The small regulatory RNA molecule MicA is involved in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium biofilm formation

Background  

LuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules.     

Potential for <Emphasis Type="Italic">luxS</Emphasis> related signalling in marine bacteria and production of autoinducer-2 in the genus <Emphasis Type="Italic">Shewanella</Emphasis>

Background  

The autoinducer-2 (AI-2) group of signalling molecules are produced by both Gram positive and Gram negative bacteria as the by-product of a metabolic transformation carried out by the LuxS enzyme. They are the only non species-specific quorum sensing compounds presently known in bacteria. The luxS gene coding for the AI-2 synthase enzyme was found in many important pathogens. Here, we surveyed its occurrence in a collection of 165 marine isolates belonging to abundant marine phyla using conserved degenerated PCR primers and sequencing of selected positive bands to determine if the presence of the luxS gene is phylogenetically conserved or dependent on the habitat.     

In <Emphasis Type="Italic">Helicobacter pylori</Emphasis> auto-inducer-2, but not LuxS/MccAB catalysed reverse transsulphuration,regulates motility through modulation of flagellar gene transcription

Background  

LuxS may function as a metabolic enzyme or as the synthase of a quorum sensing signalling molecule, auto-inducer-2 (AI-2); hence, the mechanism underlying phenotypic changes upon luxS inactivation is not always clear. In Helicobacter pylori, we have recently shown that, rather than functioning in recycling methionine as in most bacteria, LuxS (along with newly-characterised MccA and MccB), synthesises cysteine via reverse transsulphuration. In this study, we investigated whether and how LuxS controls motility of H. pylori, specifically if it has its effects via luxS-required cysteine metabolism or via AI-2 synthesis only.     

Lack of genomic evidence of AI-2 receptors suggests a non-quorum sensing role for <Emphasis Type="Italic">luxS</Emphasis> in most bacteria

Background  

Great excitement accompanied discoveries over the last decade in several Gram-negative and Gram-positive bacteria of the LuxS protein, which catalyzes production of the AI-2 autoinducer molecule for a second quorum sensing system (QS-2). Since the luxS gene was found to be widespread among the most diverse bacterial taxa, it was hypothesized that AI-2 may constitute the basis of a universal microbial language, a kind of bacterial Esperanto. Many of the studies published in this field have drawn a direct correlation between the occurrence of the luxS gene in a given organism and the presence and functionality of a QS-2 therein. However, rarely hathe existence of potential AI-2 receptors been examined. This is important, since it is now well recognized that LuxS also holds a central role as a metabolic enzyme in the activated methyl cycle which is responsible for the generation of S-adenosyl-L-methionine, the major methyl donor in the cell.     

Expression of a bacterial effector,harpin N,causes increased resistance to fire blight in <Emphasis Type="Italic">Pyrus communis</Emphasis>

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. In order to create novel mechanisms for fire blight resistance in pear, we have generated transgenic pears expressing the elicitor harpin N_ea from Erwinia amylovora under the control of the constitutive promoter CaMV35S. The transient expression of hrpN _Ea in pear cells did not provoke any apparent damage. Therefore, stable constitutive expression of hrpN _Ea was studied in seventeen transgenic clones of the very susceptible cultivar “Passe Crassane.” Most transgenic clones displayed significant reduction of susceptibility to fire blight in vitro when inoculated by E. amylovora, which was positively correlated to their degree of expression of the transgene hrpN _Ea . These results indicate that ectopic expression of a bacterial elicitor such as harpin N_ea is a promising way to improve pear resistance to fire blight.     

<Emphasis Type="Italic">luxRI</Emphasis> homologs are universally present in the genus <Emphasis Type="Italic">Aeromonas</Emphasis>

Background  

Aeromonas spp. have been regarded as "emerging pathogens". Aeromonads possess multifactorial virulence and the production of many of these virulence determinants is associated with high cell density, a phenomenon that might be regulated by quorum sensing. However, only two species of the genus are reported to possess the luxRI quorum sensing gene homologs. The purpose of this study was to investigate if the luxRI homologs are universally present in the Aeromonas strains collected from various culture collections, clinical laboratories and field studies.     

