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Porcine extraintestinal pathogenic Escherichia coli (ExPEC) is a pathogenic bacterium that causes huge economic losses to the pig farming industry and considerably threatens human health. The quorum sensing (QS) system plays a crucial role in the survival and pathogenesis of pathogenic bacteria. Hence, it is a viable approach to prevent ExPEC infection by compromising the QS system, particularly the LuxS/AI-2 system. In this study, we investigated the effects of baicalin on the LuxS/AI-2 system of ExPEC. Baicalin at concentrations of 25, 50, and 100 μg/mL significantly diminished the survival ability of ExPEC in hostile environments and could inhibit the biofilm formation and autoagglutination ability in ExPEC. Moreover, baicalin dose-dependently decreased the production of AI-2 and down-regulated the expression level of luxS in PCN033. These results suggest that baicalin can weaken the virulence of PCN033 by inhibiting the LuxS/AI-2 system. After the gene luxS was deleted, AI-2 production in PCN033 was almost completely eliminated, similar to the effect of baicalin on the production of AI-2 in PCN033. This indicates that baicalin reduced the production of AI-2 by inhibiting the expression level of luxS in ExPEC. In addition, the animal experiment further showed the potential of baicalin as a LuxS/AI-2 system inhibitor to prevent ExPEC infection. This study highlights the potential of baicalin as a natural quorum-sensing inhibitor for therapeutic applications in preventing ExPEC infection by targeting the LuxS/AI-2 system. [ABSTRACT FROM AUTHOR]

ABSTRACT Candida albicans (C. albicans) and Lactiplantibacillus plantarum subsp. plantarum (L. plantarum) are frequently identified in various niches, but their dual-species interaction, especially with C. albicans in yeast form, remains unclear. This study aimed to investigate the dual-species interaction of L. plantarum and C. albicans, including proliferation, morphology, and transcriptomes examined by selective agar plate counting, microscopy, and polymicrobial RNA-seq, respectively. Maintaining a stable and unchanged growth rate, L. plantarum inhibited C. albicans yeast cell proliferation but not hyphal growth. Combining optical microscopy and atomic force microscopy, cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed during dual-species interaction. Reduced C. albicans yeast cell proliferation in mixed culture was partially due to L. plantarum cell-free culture supernatant but not the acidic environment. Upon polymicrobial transcriptomics analysis, interesting changes were identified in both L. plantarum and C. albicans gene expression. First, two L. plantarum quorum-sensing systems showed contrary changes, with the activation of lamBDCA and repression of luxS. Second, the upregulation of stress response-related genes and downregulation of cell cycle, cell survival, and cell integrity-related pathways were identified in C. albicans, possibly connected to the stress posed by L. plantarum and the reduced yeast cell proliferation. Third, a large scale of pathogenesis and virulence factors were downregulated in C. albicans, indicating the potential interruption of pathogenic activities by L. plantarum. Fourth, partial metabolism and transport pathways were changed in L. plantarum and C. albicans. The information in this study might aid in understanding the behavior of L. plantarum and C. albicans in dual-species interaction.IMPORTANCEThe anti-Candida albicans activity of Lactiplantibacillus plantarum has been explored in the past decades. However, the importance of C. albicans yeast form and the effect of C. albicans on L. plantarum had also been omitted. In this study, the dual-species interaction of L. plantarum and C. albicans was investigated with a focus on the transcriptomes. Cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed. Upon polymicrobial transcriptomics analysis, interesting changes were identified, including contrary changes in two L. plantarum quorum-sensing systems and reduced cell survival-related pathways and pathogenesis determinants in C. albicans.

Bacteria mainly exist in the form of a biofilm in nature. The initial adhesion of bacteria starts the life cycle of biofilm formation. Initial adhesion is a process in which bacteria in a planktonic state adsorb on biotic/abiotic surfaces and begin to form biofilms under the influence of factors such as bacterial components and extracellular polymeric substances. The initial adhesion of bacteria is affected by various regulatory systems. Therefore, it is of significance to study these regulatory systems. This article reviews three major regulatory systems that affect initial bacterial adhesion, namely, cyclic diguanylate (c-di-GMP) system, two-component system and quorum sensing system, and introduces the mechanisms of how these regulatory systems regulate initial bacterial adhesion. Also, the application of controlling initial bacterial adhesion to ensure food quality is summarized and future prospects are discussed. This reviews is expected to provide a theoretical basis for protecting food quality from being harmed by bacterial biofilm, which is of practical significance for promoting the development of the food industry.

