Bacterial Signaling Molecules Research Articles

Distinctive Growth and Transcriptional Changes of the Diatom Seminavis robusta in Response to Quorum Sensing Related Compounds.

In aquatic habitats, diatoms are frequently found in association with Proteobacteria, many members of which employ cell-to-cell communication via N-acyl homoserine lactones (AHLs). It has been suggested that diatoms could distinguish between beneficial and algicidal bacteria in their surroundings by sensing AHLs. Although some microalgae can interfere with AHL signaling, e.g., by releasing AHL mimics or degrading them, molecular responses to AHLs in microalgae are still unclear. Therefore, we tested the effects of short-chained AHLs, i.e., N-hexanoyl homoserine lactone (C6-HSL), N-3-hydroxyhexanoyl homoserine lactone (OH-C6-HSL), and N-3-oxohexanoyl homoserine lactone (oxo-C6-HSL) and long-chained AHLs, i.e., N-tetradecanoyl homoserine lactone (C14-HSL), N-3-hydroxytetradecanoyl homoserine lactone (OH-C14-HSL), and N-3-oxotetradecanoyl homoserine lactone (oxo-C14-HSL), on growth of the benthic diatom Seminavis robusta. All tested short-chained AHLs did not affect diatom growth, while long-chained AHLs promoted (C14-HSL) or inhibited (OH-C14-HSL and oxo-C14-HSL) growth. To investigate the physiological effects of these long-chained AHLs in more detail, an RNA-seq experiment was performed during which S. robusta was treated with the growth-promoting C14-HSL and the growth-inhibiting oxo-C14-HSL. One tetramic acid was also tested (TA14), a structural rearrangement product of oxo-C14-HSL, which also induced growth inhibition in S. robusta. After 3 days of treatment, analysis revealed that 3,410 genes were differentially expressed in response to at least one of the compounds. In the treatment with the growth-promoting C14-HSL many genes involved in intracellular signaling were upregulated. On the other hand, exposure to growth-inhibiting oxo-C14-HSL and TA14 triggered a switch in lipid metabolism towards increased fatty acid degradation. In addition, oxo-C14-HSL led to downregulation of cell cycle genes, which is in agreement with the stagnation of cell growth in this treatment. Combined, our results indicate that bacterial signaling molecules with high structural similarity induce contrasting physiological responses in S. robusta.

The Signaling Molecule Indole Inhibits Induction of the AR2 Acid Resistance System in Escherichia coli.

Induction of the AR2 acid response system of Escherichia coli occurs at a moderately low pH (pH 5.5) and leads to high levels of resistance to pH levels below 2.5 in the presence of glutamate. Induction is mediated in part by the EvgAS two component system. Here, we show that the bacterial signaling molecule indole inhibits the induction of key promoters in the AR2 system and blocks the development of glutamate-dependent acid resistance. The addition of tryptophan, the precursor for indole biosynthesis, had the same effects, and this block was relieved in a tnaA mutant, which is unable to synthesize indole. Expression of a constitutively active EvgS protein was able to relieve the inhibition caused by indole, consistent with EvgS being inhibited directly or indirectly by indole. Indole had no effect on autophosphorylation of the isolated cytoplasmic domain of EvgS. This is consistent with a model where indole directly or indirectly affects the ability of EvgS to detect its inducing signal or to transduce this information across the cytoplasmic membrane. The inhibitory activity of indole on the AR2 system is not related to its ability to act as an ionophore, and, conversely, the ionophore CCCP had no effect on acid-induced AR2 promoter activity, showing that the proton motive force is unlikely to be a signal for induction of the AR2 system.

Characterization of Autoinducer-3 Structure and Biosynthesis in E. coli.

Escherichia coli is a common inhabitant of the human microbiota and a beacon model organism in biology. However, an understanding of its signaling systems that regulate population-level phenotypes known as quorum sensing remain incomplete. Here, we define the structure and biosynthesis of autoinducer-3 (AI-3), a metabolite of previously unknown structure involved in the pathogenesis of enterohemorrhagic E. coli (EHEC). We demonstrate that novel AI-3 analogs are derived from threonine dehydrogenase (Tdh) products and “abortive” tRNA synthetase reactions, and they are distributed across a variety of Gram-negative and Gram-positive bacterial pathogens. In addition to regulating virulence genes in EHEC, we show that the metabolites exert diverse immunological effects on primary human tissues. The discovery of AI-3 metabolites and their biochemical origins now provides a molecular foundation for investigating the diverse biological roles of these elusive yet widely distributed bacterial signaling molecules.