Role of RpoS in stress survival,synthesis of extracellular autoinducer 2, and virulence in <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

Vibrio alginolyticus, a marine bacterium, is an opportunistic pathogen capable of causing vibriosis with high mortality to fishes in the South China Sea. Stress resistance is very important for its survival in the natural environment and upon infection of the host. RpoS, an alternative sigma factor, is considered as an important regulator involved in stress response and virulence in many pathogens. In this study, the rpoS gene was cloned and characterized to evaluate the role of RpoS in V. alginolyticus. The predicted protein showed high identity with other reported rpoS gene products. The in-frame deleted mutation of rpoS in V. alginolyticus led to sensitivity of the strain to ethanol, hyperosmolarity, heat, and hydrogen peroxide challenges. Further studies showed that extracellular autoinducer 2 level, four of seven detected protease activities, and cytotoxicity of extracellular products were markedly decreased in the rpoS mutant compared with that in the wild-type strain. The results indicated that the global regulator RpoS was part of the regulatory networks of virulence and LuxS quorum sensing system.     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.     

GacS-dependent regulation of enzymic and antifungal activities and synthesis of <Emphasis Type="Italic">N</Emphasis>-acylhomoserine lactones in rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

Pseudomonas chlororaphis strain 449 isolated from the rhizosphere of maize suppresses numerous plant pathogens in vitro. The strain produces phenazine antibiotics and synthesizes at least three types of quorum sensing signaling molecules, N-acylhomoserine lactones. Here we have shown that the rhizospheric P. chlororaphis strains 449, well known strain 30–84 as well as two other P. chlororaphis strains exhibit polygalacturonase activity. Using mini-Tn5 transposon mutagenesis, four independent mutants of strain P. chlororaphis 449 with insertion of mini-Tn5 Km2 in gene gacS of two-component GacA-GacS system of global regulation were selected. All these mutant strains were deficient in production of extracellular proteinase(s), phenazines, N-acylhomoserine lactones synthesis, and did not inhibit the growth of G⁺ bacteria in comparison with the wild type strain. The P. chlororaphis 449-06 gacS ⁻ mutant studied in greater detail was deficient in polygalacturonase, pectin methylesterase activities, swarming motility and antifungal activity. It is the first time the involvement of GacA-GacS system in the regulation of enzymes of pectin metabolism, polygalacturonase and pectin methylesterase, was demonstrated in fluorescent pseudomonads.     

PCR Detection of <Emphasis Type="Italic">Serratia</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> Using Primers Targeting <Emphasis Type="Italic">pfs</Emphasis> and <Emphasis Type="Italic">luxS</Emphasis> Genes Involved in AI-2-Dependent Quorum Sensing

Quorum sensing is the ability of bacteria to communicate and coordinate behavior emitting signaling molecules. A series of primers for PCR detection of Serratia spp. has been designed using as targets the pfs and luxS genes involved in AI-2-dependent quorum sensing. The identities of the PCR products (193 and 102 bp) were confirmed by commercial sequencing. Twenty-seven Serratia strains (representing 10 different species) tested positive for the presence of the pfs and luxS genes, while a total of 7 different species of non-Serratia (25 strains) were tested and gave negative results. The sensitivity and specificity of the pfs- and luxS-based PCR assay were also checked in artificially contaminated bacterial samples. In this study we established a novel method to detect Serratia using quorum-sensing genes as diagnostic markers.     

New ubiquitous translocators: amino acid export by<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis>

Molecular access to amino acid excretion by Corynebacterium glutamicum and Escherichia coli led to the identification of structurally novel carriers and novel carrier functions. The exporters LysE, RhtB, ThrE and BrnFE each represent the protoype of new transporter families, which are in part distributed throughout all of the kingdoms of life. LysE of C. glutamicum catalytes the export of basic amino acids. The expression of the carrier gene is regulated by the cell-internal concentration of basic amino acids. This serves, for example, to maintain homoeostasis if an excess of l-lysine or l-arginine inside the cell should arise during growth on complex media. RhtB is one of five paralogous systems in E. coli, of which at least two are relevant for l-threonine production. A third system is relevant for l-cysteine production. It is speculated that the physiological function of these paralogues is related to quorum sensing. ThrE of C. glutamicum exports l-threonine and l-serine. However, a ThrE domain with a putative hydrolytic function points to an as yet unknown role of this exporter. BrnFE in C. glutamicum is a two-component permease exporting branched-chained amino acids from the cell, and an orthologue in B. subtilis exports 4-azaleucine.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

2D proteome analysis initiates new Insights on the Salmonella Typhimurium LuxS protein

Background  

Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2.     