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Aims: Quorum sensing (QS) is the intercellular communication used by bacteria to regulate collective behaviour. QS regulates the production of virulence factors in many bacterial species and is considered to be an attractive target for reducing bacterial pathogenicity. Chlorogenic acid (CA) is abundant in vegetables, fruits, and traditional Chinese medicine, and has multiple activities. This study aimed to investigate the QS quenching activity of CA against clinically isolated multidrug‐resistant Pseudomonas aeruginosa. Methods and Results: The results showed that CA inhibited the mobility of bacteria, reduced the production of pyocyanin, and inhibited the activity of elastase. Furthermore, crystal violet staining and scanning electron microscope experiments showed that CA inhibited the formation of multidrug‐resistant P. aeruginosa biofilm. CA at or below the concentration of 2560 µg/mL exerted negligible cytotoxicity to RAW264.7 cells. The study also examined the expression of QS‐related genes, including lasI, lasR, rhlI, rhlR, pqsA, and pqsR in P. aeruginosa and found that the expression of these genes was down‐regulated under CA treatment. Conclusions: The study showed that CA could be used as an anti‐virulence factor for treating clinical P. aeruginosa infection. Significance and Impact of Study: For the first time, this study took clinically isolated multidrug‐resistant P. aeruginosa as the experimental object, and suggested that CA might be an effective antimicrobial compound targeting QS in treating P. aeruginosa infection, thus providing a new therapeutic direction for treating bacterial infection and effectively alleviating bacterial resistance. [ABSTRACT FROM AUTHOR]

Abstract Background 4-Hydroxyphenylacetic acid (4HPAA) is an important building block for synthesizing drugs, agrochemicals, biochemicals, etc. 4HPAA is currently produced exclusively via petrochemical processes and the process is environmentally unfriendly and unsustainable. Microbial cell factory would be an attractive approach for 4HPAA production. Results In the present study, we established a microbial biosynthetic system for the de novo production of 4HPAA from glucose in Escherichia coli. First, we compared different biosynthetic pathways for the production of 4HPAA. The yeast Ehrlich pathway produced the highest level of 4HPAA among these pathways that were evaluated. To increase the pathway efficiency, the yeast Ehrlich pathway enzymes were directedly evolved via error-prone PCR. Two phenylpyruvate decarboxylase ARO10 and phenylacetaldehyde dehydrogenase FeaB variants that outperformed the wild-type enzymes were obtained. These mutations increased the in vitro and in vivo catalytic efficiency for converting 4-hydroxyphenylpyruvate to 4HPAA. A tunable intergenic region (TIGR) sequence was inserted into the two evolved genes to balance their expression. Regulation of TIGR for the evolved pathway enzymes further improved the production of 4HPAA, resulting in a 1.13-fold increase in titer compared with the fusion wild-type pathway. To prevent the toxicity of a heterologous pathway to the cell, an Esa quorum-sensing (QS) circuit with both activating and repressing functions was developed for inducer-free productions of metabolites. The Esa-PesaR activation QS system was used to dynamically control the biosynthetic pathway of 4HPAA in E. coli, which achieved 17.39 ± 0.26 g/L with a molar yield of 23.2% without addition of external inducers, resulting in a 46.4% improvement of the titer compared to the statically controlled pathway. Conclusion We have constructed an E. coli for 4HPAA production with the highest titer to date. This study also demonstrates that the combination of directed evolution of pathway enzymes and dynamic pathway regulation using a QS circuit is a powerful strategy of metabolic engineering for the productions of metabolites.

The quorum-sensing system, consisting of an autoregulator synthase (AfsA or Aco homolog) and an autoregulator receptor (ArpA homolog), has been reported to be universally involved in regulating secondary metabolism in streptomycetes. Although the autoregulator synthase is thought to activate antibiotic production, the activation pathway remains poorly understood. Streptomyces cyaneogriseus ssp. noncyanogenus NMWT1 produces nemadectin, which is widely used as a biopesticide and veterinary drug due to its potent nematocidal activity. Here, we identified the Aco/ArpA-like system ScyA1/ScyR1, the ArpA homolog ScyR2 and the AfsA/ArpA-like system ScyA3/ScyR3 as important regulators of nemadectin production in NMWT1. Genetic experiments revealed that these five genes positively regulate nemadectin production, with scyA1 and scyR1 having the most potent effects. Importantly, ScyA1 is an upstream regulator of scyR1 and promotes nemadectin production and sporulation by activating scyR1 transcription. Intriguingly, scyR1 silencing in NMWT1 up-regulated 12 of the 17 secondary metabolite biosynthetic core genes present in the NMWT1 genome, suggesting that ScyR1 mainly to be a repressor of secondary metabolism. In conclusion, our findings unveiled the regulatory pathways adopted by the quorum-sensing system, and provided the basis for a method to enhance antibiotic production and to activate the expression of cryptic biosynthetic gene clusters.

