an adaptive response mediated by the general stress response alternative sigma factor SigB. In order to understand the ecophysiological role of this regulator in Bacillus thuringiensis, an entomopathogenic bacterium widely used as a biopesticide, we sought to examine the fate of a ΔsigB mutant during its life cycle in the natural setting of an insect larva. This allowed us, in particular, to show that SigB was activated during infection and that it was required for the pathogenicity of B. thuringiensis via the oral route of infection.

Linking bacterial growth, survival, and multicellularity - small signaling molecules as triggers and drivers.

An overarching theme of cellular regulation in bacteria arises from the trade-off between growth and stress resilience. In addition, the formation of biofilms contributes to stress survival, since these dense multicellular aggregates, in which cells are embedded in an extracellular matrix of self-produced polymers, represent a self-constructed protective and homeostatic 'niche'. As shown here for the model bacterium Escherichia coli, the inverse coordination of bacterial growth with survival and the transition to multicellularity is achieved by a highly integrated regulatory network with several sigma subunits of RNA polymerase and a small number of transcriptional hubs as central players. By conveying information about the actual (micro)environments, nucleotide second messengers such as cAMP, (p)ppGpp, and in particular c-di-GMP are the key triggers and drivers that promote either growth or stress resistance and organized multicellularity in a world of limited resources.

Two paralogous EcfG σ factors hierarchically orchestrate the activation of the General Stress Response in Sphingopyxis granuli TFA.

Under ever-changing environmental conditions, the General Stress Response (GSR) represents a lifesaver for bacteria in order to withstand hostile situations. In α-proteobacteria, the EcfG-type extracytoplasmic function (ECF) σ factors are the key activators of this response at the transcriptional level. In this work, we address the hierarchical function of the ECF σ factor paralogs EcfG1 and EcfG2 in triggering the GSR in Sphingopyxis granuli TFA and describe the role of EcfG2 as global switch of this response. In addition, we define a GSR regulon for TFA and use in vitro transcription analysis to study the relative contribution of each EcfG paralog to the expression of selected genes. We show that the features of each promoter ultimately dictate this contribution, though EcfG2 always produced more transcripts than EcfG1 regardless of the promoter. These first steps in the characterisation of the GSR in TFA suggest a tight regulation to orchestrate an adequate protective response in order to survive in conditions otherwise lethal.

Generally Stressed Out Bacteria: Environmental Stress Response Mechanisms in Gram-Positive Bacteria.

The ability to monitor the environment for toxic chemical and physical disturbances is essential for bacteria that live in dynamic environments. The fundamental sensing mechanisms and physiological responses that allow bacteria to thrive are conserved even if the molecular components of these pathways are not. The bacterial general stress response (GSR) represents a conceptual model for how one pathway integrates a wide range of environmental signals, and how a generalized system with broad molecular responses is coordinated to promote survival likely through complementary pathways. Environmental stress signals such as heat, osmotic stress, and pH changes are received by sensor proteins that through a signaling cascade activate the sigma factor, SigB, to regulate over 200 genes. Additionally, the GSR plays an important role in stress priming that increases bacterial fitness to unrelated subsequent stressors such as oxidative compounds. While the GSR response is implicated during oxidative stress, the reason for its activation remains unknown and suggests crosstalk between environmental and oxidative stress sensors and responses to coordinate antioxidant functions. Systems levels studies of cellular responses such as transcriptomes, proteomes, and metabolomes of stressed bacteria and single-cell analysis could shed light into the regulated functions that protect, remediate, and minimize damage during dynamic environments. This perspective will focus on fundamental stress sensing mechanisms and responses in Gram-positive bacterial species to illustrate their commonalities at the molecular and physiological levels; summarize exciting directions; and highlight how system-level approaches can help us understand bacterial physiology.

A novel mechanism of RyeA/SraC induction under acid stress.