Chemiluminescent sensors for quantitation of the bacterial second messenger cyclic di-GMP.

Chemiluminescent biosensors have been developed and broadly applied to mammalian cell systems for studying intracellular signaling networks. For bacteria, biosensors have largely relied on fluorescence-based systems for quantitating signaling molecules, but these designs can encounter issues in complex environments due to their reliance on external illumination. In order to circumvent these issues, we designed the first ratiometric chemiluminescent biosensors for studying a key bacterial second messenger, cyclic di-GMP. We have shown recently that these biosensors function both in vitro and in vivo for detecting changes in cyclic di-GMP levels. In this chapter, we present a practical and broadly applicable method for high-throughput quantitation of cyclic di-GMP in bacterial cell extracts using the high affinity biosensor tVYN-TmΔ that could serve as the "Bradford assay" equivalent for this bacterial signaling molecule.

Phenotypic-genotypic analysis of GGDEF/EAL/HD-GYP domain-encoding genes in Pseudomonas putida.

Cyclic diguanylate (c-di-GMP) is a broadly conserved bacterial signalling molecule that modulates diverse cellular processes, such as biofilm formation, colony morphology and swimming motility. The intracellular level of c-di-GMP is controlled by diguanylate cyclases (DGCs) with GGDEF domain and phosphodiesterases (PDEs) with either EAL or HD-GYP domain. Pseudomonas putida KT2440 has a large group of genes on its genome encoding proteins with GGDEF/EAL/HD-GYP domains. However, phenotypic-genotypic correlation and c-di-GMP metabolism of these genes were largely unknown. Herein, by systematically constructing deletion mutants/overexpression strains of the 42 predicted c-di-GMP metabolism-related genes and analysing the phenotypes, we preliminarily revealed the role of each gene in biofilm formation, colony morphology and swimming motility. Subsequent results from protein sequence alignments and cellular c-di-GMP assessment indicated that 25 out of the 42 genes were likely to encode DGCs, nine genes were predicted to encode PDEs, four genes encoded bifunctional enzymes and the other four genes encoded enzymatically inactive proteins. This study offers a basic understanding of the roles of these 42 genes and can serve as a toolkit for investigators to further elucidate the functions of these GGDEF and EAL/HD-GYP domain-containing proteins.

New sources of Sym2A allele in the pea (Pisum sativum L.) carry the unique variant of candidate LysM-RLK gene LykX.

At the onset of legume-rhizobial symbiosis, the mutual recognition of partners occurs based on a complicated interaction between signal molecules and receptors. Bacterial signal molecules named Nod factors (“nodulation factors”) are perceived by the plant LysM-containing receptor-like kinases (LysM-RLKs) that recognize details of its structure (i.e., unique substitutions), thus providing the conditions particular to symbiosis. In the garden pea (Pisum sativum L.), the allelic state of Sym2 gene has long been reported to regulate the symbiotic specificity: for infection to be successful, plants with the Sym2A allele (for “Sym2 Afghan”, as these genotypes originate mostly from Afghanistan) require an additional acetylation of the Nod factor which is irrelevant for genotypes with the Sym2E allele (for “Sym2 European”). Despite being described about 90 years ago, Sym2 has not yet been cloned, though phenotypic analysis suggests it probably encodes a receptor for the Nod factor. Recently, we described a novel pea gene LykX (PsLykX) from the LysM-RLK gene family that demonstrates a perfect correlation between its allelic state and the symbiotic specificity of the Sym2A-type. Here we report on a series of Middle-Eastern pea genotypes exhibiting the phenotype of narrow symbiotic specificity discovered in the VIR plant genetic resources gene bank (Saint-Petersburg, Russia). These genotypes are new sources of Sym2A, as has been confirmed by an allelism test with Sym2A pea cv. Afghanistan. Within these genotypes, LykX is present either in the allelic state characteristic for cv. Afghanistan, or in another, minor allelic state found in two genotypes from Tajikistan and Turkmenistan. Plants carrying the second allele demonstrate the same block of rhizobial infection as cv. Afghanistan when inoculated with an incompatible strain. Intriguingly, this “Tajik” allele of LykX differs from the “European” one by a single nucleotide polymorphism leading to an R75P change in the receptor part of the putative protein. Thus, our new data are in agreement with the hypothesis concerning the identity of LykX and the elusive Sym2 gene.