The GntR family regulators are widely distributed in bacteria and play critical roles in metabolic processes and bacterial pathogenicity. In this study, we describe a GntR family protein encoded by PA4132 that we named MpaR (MvfR‐mediated PQS and anthranilate regulator) for its regulation of Pseudomonas quinolone signal (PQS) production and anthranilate metabolism in Pseudomonas aeruginosa. The deletion of mpaR increased biofilm formation and reduced pyocyanin production. RNA sequencing analysis revealed that the mRNA levels of antABC encoding enzymes for the synthesis of catechol from anthranilate, a precursor of the PQS, were most affected by mpaR deletion. Data showed that MpaR directly activates the expression of mvfR, a master regulator of pqs system, and subsequently promotes PQS production. Accordingly, deletion of mpaR activates the expression of antABC genes, and thus, increases catechol production. We also demonstrated that MpaR represses the rhl quorum‐sensing (QS) system, which has been shown to control antABC activity. These results suggested that MpaR function is integrated into the QS regulatory network. Moreover, mutation of mpaR promotes bacterial survival in a mouse model of acute pneumonia infection. Collectively, this study identified a novel regulator of pqs system, which coordinately controls anthranilate metabolism and bacterial virulence in P. aeruginosa. [ABSTRACT FROM AUTHOR]

Objective To screen out the potential regulatory genes of suppressor of cell division ( Sdi A) in Esche richia coli. Methods E.coli SMlOll.pir wild strain, E.coli SMlOll.pir Sdi A mutant strain, and E.coli SMlOll.pir Sdi A overexpression strain were treated with N-acyl-L-homoserine lactones ( AHLs) signal molecules and recorded as the AHLs treatment group. On the other hand, the three strains were treated with equal amount of DMSO, which were recorded as the non-AHLs treatment group. After all the strains were sequenced, the differentially expressed genes were screened out by edgeR software. We selected the common differentially expressed genes between E.coli SMlOll.pir Sdi A mutant strain and E.coli SMlOll.pir wild strain and between the E.coli SMlOll.pir Sdi A overexpression strain and E.coli SMlOll.pir Sdi A mu tant strain as the potential regulatory genes of Sdi A with or without AHLs signaling molecules, and observed the changes in the potential regulatory genes of Sdi A with or without AHLs signaling molecules. Results In the non-AHLs treatment group, the common differentially expressed genes between E. coli SMlOll.pir Sdi A mutant strain and E.coli SMlOll.pir wild strain and between the E.coli SMlOll.pir Sdi A overexpression strain and E. coli SMlOll.pir Sdi A mutant strain were narH, narJ, narl, yeeE, yeeR, paaE and flu. In the AHLs treatment group, the common differentially expressed genes between E.coli SMlOll.pir Sdi A mutant strain and E.coli SMlOll.pir wild strain, and between the E.coli SMlOll.pir Sdi A overexpres sion strain and E.coli SMlOll.pir Sdi A mutant strain were hep, ytfE, her, hmp, narH, narJ, narl, yeeE, yeeR, flu, ydiV and hdhA. According to the comparison, narH, narJ, narl, yeeE, yeeR and flu genes were the potential regulatory genes of Sdi A, and meanwhile, hep, ytfE, her, hmp, ydiV, hdhA genes were the potential regulatory genes of Sdi A in the presence of AHLs signaling molecules. Conclusion In Escherichia coli, the potential regulatory genes of Sdi A are narH, narJ, narl, yeeE, yeeR, flu, paaE, hep, ytfE, her, hmp, ydiV, and hdhA, and the regulation on hep, ytfE, her, hmp, ydiV and hdhA gene still mainly depends on AHLs signaling molecules. [ABSTRACT FROM AUTHOR]

The main aim of this paper is to study the oscillatory behaviors of gene expression networks in quorum-sensing system with time delay. The stability of the unique positive equilibrium and the existence of Hopf bifurcation are investigated by choosing the time delay as the bifurcation parameter and by applying the bifurcation theory. The explicit criteria determining the direction of Hopf bifurcation and the stability of bifurcating periodic solutions are developed based on the normal form theory and the center manifold theorem. Numerical simulations demonstrate good agreements with the theoretical results. Results of this paper indicate that the time delay plays a crucial role in the regulation of the dynamic behaviors of quorum-sensing system. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an opportunistic pathogen with high clinical relevance for hospital infections of patients. Accumulating DNA sequencing results of clinical P. aeruginosa isolates have revealed frequent mutations in lasR gene, which encodes the highest arches component of quorum-sensing system (QS). We analyzed the sequencing data of lasR gene from a large collection of cystic fibrosis (CF) P. aeruginosa isolates. Our systematical analyses revealed that single nucleotide polymorphisms (SNPs) selection in lasR gene were largely constrained by codon-usage frequency. As a whole, SNP-substituted codons encoding unconserved amino acid resulted in unfavored codons with relatively low codon-usage frequency, while those associating with conserved amino acid were not strictly regulated in such way. These SNPs substitutions gives rise to diverse functional LasR isoforms and contributes to the relative growth fitness of recombinant lasR variant strains. Our survey reveals a novel pattern of SNPs selections in lasR gene of CF isolates. Our findings could be served as a powerful resource for understanding adaptive mechanism of clinical isolates under environmental constrains and developing anti-bacteria drugs for CF patients. [ABSTRACT FROM AUTHOR]