RyeA/SraC is a cis-encoded small RNA (sRNA), which act as an anti-toxin to RpoS-regulated RyeB toxin in Escherichia coli. Ectopic expression of RyeA was reported to diminish the RyeB accumulation by serving as a RNA trap. Lower abundance of RyeA in the early exponential growth phase turned out to be the outcome of its degradation by RNase BN/Z. In the current study, we show that RyeA is an acid stress inducible sRNA, and global stress responsive factor RpoS appeared to be inessential in RyeA induction. Although, ryeB-pphA dicistronic transcript at low pH condition was stimulated by ∼4-fold, however, RyeB population was found to be decreased by > 50% under the same condition by the decoy action of enhanced RyeA accumulation. Investigation of the mechanism of RyeA induceduction at low pH in the exponential phase, revealed that RNase BN/Z, which catabolizes RyeA in the exponential phase, appeared to be highly sensitive to low pH stress. Both mRNA and protein level of RNase BN transpired to be decreased to <10% of their initial population. The expression of RyeA under acid stress is regulated by a feed-forward mechanism to normalize the RyeB profusion.

EcAMSat spaceflight measurements of the role of σs in antibiotic resistance of stationary phase Escherichia coli in microgravity.

We report the results of the EcAMSat (Escherichia coli Antimicrobial Satellite) autonomous space flight experiment, investigating the role of σs in the development of antibiotic resistance in uropathogenic E. coli (UPEC) in microgravity (µ-g). The presence of σs, encoded by the rpoS gene, has been shown to increase antibiotic resistance in Earth gravity, but it was unknown if this effect occurs in µ-g. Two strains, wildtype (WT) UPEC and its isogenic ΔrpoS mutant, were grown to stationary phase aboard EcAMSat, an 11-kg small satellite, and in a parallel ground-based control experiment; cell growth rates for the two strains were found to be unaltered by µ-g. After starvation for over 24 h, stationary-phase cells were incubated with three doses of gentamicin (Gm), a common treatment for urinary tract infections (which have been reported in astronauts). Cellular metabolic activity was measured optically using the redox-based indicator alamarBlue (aB): both strains exhibited slower metabolism in µ-g, consistent with results from previous smallsat missions. The results also showed that µ-g did not enhance UPEC resistance to Gm; in fact, both strains were more susceptible to Gm in µ-g. It was also found, via a second ground-control experiment, that multi-week storage in the payload hardware stressed the cells, potentially obscuring small differential effects of the antibiotic between WT and mutant and/or between µ-g and ground. Overall, results showed that the ∆rpoS mutant was 34-37% less metabolically active than the WT for four different sets of conditions: ground without Gm, ground with Gm; µ-g without Gm, µ-g with Gm. We conclude therefore that the rpoS gene and its downstream products are important therapeutic targets for treating bacterial infections in space, much as they are on the ground.

Pseudomonas syringae AlgU Downregulates Flagellin Gene Expression, Helping Evade Plant Immunity.

Flagella power bacterial movement through liquids and over surfaces to access or avoid certain environmental conditions, ultimately increasing a cell's probability of survival and reproduction. In some cases, flagella and chemotaxis are key virulence factors enabling pathogens to gain entry and attach to suitable host tissues. However, flagella are not always beneficial; both plant and animal immune systems have evolved receptors to sense the proteins that make up flagellar filaments as signatures of bacterial infection. Microbes poorly adapted to avoid or counteract these immune functions are unlikely to be successful in host environments, and this selective pressure has driven the evolution of diverse and often redundant pathogen compensatory mechanisms. We tested the role of AlgU, the Pseudomonas extracytoplasmic function sigma factor σE/σ22 ortholog, in regulating flagellar expression in the context of Pseudomonas syringae-plant interactions. We found that AlgU is necessary for downregulating bacterial flagellin expression in planta and that this results in a corresponding reduction in plant immune elicitation. This AlgU-dependent regulation of flagellin gene expression is beneficial to bacterial growth in the course of plant infection, and eliminating the plant's ability to detect flagellin makes this AlgU-dependent function irrelevant for bacteria growing in the apoplast. Together, these results add support to an emerging model in which P. syringae AlgU functions at a key control point that serves to optimize the expression of bacterial functions during host interactions, including minimizing the expression of immune elicitors and concomitantly upregulating beneficial virulence functions.IMPORTANCE Foliar plant pathogens, like Pseudomonas syringae, adjust their physiology and behavior to facilitate host colonization and disease, but the full extent of these adaptations is not known. Plant immune systems are triggered by bacterial molecules, such as the proteins that make up flagellar filaments. In this study, we found that during plant infection, AlgU, a gene expression regulator that is responsive to external stimuli, downregulates expression of fliC, which encodes the flagellin protein, a strong elicitor of plant immune systems. This change in gene expression and resultant change in behavior correlate with reduced plant immune activation and improved P. syringae plant colonization. The results of this study demonstrate the proximate and ultimate causes of flagellar regulation in a plant-pathogen interaction.