Mechanisms by Which Small RNAs Affect Bacterial Activity

The oral cavity contains a distinct habitat that supports diverse bacterial flora. Recent observations have provided additional evidence that sRNAs are key regulators of bacterial physiology and pathogenesis. These sRNAs have been divided into 5 functional groups: cis-encoded RNAs, trans-encoded RNAs, RNA regulators of protein activity, bacterial CRISPR (clustered regularly interspaced short palindromic repeat) RNAs, and a novel category of miRNA-size small RNAs (msRNAs). In this review, we discuss a critical group of key commensal and opportunistic oral pathogens. In general, supragingival bacterial sRNAs function synergistically to fine-tune the regulation of cellular processes and stress responses in adaptation to environmental changes. Particularly in the cariogenic bacteria Streptococcus mutans, both the antisense vicR RNA and msRNA1657 can impede the metabolism of bacterial exopolysaccharides, prevent biofilm formation, and suppress its cariogenicity. In Enterococcus faecalis, selected sRNAs control the expression of proteins involved in diverse cellular processes and stress responses. In subgingival plaques, sRNAs from periodontal pathogens can function as novel bacterial signaling molecules that mediate bacterial-human interactions in periodontal homeostasis. In Porphyromonas gingivalis, the expression profiles of putative sRNA101 and sRNA42 were found to respond to hemin availability after hemin starvation. Regarding Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), a major periodontal pathogen associated with aggressive periodontitis, the predicted sRNAs interact with several virulence genes, including those encoding leukotoxin and cytolethal distending toxin. Furthermore, in clinical isolates, these associated RNAs could be explored not only as potential biomarkers for oral disease monitoring but also as alternative types of regulators for drug design. Thus, this emerging subspecialty of bacterial regulatory RNAs could reshape our understanding of bacterial gene regulation from their key roles of endogenous regulatory RNAs to their activities in pathologic processes.

Nitric Oxide Enters Quorum Sensing via the H-NOX Signaling Pathway in Vibrio parahaemolyticus.

Nitric oxide (NO) plays a major role in the regulation of mammalian biological functions. In recent years, NO has also been implicated in bacterial life cycles, including in the regulation of biofilm formation, and the metabolism of the bacterial second messenger signaling molecule cyclic-di-GMP. In a previous study, we reported the discovery of an NO-responsive quorum sensing (QS) circuit in Vibrio harveyi. Here, we characterize the homologous QS pathway in Vibrio parahaemolyticus. Spectroscopic analysis shows V. parahaemolyticus H-NOX is an NO sensory protein that binds NO in 5/6-coordinated mixed manner. Further, we demonstrate that through ligation to H-NOX, NO inhibits the autophosphorylation activity of an H-NOX-associated histidine kinase (HqsK; H-NOX-associated quorum sensing kinase) that transfers phosphate to the Hpt (histidine-containing phosphotransfer protein) protein LuxU. Indeed, among the three Hpt proteins encoded by V. parahaemolyticus, HqsK transfers phosphate only to the QS-associated phosphotransfer protein LuxU. Finally, we show that NO promotes transcription of the master quorum sensing regulatory gene opaR at low cell density.

Probiotics and Psychobiotics: the Role of Microbial Neurochemicals.

In light of recent data, microorganisms should be construed as organisms that are capable of communication and collective behaviors. Microbial communication signals are involved both in interactions among microbial cells within microbial social systems, including the human body-inhabiting microconsortium, and the dialog between the microbiota and the host organism. The microbiota inhabits various niches of the host organism, especially the gastrointestinal (GI) tract. Microorganisms release diverse signal molecules and, in addition, specifically respond to host signals. This enables them to constantly interact with the nervous system including the brain and the immune system of the host organism. Evolutionarily conserved signals that are involved in the communication between microbiota and the host include neuroactive substances (neurochemicals) such as peptides, amino acids, biogenic amines, short-chain fatty acids, and gaseous substances. This ongoing dialog may either stabilize the host's physical and mental health state or, alternatively, cause serious health problems. Attempts are made to correct imbalances in the brain-gut-microbiota axis with probiotics including their subgroup called psychobiotics that release neuroactive substances directly influencing the human brain, psyche, and behavior. A number of recent review works address the microbiota-host system and its communication signals. Some of the publications focus on the involvement of neurochemicals in the bidirectional communication within the host-microbiota system. However, this work concentrates on the impact of bacterial cell components, metabolites, and signal molecules as promising alternatives to the currently widespread probiotics that have both advantages and disadvantages. Such biologically active agents of microbial origin are referred to as postbiotics or, alternatively, metabiotics (the term preferred in this work).