Most of the infections caused by multi-drug resistant (MDR) P. aeruginosa strains are extremely difficult to be treated with conventional antibiotics. Biofilm formation and efflux pumps are recognized as the major antibiotic resistance mechanisms in MDR P. aeruginosa. Biofilm formation by P. aeruginosa depends mainly on the cell-to-cell communication quorum-sensing (QS) systems. Titanium dioxide nanoparticles (TDN) have been used as antimicrobial agents against several microorganisms but have not been reported as an anti-QS agent. This study aims to evaluate the impact of titanium dioxide nanoparticles (TDN) on QS and efflux pump genes expression in MDR P. aeruginosa isolates. The antimicrobial susceptibility of 25 P. aeruginosa isolates were performed by Kirby–Bauer disc diffusion. Titanium dioxide nanoparticles (TDN) were prepared by the sol gel method and characterized by different techniques (DLS, HR-TEM, XRD, and FTIR). The expression of efflux pumps in the MDR isolates was detected by the determination of MICs of different antibiotics in the presence and absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Biofilm formation and the antibiofilm activity of TDN were determined using the tissue culture plate method. The effects of TDN on the expression of QS genes and efflux pump genes were tested using real-time polymerase chain reaction (RT-PCR). The average size of the TDNs was 64.77 nm. It was found that TDN showed a significant reduction in biofilm formation (96%) and represented superior antibacterial activity against P. aeruginosa strains in comparison to titanium dioxide powder. In addition, the use of TDN alone or in combination with antibiotics resulted in significant downregulation of the efflux pump genes (MexY, MexB, MexA) and QS-regulated genes (lasR, lasI, rhll, rhlR, pqsA, pqsR) in comparison to the untreated isolate. TDN can increase the therapeutic efficacy of traditional antibiotics by affecting efflux pump expression and quorum-sensing genes controlling biofilm production.

Plantaricin is a polypeptide bacteriocin composed of amino acids. It is a kind of important bacteriocin,with various types,broad anti-bacteria spectrum,tolerance to drug,and high safety. Therefore,it has a potential application in the fields of food,feed and medicine. However,its application in food and medicine is restricted due to the low yield. In this paper,the latest research on regulatory mechanism of plantaricin synthesis is introduced from the aspects of plantaricin species,quorum-sensing system and plantaricin gene clusters. [ABSTRACT FROM AUTHOR]

Emergence of antibiotic-resistant bacteria constitutes an increasing threat to human health. For example, treatment options for Staphylococcus aureus infections is declining with the worldwide spreading of highly virulent community-associated methicillin-resistant S. aureus (CA-MRSA) strains. Anti-virulence therapy has been proposed as an alternative treatment strategy, as it typically involves inhibition of expression of virulence factors rather than direct bacterial killing, thereby attenuating the risk of resistance development. An intriguing target is the agr quorum-sensing system, which is a major inducer of virulence in CA-MRSA upon activation by agr -encoded staphylococcal autoinducing peptides (AIPs). In the present work a previously identified lactam hybrid analogue based on the marine depsipeptide solonamide B and the general structure of AIPs was investigated with respect to structure–function relationships. An array of 27 analogues exploring expansion of ring size, type of side chain, amino acid substitutions, and stereochemistry was designed and tested for AgrC-inhibitory activity. Interestingly, it was found that an analogue derived from the mirror image of the original hit proved to be the hitherto most efficient AgrC inhibitor resembling solonamide B in amino acid sequence. This and closely related compounds were 20- to 40-fold more potent in AgrC inhibition than the starting hit compound. [ABSTRACT FROM AUTHOR]

Many Gram-negative bacteria can regulate gene expression in a cell density-dependent manner via quorum-sensing systems using N-acyl-homoserine lactones (AHLs), which are typical quorum-sensing signaling molecules, and thus modulate physiological characteristics. N-acyl-homoserine lactones are small chemical molecules produced at low concentrations by bacteria and are, therefore, difficult to detect. Here, a biosensor system method and liquid chromatography-tandem mass spectrometry were combined to detect and assay AHL production. As demonstrated by liquid chromatography-tandem mass spectrometry, Gluconacetobacter xylinus CGMCC No. 2955, a Gram-negative acetic acid-producing bacterium and a typical bacterial cellulose (BC) biosynthesis strain, produces six different AHLs, including N-acetyl-homoserine lactone, N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, N-3-oxo-decanoyl-homoserine lactone, N-dodecanoyl-homoserine lactone, and N-tetradecanoyl-homoserine lactone. Gluconacetobacter sp. strain SX-1, another Gram-negative acetic acid-producing bacterium, which can synthesize BC, produces seven different AHLs including N-acetyl-homoserine lactone, N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, N-3-oxo-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-dodecanoyl-homoserine lactone, and N-tetradecanoyl-homoserine lactone. These results lay the foundation for investigating the relationship between BC biosynthesis and quorum-sensing systems.