Tripartite Regulation of the glpFKD Operon Involved in Glycerol Catabolism by GylR, Crp, and SigF in Mycobacterium smegmatis.

The glpD (MSMEG_6761) gene encoding glycerol-3-phosphate dehydrogenase was shown to be crucial for M. smegmatis to utilize glycerol as the sole carbon source. The glpD gene likely forms the glpFKD operon together with glpF and glpK, encoding a glycerol facilitator and glycerol kinase, respectively. The gylR (MSMEG_6757) gene, whose product belongs to the IclR family of transcriptional regulators, was identified 182 bp upstream of glpF It was demonstrated that GylR serves as a transcriptional activator and is involved in the induction of glpFKD expression in the presence of glycerol. Three GylR-binding sites with the consensus sequence (GKTCGRC-N3-GYCGAMC) were identified in the upstream region of glpF by DNase I footprinting analysis. The presence of glycerol-3-phosphate was shown to decrease the binding affinity of GylR to the glpF upstream region with changes in the quaternary structure of GylR from tetramer to dimer. Besides GylR, cAMP receptor protein (Crp) and an alternative sigma factor, SigF, are also implicated in the regulation of glpFKD expression. Crp functions as a repressor, while SigF induces expression of glpFKD under energy-limiting conditions. In conclusion, we suggest here that the glpFKD operon is under the tripartite control of GylR, SigF, and Crp, which enables M. smegmatis to integrate the availability of glycerol, cellular energy state, and cellular levels of cAMP to exquisitely control expression of the glpFKD operon involved in glycerol metabolism.IMPORTANCE Using genetic approaches, we first revealed that glycerol is catabolized through the glycolytic pathway after conversion to dihydroxyacetone phosphate in two sequential reactions catalyzed by glycerol kinase (GlpK) and flavin adenine dinucleotide (FAD)-containing glycerol-3-phosphate dehydrogenase (GlpD) in M. smegmatis Our study also revealed that in addition to the GylR transcriptional activator that mediates the induction of the glpFKD operon by glycerol, the operon is regulated by SigF and Crp, which reflect the cellular energy state and cAMP level, respectively.

Group 2 Sigma Factors are Central Regulators of Oxidative Stress Acclimation in Cyanobacteria.

Regulatory σ factors of the RNA polymerase (RNAP) adjust gene expression according to environmental cues when the cyanobacterium Synechocystis sp. PCC 6803 acclimates to suboptimal conditions. Here we show central roles of the non-essential group 2 σ factors in oxidative stress responses. Cells missing all group 2 σ factors fail to acclimate to chemically induced singlet oxygen, superoxide or H2O2 stresses, and lose pigments in high light. SigB and SigD are the major σ factors in oxidative stress, whereas SigC and SigE play only minor roles. The SigD factor is up-regulated in high light, singlet oxygen and H2O2 stresses, and overproduction of the SigD factor in the ΔsigBCE strain leads to superior growth of ΔsigBCE cells in those stress conditions. Superoxide does not induce the production of the SigD factor but instead SigB and SigC factors are moderately induced. The SigB factor alone in ΔsigCDE can support almost as fast growth in superoxide stress as the full complement of σ factors in the control strain, but an overdose of the stationary phase-related SigC factor causes growth arrest of ΔsigBDE in superoxide stress. A drastic decrease of the functional RNAP limits the transcription capacity of the cells in H2O2 stress, which explains why cyanobacteria are sensitive to H2O2. Formation of RNAP-SigB and RNAP-SigD holoenzymes is highly enhanced in H2O2 stress, and cells containing only SigB (ΔsigCDE) or SigD (ΔsigBCE) show superior growth in H2O2 stress.