Recombinant BCG With Bacterial Signaling Molecule Cyclic di-AMP as Endogenous Adjuvant Induces Elevated Immune Responses After Mycobacterium tuberculosis Infection.

Bacillus Calmette-Guerin (BCG) is a live attenuated vaccine against tuberculosis (TB) and remains the most commonly used vaccine worldwide. However, BCG has varied protective efficiency in adults and has safety concerns in immunocompromised population. Thus, effective vaccines are necessary for preventing the prevalence of TB. Cyclic di-AMP (c-di-AMP) is a bacterial second messenger which regulates various cellular processes and host immune response. Previous work found that c-di-AMP regulates bacterial physiological function, pathogenicity and host type I IFN response. In this study, we constructed a recombinant BCG (rBCG) by overexpressing DisA, the diadenylate cyclase of Mycobacterium tuberculosis (Mtb), and observed the physiological changes of rBCG-DisA. The immunological characteristics of rBCG-DisA were investigated on humoral and cellar immune responses in a mice infection model. Our study demonstrated that overexpression of DisA in BCG does not affect the growth but reduces the length of BCG. rBCG-DisA-immunized mice show similar humoral and cellar immune responses in BCG-immunized mice. After Mtb infection, the splenic lymphocytes from both BCG and rBCG-DisA-immunized mice produced more IFN-γ, IL-2, and IL-10 than the un-immunized (UN) mice, while the cytokine levels of the rBCG-DisA group increased significantly than those of the BCG group. The transcription of IFN-β, IL-1β and autophagy related genes (Atgs) were up-regulated in macrophages after treated with c-di-AMP or bacterial infection. The productions of IL-6 were increased after Mtb challenge, especially in the rBCG-DisA-immunized mice. Strikingly, H3K4me3, the epigenetic marker of innate immune memory, was found in both two immunized groups, and the rBCG-DisA group showed stronger expression of H3K4me3 than that of BCG. In addition, the pathological changes of rBCG-DisA immunized mice were similar to that of BCG-immunized mice. The bacterial burdens in the lungs and spleens of BCG- and rBCG-DisA-immunized mice were significantly decreased, but there was no significant difference between the two immunized groups. Together, these results suggested that compared to BCG, rBCG-DisA vaccination, induces stronger immune responses but did not provided additional protection against Mtb infection in this study, which may be related to the innate immunity memory. Hence, c-di-AMP is a promising immunomodulator for a further developed BCG as a better vaccine.

Cell density-dependent auto-inducible promoters for expression of recombinant proteins in Pseudomonas putida.

SummaryInducible promoters such as Plac are of limited usability for industrial protein production with Pseudomonas putida. We therefore utilized cell density‐dependent auto‐inducible promoters for recombinant gene expression in P. putida KT2440 based on the RoxS/RoxR Quorum Sensing (QS) system of the bacterium. To this end, genetic regions upstream of the RoxS/RoxR‐regulated genes ddcA (PR ox132) and PP_3332 (PR ox306) were inserted into plasmids that mediated the expression of superfolder green fluorescent protein (sfGFP) and surface displayed mCherry, confirming their promoter functionalities. Mutation of the Pribnow box of PR ox306 to the σ70 consensus sequence (PR ox3061) resulted in a more than threefold increase of sfGFP production. All three promoters caused cell density‐dependent expression, starting transcription at optical densities (OD 578) of approximately 1.0 (PR ox132, PR ox306) or 0.7 (PR ox3061) as determined by RT‐qPCR. The QS dependency of PR ox306 was further shown by cultivating P. putida in media that had already been used for cultivation and thus contained bacterial signal molecules. The longer P. putida had grown in these media before, the earlier protein expression in freshly inoculated P. putida appeared with PR ox306. This confirmed previous findings that a bacterial compound accumulates within the culture and induces protein expression.