Detection of quorum-sensing-related molecules in <Emphasis Type="Italic">Vibrio scophthalmi</Emphasis>

Background  

Cell-to-cell communication (also referred to as quorum sensing) based on N-acyl-homoserine lactones (AHLs) is a widespread response to environmental change in Gram-negative bacteria. AHLs seem to be highly variable, both in terms of the acyl chain length and in the chemical structure of the radicals. Another quorum sensing pathway, the autoinducer-2-based system, is present both in Gram-positive and Gram-negative bacteria. In this study the presence of signal molecules belonging to both quorum sensing signalling pathways was analysed in the marine symbiotic species Vibrio scophthalmi.     

Rice bacterial blight pathogen <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> produces multiple DSF-family signals in regulation of virulence factor production

Background  

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease. Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence. Genetic and genomics evidence suggest that Xoo might use the diffusible signal factor (DSF) type quorum sensing (QS) system to regulate the virulence factor production. However, little is known about the chemical structure of the DSF-like signal(s) produced by Xoo and the factors influencing the signal production.     

Identification of a suppressor gene for the arginine-auxotrophic <Emphasis Type="Italic">argJ</Emphasis> mutation in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

We recently proposed a metabolic engineering strategy for l-ornithine production based on the hypothesis that an increased intracellular supply of N-acetylglutamate may further enhance l-ornithine production in a well-defined recombinant strain of Corynebacterium glutamicum. In this work, an argJ-deficient arginine auxotrophic mutant of C. glutamicum is suppressed by a different locus of C. glutamicum ATCC13032. Overexpression of the NCgl1469 open reading frame (ORF), exhibiting N-acetylglutamate synthase (NAGS) activity, was able to complement the C. glutamicum arginine-auxotrophic argJ strain and showed increased NAGS activity from 0.03 to 0.17 units mg⁻¹ protein. Additionally, overexpression of the NCgl1469 ORF resulted in a 39% increase in excreted l-ornithine. These results indicate that the intracellular supply of N-acetylglutamate is a rate-limiting step during l-ornithine production in C. glutamicum.     

Identification and Characterization of <Emphasis Type="Italic">sanH</Emphasis> and <Emphasis Type="Italic">sanI</Emphasis> Involved in the Hydroxylation of Pyridyl Residue During Nikkomycin Biosynthesis in <Emphasis Type="Italic">Streptomyces ansochromogenes</Emphasis>

Nikkomycins are highly potent inhibitors of chitin synthase. The nikkomycin biosynthetic gene cluster has been cloned from Streptomyces asochromogenes. Two cytochrome P450 monooxygenase genes (sanQ, sanH) and one ferredoxin gene (sanI) were found in the cluster. It was reported that SanQ is involved in the hydroxylation of l-His, a key step in 4-formyl-4-imidazolin-2-one base biosynthesis. Here, we have studied the function of sanH and sanI. Disruption of sanH abolished the production of nikkomycin X and Z, but it accumulated one dominant component nikkomycin Lx, which is the nikkomycin X analog lacking the hydroxy group at the pyridyl residue. The sanI disruption mutant accumulated predominantly nikkomycin Lx in addition to nikkomycin X and Z. The nikkomycin production profile of the sanH and sanI double disruption mutant was the same as that of the sanH disruption mutant. These results confirmed that SanH is essential for the hydroxylation of pyridyl residue in nikkomycin biosynthesis of S. ansochromogenes and first demonstrated that SanI is an effective electron donor for SanH, but not for SanQ in vivo.