In response to a high concentration of glucose, Bacillus subtilis , a microbial chassis for producing many industrial metabolites, rapidly takes up glucose using the phosphotransferase system (PTS), leading to overflow metabolism, a common phenomenon observed in many bacteria. Although overflow metabolism affects cell growth and reduces the production of many metabolites, effective strategies that reduce overflow metabolism while maintaining normal cell growth remain to be developed. Here, we used a quorum sensing (QS)-mediated circuit to tune the glucose uptake rate and thereby relieve overflow metabolism in an engineered B. subtilis for producing d-pantothenic acid (DPA). A low-efficiency non-PTS system was used for glucose uptake at the early growth stages to avoid a rapid glycolytic flux, while an efficient PTS system, which was activated by a QS circuit, was automatically activated at the late growth stages after surpassing a threshold cell density. This strategy was successfully applied as a modular metabolic engineering process for the high production of DPA. By enhancing the translation levels of key enzymes (3-methyl-2-oxobutanoate hydroxymethytransferase, pantothenate synthetase, aspartate 1-decarboxylase proenzyme, 2-dehydropantoate 2-reductase, dihydroxy-acid dehydratase, and acetolactate synthase) with engineered 5'-untranslated regions (UTRs) of mRNAs, the metabolic flux was promoted in the direction of DPA production, elevating the yield of DPA to 5.11 g/L in shake flasks. Finally, the engineered B. subtilis produced 21.52 g/L of DPA in fed-batch fermentations. Our work not only revealed a new strategy for reducing overflow metabolism by adjusting the glucose uptake rate in combination with promoting the translation of key metabolic enzymes through engineering the 5'-UTR of mRNAs but also showed its power in promoting the bioproduction of DPA in B. subtilis , exhibiting promising application prospects.

The factors regulating the assembly of the three-dimensional structure of Streptococcus mutans biofilms remain obscure. Polyamines are essential in biofilm formation of certain bacteria. Norspermidine, an unusual polyamine, has been a controversial polyamine that can lead to biofilm disassembly. However, the role of norspermidine in S. mutans b iofilms r emains u nknown. T herefore, t he present study investigated the impact of norspermidine on S. mutans biofilms. The different architectures of the biofilms in norspermidine and control groups indicated that the basic units, bacteria-exopolysaccharide units (BEUs), represent the exopolysaccharide (EPS) and bacterial assembly pattern in S. mutans biofilms. In addition, norspermidine inhibited S. mutans biofilm formation and changed the basic composition of the biofilm, which led to an unusual EPS architecture. Therefore, 5 mM norspermidine inhibited biofilm formation both by decreasing the rate of cell viability and changing the biofilm structure. Gene-expression microarray analysis indicated that the formation of an irregular architecture in the norspermidine group was potentially attributable to the downregulation of elements of the quorum-sensing system (by 2.7-15-fold). The present study suggested that the BEUs are a basic structure of S. mutans biofilm and its assembly is regulated majorly by the quorum-sensing system. Norspermidine can lead to structure change in BEUs by influencing S. mutans quorum-sensing system. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Antimicrobial sensitivity of Streptococcus mutans can be modulated by putative bacteriocin immunity proteins. Bacteria use a quorum sensing (QS) system to regulate physiological activities including bacteriocin production, antimicrobial response, and biofilm formation. QS system of S. mutans is dependent on competence stimulating peptide (CSP), whose precursor is encoded by comC. However, whether bacteriocin immunity proteins play a role in QS system regulated S. mutans antimicrobial sensitivity is still unknown. We hypothesize that bacteriocin immunity proteins encoded by immA and immB play roles in QS regulated antimicrobial sensitivity in S. mutans UA159. Design: In this study, sensitivity of S. mutans UA 159 comC mutant in planktonic and biofilm states to clinically used antimicrobials was investigated by the plate count method and confocal laser scanning microscopy. The effect of immA and immB inactivation on S. mutans antimicrobial sensitivity was studied. The expression of immA and immB in S. mutans comC mutant before and after chlorhexidine (CHX) treatment was also examined. Results: It was found that comC, immA and immB mutation resulted in enhanced antimicrobial sensitivity to sodium fluoride (NaF), CHX and ampicillin (AMP) in planktonic states. After 2% CHX treatment, the live/dead cell ratio in comC mutant and wild strain biofilms decreased 67% and 39% (P <0.05). The expression of immA and immB was up-regulated in wild strain after CHX treatment, while the up-regulation of immB was largely inhibited in comC mutant (P <0.05). Conclusions: These findings suggest that the effect of S. mutans UA159 comC mutation on antimicrobial sensitivity can be due, in part, to attenuation of the expression of the bacteriocin immunity proteins related genes. [Copyright &y& Elsevier]