Intestinal lysozyme liberates Nod1 ligands from microbes to direct insulin trafficking in pancreatic beta cells

Long-range communication between intestinal symbiotic bacteria and extra-intestinal organs can occur through circulating bacterial signal molecules, through neural circuits, or through cytokines or hormones from host cells. Here we report that Nod1 ligands derived from intestinal bacteria act as signal molecules and directly modulate insulin trafficking in pancreatic beta cells. The cytosolic peptidoglycan receptor Nod1 and its downstream adapter Rip2 are required for insulin trafficking in beta cells in a cell-autonomous manner. Mechanistically, upon recognizing cognate ligands, Nod1 and Rip2 localize to insulin vesicles, recruiting Rab1a to direct insulin trafficking through the cytoplasm. Importantly, intestinal lysozyme liberates Nod1 ligands into the circulation, thus enabling long-range communication between intestinal microbes and islets. The intestine-islet crosstalk bridged by Nod1 ligands modulates host glucose tolerance. Our study defines a new type of inter-organ communication based on circulating bacterial signal molecules, which has broad implications for understanding the mutualistic relationship between microbes and host.

Functional Specialization in Vibrio cholerae Diguanylate Cyclases: Distinct Modes of Motility Suppression and c-di-GMP Production.

Vibrio cholerae biofilm formation and associated motility suppression are correlated with increased concentrations of cyclic diguanylate monophosphate (c-di-GMP), which are in turn driven by increased levels and/or activity of diguanylate cyclases (DGCs). To further our understanding of how c-di-GMP modulators in V. cholerae individually and collectively influence motility with cellular resolution, we determined how DGCs CdgD and CdgH impact intracellular c-di-GMP levels, motility, and biofilm formation. Our results indicated that CdgH strongly influences swim speed distributions; cells in which cdgH was deleted had higher average swim speeds than wild-type cells. Furthermore, our results suggest that CdgD, rather than CdgH, is the dominant DGC responsible for postattachment c-di-GMP production in biofilms. Lipopolysaccharide (LPS) biosynthesis genes were found to be extragenic bypass suppressors of the motility phenotypes of strains ΔcdgD and ΔcdgH We compared the motility regulation mechanism of the DGCs with that of Gmd, an LPS O-antigen biosynthesis protein, and discovered that comodulation of c-di-GMP levels by these motility effectors can be positively or negatively cooperative rather than simply additive. Taken together, these results suggest that different environmental and metabolic inputs orchestrate DGC responses of V. cholerae via c-di-GMP production and motility modulation.IMPORTANCE Cyclic diguanylate monophosphate (c-di-GMP) is a broadly conserved bacterial signaling molecule that affects motility, biofilm formation, and virulence. Although it has been known that high intracellular concentrations of c-di-GMP correlate with motility suppression and biofilm formation, how the 53 predicted c-di-GMP modulators in Vibrio cholerae collectively influence motility is not understood in detail. Here we used a combination of plate assays and single-cell tracking methods to correlate motility and biofilm formation outcomes with specific enzymes involved in c-di-GMP synthesis in Vibrio cholerae, the causative agent of the disease cholera.

An Improved Strategy for the Chemical Synthesis of 3',5'-Cyclic Diguanylic Acid.

The physiological functions of c-di-GMP and its involvement in many key processes led to its recognition as a major and ubiquitous bacterial second messenger. Aside from being a bacterial signaling molecule, c-di-GMP is also an immunostimulatory molecule capable of inducing innate and adaptive immune responses through maturation of immune mammalian cells. Given the broad biological functions of c-di-GMP and its potential applications as a nucleic-acid-based drug, the chemical synthesis of c-di-GMP has drawn considerable interest. An improved phosphoramidite approach to the synthesis of c-di-GMP is reported herein. The synthetic approach is based on the use of a 5'-O-formyl protecting group, which can be rapidly and chemoselectively cleaved from a key dinucleotide phosphoramidite intermediate to enable a cyclocondensation reaction leading to a fully protected c-di-GMP product in a yield ∼80%. The native c-di-GMP is isolated, after complete deprotection, in an overall yield of 36% based on the commercial ribonucleoside used as starting material. © 2019 by John Wiley & Sons, Inc.

The DELLA Proteins Influence the Expression of Cytokinin Biosynthesis and Response Genes During Nodulation.