RpoS controls the expression and the transport of the AlgE1-7 epimerases in Azotobacter vinelandii.

Azotobacter vinelandii produces differentiated cells, called cysts, surrounded by two alginate layers, which are necessary for their desiccation resistance. This alginate contains variable proportions of guluronate residues, resulting from the activity of seven extracytoplasmic epimerases, AlgE1-7. These enzymes are exported by a system secretion encoded by the eexDEF operon; mutants lacking the AlgE1-7 epimerases, the EexDEF or the RpoS sigma factor produce alginate, but are unable to form desiccation resistant cysts. Herein, we found that RpoS was required for full transcription of the algE1-7 and eexDEF genes. We found that the AlgE1-7 protein levels were diminished in the rpoS mutant strain. In addition, the alginate produced in the absence of RpoS was more viscous in the presence of proteases, a phenotype similar to that of the eexD mutant. Primer extension analysis located two promoters for the eexDEF operon, one of them was RpoS-dependent. Thus, during encysting conditions, RpoS coordinates the expression of both the AlgE1-7 epimerases and the EexDEF protein complex responsible for their transport.

Critical role for a promoter discriminator in RpoS control of virulence in Edwardsiella piscicida.

Edwardsiella piscicida is a leading fish pathogen that causes significant economic loses in the aquaculture industry. The pathogen depends on type III and type VI secretion systems (T3/T6SS) for growth and virulence in fish and the expression of both systems is controlled by the EsrB transcription activator. Here, we performed a Tn-seq-based screen to uncover factors that govern esrB expression. Unexpectedly, we discovered that RpoS antagonizes esrB expression and thereby inhibits production of E. piscicida's T3/T6SS. Using in vitro transcription assays, we showed that RpoS can block RpoD-mediated transcription of esrB. ChIP-seq- and RNA-seq-based profiling, as well as mutational and biochemical analyses revealed that RpoS-repressed promoters contain a -6G in their respective discriminator sequences; moreover, this -6G proved critical for RpoS to inhibit esrB expression. Mutation of the RpoS R99 residue, an amino acid that molecular modeling predicts interacts with -6G in the esrB discriminator, abolished RpoS' capacity for repression. In a turbot model, an rpoS deletion mutant was attenuated early but not late in infection, whereas a mutant expressing RpoSR99A exhibited elevated fitness throughout the infection period. Collectively, these findings deepen our understanding of how RpoS can inhibit gene expression and demonstrate the temporal variation in the requirement for this sigma factor during infection.

Analysis of two quorum sensing-deficient isolates of Pseudomonas aeruginosa.

Three strains of Pseudomonas aeruginosa were isolated: wild-type (WT, NO4) showed normal quorum sensing (QS), whereas QSD3 and QSD7 were QS-deficient (QSD) containing limited N-butyryl homoserine lactone (C4-HSL). The autoinducer activity produced by NO4 was found to be at least 50-fold higher than those by the QSD3 and the QSD7 strains. The QSDs produced lower levels of phenazine compounds (pyocyanin), siderophores (pyoverdine) and biosurfactants (rhamnolipids) than NO4. Therefore, the swarming motility and the swimming motility of the QSD3 and the QSD7 strains also decreased. Treatment with exogenous C4-HSL completely restored rhamnolipid production in both QSDs, suggesting that the biosynthesis of C4-HSL is defective. However, the biofilm production of the QSDs reached much higher levels than those of wild-types (NO4 and P. aeruginosa PAO1). And both QSD strains were more resistant than wild-type cell (NO4) against kanamycin and tobramycin. The RpoS gene, which function is related with QS, is point-nonsense mutated in QSD3 strain. But eleven QS-related genes in QSD3 were not mutated, compared to those of PAO1, which carries intact QS genes and is used as a positive control. This study is helpful in the development of novel approaches in the treatment of P. aeruginosa infections.