Biofilms of a number of gram-positive and gram-negative bacteria (both environmental strains from the stratal waters of oil fields and collection strains) were found to exhibit higher resistance to extreme physicochemical factors (unfavorable temperature, pH, and salt concentration) than planktonic cultures. The extracellular polymers forming the structure of the biofilm matrix were shown to contribute significantly to this resistance, since suppression of matrix formation by subbacteriostatic concentrations of azithromycin (for Pseudomonas acephalitica) or mutation in the cvil gene encoding N-hexanoyl homoserine lactone synthetase (for Chromobacterium violaceum CV026) resulted in the resistance of biofilms being decreased almost to the level of planktonic cultures. The role of the biofilm matrix for bacterial survival under extreme conditions is discussed. [ABSTRACT FROM AUTHOR]

The basis of the bactericidal action of antibiotics and the mechanisms of antibiotic tolerance are largely unknown. To elucidate one of the mechanisms of antibiotic tolerance, the present study investigated the role of Pseudomonas aeruginosa quorum sensing (QS) and the rpoS gene in antibiotic tolerance. The survival rates of the lasR and lasI mutants were observed to be lower than that of the parental strain in time-dependent killing studies with 8 μg mL−1 ofloxacin, but the survival rates of the rhlR and rhlI mutants were not different from that of the parental strain. Moreover, a lasR-overexpressing strain was more tolerant to ofloxacin than the parental strain, but this was not the case for an rhlR-overexpressing strain. The mRNA expression levels of lasR, lasI, and rpoS in the wild-type strain in the presence of bactericidal concentration of ofloxacin were lower than that in the absence of ofloxacin. In addition, the significant loss of antibiotic tolerance in the lasR mutant was recovered by the overexpression of rpoS. These results suggest that the Las QS system in P. aeruginosa is involved in the development of ofloxacin tolerance, and the tolerance induced by the Las-system is regulated by rpoS gene. [ABSTRACT FROM AUTHOR]

Abstract: Methicillin resistant Staphylococcus aureus (MRSA) is a major human pathogen with many clinical aspects. In S. aureus, the accessory gene regulator (agr) globally controls the production of virulence factors. There are four agr groups. Our study was done to define the agr specificity of MRSA circulating at Charles Nicolle hospital and to investigate a possible relationship between agr groups and human disease types. From January 2004 to June 2005, a total of 57 MRSA isolated from individual hospitalized patients were collected, representing 12% of the total S. aureus isolates. The isolates were identified by conventional methods. Methicillin resistance was detected by oxacillin and cefoxitin disks and confirmed by the amplification of mecA gene by PCR. The agr groups were identified by multiplex PCR. All the strains were recovered from different wards: medicine (57.8%) especially from dermatology (56.2%), surgery (28%) and pediatrics (7%). Cutaneous pus (36.84%) and blood culture (35.08%) represented the main specimens. The agr groups were distributed as follow nine (15.7%) belonged to group I, two (3.5%) belonged to group II and 23 (40.3%) belonged to group III. For 23 strains, the agr group was not identified. A relationship between agr group and type of disease was observed: agr group III strains were associated with non invasive infections (P =0.02) and agr group I strains with invasive infections especially bacteremia (P =0.002). [Copyright &y& Elsevier]

Vibrio vulnificus is ubiquitous in aquatic environments; however, it occasionally causes serious and often fatal infections in humans. These include invasive septicemia contracted through consumption of raw seafood, as well as wound infections acquired through contact with brackish or marine waters. In most cases of septicemia, the patients have underlying disease(s), such as liver dysfunction or alcoholic cirrhosis, and the secondary skin lesions including cellulitis, edema and hemorrhagic bulla appear on the limbs. Although V. Vul produces various virulent factors including polysaccharide capsule, type IV pili, hemolysin and proteolytic enzymes, the 45-kDa metalloprotease may be a causative factor of the skin lesions, because the purified protease enhances vascular permeability through generation of chemical mediators and also induces serious hemorrhagic damage through digestion of the vascular basement membrane. As well as other bacteria, V. Vul can regulate the protease production through the quorum-sensing system depending on bacterial cell density. However, this system operates efficiently at 25°C, but not at 37°C. Therefore, V. vulnificus may produce sufficient amounts of the protease only in the interstitial tissue of the limbs, in which temperature is lower than the internal temperature of the human body. [ABSTRACT FROM AUTHOR]

The bioluminescence assay using Vibrio harveyi BB170 was used to examine quorum-sensing autoinducer 2 (AI-2) activity from cell-free culture fluids of rumen bacteria. The assay showed that the culture fluids of four species of rumen bacteria, Butyrivibrio fibrisolvens, Eubacterium ruminantium, Ruminococcus flavefaciens, and Succinimonas amylolytica, contained AI-2-like molecules. Furthermore, homologues for luxS genes were detected in rumen fluids collected from three cows and in bacterial cells of P. ruminicola subsp. ruminicola and R. flavefaciens. These findings suggest that the quorum-sensing system mediated by AI-2 is present in the rumen. [Copyright &y& Elsevier]