The key event that initiates nodule organogenesis is the perception of bacterial signal molecules, the Nod factors, triggering a complex of responses in epidermal and cortical cells of the root. The Nod factor signaling pathway interacts with plant hormones, including cytokinins and gibberellins. Activation of cytokinin signaling through the homeodomain-containing transcription factors KNOX is essential for nodule formation. The main regulators of gibberellin signaling, the DELLA proteins are also involved in regulation of nodule formation. However, the interaction between the cytokinin and gibberellin signaling pathways is not fully understood. Here, we show in Pisum sativum L. that the DELLA proteins can activate the expression of KNOX and BELL transcription factors involved in regulation of cytokinin metabolic and response genes. Consistently, pea la cry-s (della1 della2) mutant showed reduced ability to upregulate expression of some cytokinin metabolic genes during nodulation. Our results suggest that DELLA proteins may regulate cytokinin metabolism upon nodulation.

In vitro Interactions of Pseudomonas aeruginosa With Scedosporium Species Frequently Associated With Cystic Fibrosis.

Members of the Scedosporium apiospermum species complex are the second most frequently isolated pathogens after Aspergillus fumigatus from cystic fibrosis (CF) patients with fungal pulmonary infections. Even so, the main risk factors for the infection are unrevealed. According to previous studies, bacterial infections might reduce the risk of a fungal infection, but an antibacterial therapy may contribute to the airway colonization by several fungal pathogens. Furthermore, corticosteroids, which are often used to reduce lung inflammation in children and adults with CF, are also proved to enhance the growth of A. fumigatus in vitro. Considering all the above discussed points, we aimed to test how Pseudomonas aeruginosa influences the growth of scedosporia and to investigate the potential effect of commonly applied antibacterial agents and corticosteroids on Scedosporium species. Direct interactions between fungal and bacterial strains were tested using the disk inhibition method. Indirect interactions via volatile compounds were investigated by the plate-in-plate method, while the effect of bacterial media-soluble molecules was tested using a modified cellophane assay and also in liquid culture media conditioned by P. aeruginosa. To test the effect of bacterial signal molecules, antibacterial agents and corticosteroids on the fungal growth, the broth microdilution method was used. We also investigated the germination ability of Scedosporium conidia in the presence of pyocyanin and diffusible signal factor by microscopy. According to our results, P. aeruginosa either inhibited or enhanced the growth of scedosporia depending on the culture conditions and the mode of interactions. When the two pathogens were cultured physically separately from each other in the plate-in-plate tests, the presence of the bacteria was able to stimulate the growth of several fungal isolates. While in direct physical contact, bacterial strains inhibited the fungal growth. This effect might be attributed to bacterial signal molecules, which also proved to inhibit the germination and growth of scedosporia. In addition, antibacterial agents showed growth-promoting, while corticosteroids exhibited growth inhibitory effect on several Scedosporium isolates. These data raise the possibility that a P. aeruginosa infection or a previously administered antibacterial therapy might be able to increase the chance of a Scedosporium colonization in a CF lung.

CuO nanoparticles as haloperoxidase-mimics: Chloride-accelerated heterogeneous Cu-Fenton chemistry for H2O2 and glucose sensing

CuO nanoparticles (CuO NPs) were demonstrated as a novel nanoenzyme, i.e., haloperoxidase mimic, which catalyzes the oxidation of halide ions to form reactive halogen species (RHS). Two functions of CuO NPs have been demonstrated in the work. First, RHS halogenate bacterial signaling molecules, thus preventing biofilm formation. Second, CuO is used in sensitive colorimetric assays for H2O2 and glucose.

Preliminary study testing the effects of tea and coffee on sludge characteristics and N-butyryl-l-homoserine lactone in an MBR system