Over 2,800 Tn5 insertion mutants of Salmonella enterica sv. Typhimurium were screened for the loss of ability to suppress the multiplication of a spectinomycin-resistant (Spcr) but otherwise isogenic S. enterica sv. Typhimurium strain, when the Spcr mutant was added to 24-h LB broth cultures of the mutants. Selected "growth non-suppressive" (GNS) mutants were defective in respiration (insertions in arcA and fnr), amino acid biosynthesis (aroA and aroD), nutrient uptake and its regulation (tdcC and crp), and chemotaxis (fliD). In the last GNS mutant, the transposon inactivated yhjH, an ORF with unknown function which shows sequence similarity to di-guanylate cyclase and to novel two-component signal transduction proteins. In newly hatched chickens, all of the mutants, with the exception of the fliD mutant, were also unable to suppress colonization of the alimentary tract by the parent strain inoculated 1 day later. Defined mutations in luxS or sdiA, genes which contribute to quorum sensing in S. enterica sv. Typhimurium, had no effect on the stationary-phase growth suppression. Analysis of a transcriptional fusion construct indicated that yhjH was moderately expressed in the exponential phase of growth and up-regulated upon entry into stationary phase. Expression of yhjH was also considerably suppressed by the addition of supernatant from a 24-h stationary-phase S. enterica sv. Typhimurium culture, suggesting that the gene belongs to a new sensing and signaling regulatory pathway in S. enterica sv. Typhimurium. [ABSTRACT FROM AUTHOR]

The bioluminescence assay system using Vibrio harveyi reporter strains were used to examine quorum-sensing autoinducer (AI) activity from Mannheimia haemolytica A1 cell-free culture supernatant. We showed that M. haemolytica A1 cell-free culture supernatant contains molecules that can stimulate the quorum-sensing system that regulates the expression of the luciferase operon in V. harveyi. Specifically, M. haemolytica A1 can stimulate only the quorum system 2 but not system 1, suggesting that the culture supernatant only contains molecules similar to AI-2 of V. harveyi. The bioluminescence assay was also used to show that culture supernatants from related Pasteurellaceae organisms, Pasteurella multocida, Pasteurella trehalosi, Actinobacillus suis and Actinobacillus pleuropneumoniae, also contain AI-2-like molecules. This is consistent with the presence of a luxS homolog in the genomes of P. multocida and A. pleuropneumoniae. A luxS homolog was cloned by PCR from M. haemolytica A1 using sequencing data from the ongoing genome sequencing project. The cloned luxSM.h. was able to complement AI-2 production in the Escherichia coli DH5α luxS mutant. This is the first report of a quorum-sensing activity in M. haemolytica A1 and suggests that this bacterium utilizes this mechanism to regulate expression of genes under specific conditions. [Copyright &y& Elsevier]

Metabolic regulation strategies have been developed to redirect metabolic fluxes to production pathways. However, it is difficult to screen out target genes that, when repressed, improve yield without affecting cell growth. Here, we report a strategy using a quorum-sensing system to control small RNA transcription, allowing cell-density-dependent repression of target genes. This strategy is shown with convenient operation, dynamic repression, and availability for simultaneous regulation of multiple genes. The parameters A i , A m , and R A (3-oxohexanoyl-homoserine lactone [AHL] concentrations at which half of the maximum repression and the maximum repression were reached and value of the maximum repression when AHL was added manually, respectively) are defined and introduced to characterize repression curves, and the variant LuxR I58N is identified as the most suitable tuning factor for shake flask culture. Moreover, it is shown that dynamic overexpression of the Hfq chaperone is the key to combinatorial repression without disruptions on cell growth. To show a broad applicability, the production titers of pinene, pentalenene, and psilocybin are improved by 365.3%, 79.5%, and 302.9%, respectively, by applying combinatorial dynamic repression. [Display omitted] • A strategy to repress essential genes without disrupting cell growth in bacteria • LuxR I58N is the most suitable transcriptional factor for tuning the repression • Fine-tuned Hfq level is the key to effective repression of multiple genes • NADPH is a limiting substrate for pinene and pentalenene production Bao et al. develop a regulation strategy to simultaneously repress the expression of multiple genes at posttranscriptional level by using a quorum-sensing system to control bacterial small RNA. This strategy is shown with convenient operation, dynamic repression, and generalizability for diverse products. [ABSTRACT FROM AUTHOR]

Most of the infections caused by multi-drug resistant (MDR) P. aeruginosa strains are extremely difficult to be treated with conventional antibiotics. Biofilm formation and efflux pumps are recognized as the major antibiotic resistance mechanisms in MDR P. aeruginosa. Biofilm formation by P. aeruginosa depends mainly on the cell-to-cell communication quorum-sensing (QS) systems. Titanium dioxide nanoparticles (TDN) have been used as antimicrobial agents against several microorganisms but have not been reported as an anti-QS agent. This study aims to evaluate the impact of titanium dioxide nanoparticles (TDN) on QS and efflux pump genes expression in MDR P. aeruginosa isolates. The antimicrobial susceptibility of 25 P. aeruginosa isolates were performed by Kirby–Bauer disc diffusion. Titanium dioxide nanoparticles (TDN) were prepared by the sol gel method and characterized by different techniques (DLS, HR-TEM, XRD, and FTIR). The expression of efflux pumps in the MDR isolates was detected by the determination of MICs of different antibiotics in the presence and absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Biofilm formation and the antibiofilm activity of TDN were determined using the tissue culture plate method. The effects of TDN on the expression of QS genes and efflux pump genes were tested using real-time polymerase chain reaction (RT-PCR). The average size of the TDNs was 64.77 nm. It was found that TDN showed a significant reduction in biofilm formation (96%) and represented superior antibacterial activity against P. aeruginosa strains in comparison to titanium dioxide powder. In addition, the use of TDN alone or in combination with antibiotics resulted in significant downregulation of the efflux pump genes (MexY, MexB, MexA) and QS-regulated genes (lasR, lasI, rhll, rhlR, pqsA, pqsR) in comparison to the untreated isolate. TDN can increase the therapeutic efficacy of traditional antibiotics by affecting efflux pump expression and quorum-sensing genes controlling biofilm production. [ABSTRACT FROM AUTHOR]

Many Gram-negative bacteria can regulate gene expression in a cell density-dependent manner via quorum-sensing systems using N-acyl-homoserine lactones (AHLs), which are typical quorum-sensing signaling molecules, and thus modulate physiological characteristics. N-acyl-homoserine lactones are small chemical molecules produced at low concentrations by bacteria and are, therefore, difficult to detect. Here, a biosensor system method and liquid chromatography-tandem mass spectrometry were combined to detect and assay AHL production. As demonstrated by liquid chromatography-tandem mass spectrometry, Gluconacetobacter xylinus CGMCC No. 2955, a Gram-negative acetic acid-producing bacterium and a typical bacterial cellulose (BC) biosynthesis strain, produces six different AHLs, including N-acetyl-homoserine lactone, N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, N-3-oxo-decanoyl-homoserine lactone, N-dodecanoyl-homoserine lactone, and N-tetradecanoyl-homoserine lactone. Gluconacetobacter sp. strain SX-1, another Gram-negative acetic acid-producing bacterium, which can synthesize BC, produces seven different AHLs including N-acetyl-homoserine lactone, N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, N-3-oxo-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-dodecanoyl-homoserine lactone, and N-tetradecanoyl-homoserine lactone. These results lay the foundation for investigating the relationship between BC biosynthesis and quorum-sensing systems. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen of humans, particularly those with cystic fibrosis. As a global regulator, RpoN controls a group of virulence-related factors and quorum-sensing (QS) genes in P. aeruginosa. To gain further insights into the direct targets of RpoN in vivo, the present study focused on identifying the direct targets of RpoN regulation in QS and the type VI secretion system (T6SS). We performed chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) that identified 1,068 binding sites of RpoN, mostly including metabolic genes, a group of genes in QS (lasI, rhlI, and pqsR) and the T6SS (hcpA and hcpB). The direct targets of RpoN have been verified by electrophoretic mobility shifts assays (EMSA), lux reporter assay, reverse transcription-quantitative PCR, and phenotypic detection. The ΔrpoN::Tc mutant resulted in the reduced production of pyocyanin, motility, and proteolytic activity. However, the production of rhamnolipids and biofilm formation were higher in the ΔrpoN::Tc mutant than in the wild type. In summary, the results indicated that RpoN had direct and profound effects on QS and the T6SS. [ABSTRACT FROM AUTHOR]

Synthetic oscillators are gene circuits in which the protein expression will change over time. The delay of transcription, translation, and protein folding is used to form this kind of behavior. Here, we tried to design a synthetic oscillator by a negative feedback combined with a positive feedback. With the mutant promoter PLacC repressed by LacIq and PLux activated by AHL-bound LuxR, two gene circuits, Os-LAA and Os-ASV, were constructed and introduced into LacI-deleted E. coli DH5α cells. When glucose was used as the carbon source, a low level of fluorescence was detected in the culture, and the bacteria with Os-ASV showed no oscillation, whereas a small portion of those carrying Os-LAA demonstrated oscillation behavior with a period of about 68.3 ± 20 min. When glycerol was used as the carbon source, bacteria with Os-ASV demonstrated high fluorescence value and oscillation behavior with the period of about 121 ± 21 min.