ABSTRACTMembrane bioreactors (MBRs) are rapidly developing systems widely used for the treatment of municipal and industrial wastewater prior to their discharge into the environment. Membrane fouling is the most important challenge in MBRs, because it negatively affects membrane performance, resulting in low permeate flux, higher transmembrane pressure and frequent membrane cleaning/replacement. In this study, the effect of tea and coffee on sludge characteristics, membrane fouling and the bacterial signalling molecule N-butyryl-l-homoserine lactone (C4-HSL) in a membrane bioreactor system were investigated. For this purpose, four different continuously operated membrane bioreactors, treating synthetic domestic wastewater and dairy wastewater were operated in parallel under the same conditions. The results indicated that the soluble microbial products (SMP) and the extracellular polymeric substances (EPS) in the reactors showed similar trends for both synthetic domestic and dairy wastewater, where protein levels were higher than carbohydrate levels. Other parameters such as floc size, zeta potential, hydrophobicity, sludge volume index and capillary suction time were also analysed prior to and after the addition of coffee and tea. Analysis of the wastewater samples by GC-MS revealed that coffee and tea significantly enhance the extraction efficiency of C4-HSL. The results obtained in this study can serve as a good basis for further research.

Characterization of a novel N-acylhomoserine lactonase, AidP, from Antarctic Planococcus sp.

BackgroundN-acylhomoserine lactones (AHLs) are well-studied signalling molecules produced by some Gram-negative Proteobacteria for bacterial cell-to-cell communication or quorum sensing. We have previously demonstrated the degradation of AHLs by an Antarctic bacterium, Planococcus versutus L10.15T, at low temperature through the production of an AHL lactonase. In this study, we cloned the AHL lactonase gene and characterized the purified novel enzyme.ResultsRapid resolution liquid chromatography analysis indicated that purified AidP possesses high AHL-degrading activity on unsubstituted, and 3-oxo substituted homoserine lactones. Liquid chromatography–mass spectrometry analysis confirmed that AidP functions as an AHL lactonase that hydrolyzes the ester bond of the homoserine lactone ring of AHLs. Multiple sequence alignment analysis and phylogenetic analysis suggested that the aidP gene encodes a novel AHL lactonase enzyme. The amino acid composition analysis of aidP and the homologous genes suggested that it might be a cold-adapted enzyme, however, the optimum temperature is 28 °C, even though the thermal stability is low (reduced drastically above 32 °C). Branch-site analysis of several aidP genes of Planococcus sp. branch on the phylogenetic trees also showed evidence of episodic positive selection of the gene in cold environments. Furthermore, we demonstrated the effects of covalent and ionic bonding, showing that Zn2+ is important for activity of AidP in vivo. The pectinolytic inhibition assay confirmed that this enzyme attenuated the pathogenicity of the plant pathogen Pectobacterium carotovorum in Chinese cabbage.ConclusionWe demonstrated that AidP is effective in attenuating the pathogenicity of P. carotovorum, a plant pathogen that causes soft-rot disease. This anti-quorum sensing agent is an enzyme with low thermal stability that degrades the bacterial signalling molecules (AHLs) that are produced by many pathogens. Since the enzyme is most active below human body temperature (below 28 °C), and lose its activity drastically above 32 °C, the results of a pectinolytic inhibition assay using Chinese cabbage indicated the potential of this anti-quorum sensing agent to be safely applied in the field trials.

Physiological response of Lactuca sativa exposed to 2-nonanone emitted by Bacillus sp. BCT9

Volatile organic compounds (VOCs) released from bacterial species have been reported as plant growth inducers. In this sense, Lactuca sativa was used as model vegetable to prospect the effects of 2-nonanone released by Bacillus sp. BCT9 at cellular and organ structure level, so we present preliminary results about the physiological effects. In this study, 2-day-old L. sativa were exposed to 2-nonanone for 10 days under two delivery systems: 1) 2-nonanone (abrupt delivery) and 2) 2-nonanone + lanolin (controlled delivery). The X-ray elemental microanalysis, scanning electron and confocal laser microscopies techniques were used to evaluate physiological changes “in vivo” conditions. The results indicated that 2-nonanone increased root and shoot length independently of 2-nonanone delivery system after 7 days of exposition. Additionally, 2-nonanone elicited the increase of anthocyanin and not affects chlorophyll content and electrolyte leakage percentage. The abrupt delivery elicited the increase of both length and density of root hair without causing changes in size of cell epidermis, while controlled delivery induced stomatal opening. Besides, 2-nonanone exposition did not modify the composition and distribution of carbon, nitrogen, phosphorus, potassium, and chlorine in the surface of plant tissue. The results suggested that 2-nonanone acts as a bacterial signal molecule to elicit changes related to root development without damaging the external morphology while epidermal cells at leaf level are not affected, suggesting that 2-nonanone can be an important tool to apply to vegetables.