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群体感应(Quorum sensing,QS)是近来受到广泛关注的一种细菌群体行为调控机制,通过感应一些信号分子如酰基高丝氨酸环内酯(acyl-homoserine lactone,AHL)来判断菌群密度和周围环境变化,假单胞菌中同样也有AHL信号分子,当信号达到一定的浓度阈值时,能启动菌体中相关基因的表达来适应环境中的变化,从而调节菌体的群体行为(如致病性及群体生长调节)。众多报道说明了假单胞菌的群体感应调节系统是由一些全面的调节子所调控的。本文系统介绍了假单胞菌群体感应调控系统,并分析假单胞菌在该系统中复杂的应答反应。 相似文献
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Aims: To investigate if Burkholderia glumae can produce rhamnolipids, define a culture medium for good production yields, analyse their composition and determine their tensioactive properties. Methods and Results: Burkholderia glumae AU6208 produces a large spectrum of mono‐ and di‐rhamnolipid congeners with side chains varying between C12‐C12 and C16‐C16, the most abundant being Rha‐Rha‐C14‐C14.The effects on rhamnolipid production of the cultivation temperature, nitrogen and carbon source were investigated. With urea as the nitrogen source and canola oil as the carbon source, a production of 1000·7 mg l?1 was reached after 6 days. These rhamnolipids display a critical micelle concentration of 25–27 mg l?1 and decrease the interfacial tension against hexadecane from 40 to 1·8 mN m?1. They also have excellent emulsifying properties against long chain alkanes. Conclusions: Burkholderia glumae AU6208 can produce considerable amounts of rhamnolipids. They are produced as diversified mixtures of congeners. Their side chains are longer than those normally produced by those of Pseudomonas aeruginosa. They also present excellent tensioactive properties. Significance and Impact of the Study: In contrast with the classical rhamnolipid producer Ps. aeruginosa, B. glumae is not a pathogen to humans. This work shows that the industrial production of rhamnolipids with this species could be easier than with Ps. aeruginosa. 相似文献
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Christian Garde Michael Givskov Morten Hentzer Kim Sneppen Thomas Sams 《Journal of molecular biology》2010,396(4):849-857
We present detailed results on the C4-HSL-mediated quorum sensing (QS) regulatory system of the opportunistic Gram-negative bacterium Aeromonas hydrophila. This bacterium contains a particularly simple QS system that allows for a detailed modeling of kinetics. In a model system (i.e., the Escherichia coli monitor strain MH205), the C4-HSL production of A. hydrophila is interrupted by fusion of gfp(ASV). In the present in vitro study, we measure the response of the QS regulatory ahyRI locus in the monitor strain to predetermined concentrations of C4-HSL signal molecules. A minimal kinetic model describes the data well. It can be solved analytically, providing substantial insight into the QS mechanism: at high concentrations of signal molecules, a slow decay of the activated regulator sets the timescale for the QS regulation loop. Slow saturation ensures that, in an A. hydrophila cell, the QS system is activated only by signal molecules produced by other A. hydrophila cells. Separate information on the ahyR and ahyI loci can be extracted, thus allowing the probe to be used in identifying the target when testing QS inhibitors. 相似文献
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Quorum Sensing in Burkholderia cepacia: Identification of the LuxRI Homologs CepRI 总被引：6，自引：0，他引：6 
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下载免费PDF全文 Shawn Lewenza Barbara Conway E. P. Greenberg Pamela A. Sokol 《Journal of bacteriology》1999,181(3):748-756
Burkholderia cepacia has emerged as an important pathogen in patients with cystic fibrosis. Many gram-negative pathogens regulate the production of extracellular virulence factors by a cell density-dependent mechanism termed quorum sensing, which involves production of diffusible N-acylated homoserine lactone signal molecules, called autoinducers. Transposon insertion mutants of B. cepacia K56-2 which hyperproduced siderophores on chrome azurol S agar were identified. One mutant, K56-R2, contained an insertion in a luxR homolog that was designated cepR. The flanking DNA region was used to clone the wild-type copy of cepR. Sequence analysis revealed the presence of cepI, a luxI homolog, located 727 bp upstream and divergently transcribed from cepR. A lux box-like sequence was identified upstream of cepI. CepR was 36% identical to Pseudomonas aeruginosa RhlR and 67% identical to SolR of Ralstonia solanacearum. CepI was 38% identical to RhlI and 64% identical to SolI. K56-R2 demonstrated a 67% increase in the production of the siderophore ornibactin, was protease negative on dialyzed brain heart infusion milk agar, and produced 45% less lipase activity in comparison to the parental strain. Complementation of a cepR mutation restored parental levels of ornibactin and protease but not lipase. An N-acylhomoserine lactone was purified from culture fluids and identified as N-octanoylhomoserine lactone. K56-I2, a cepI mutant, was created and shown not to produce N-octanoylhomoserine lactone. K56-I2 hyperproduced ornibactin and did not produce protease. These data suggest both a positive and negative role for cepIR in the regulation of extracellular virulence factor production by B. cepacia. 相似文献
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Quorum sensing in Serratia marcescens, which uses two types of signaling molecules–N-acyl homoserine lactones and furanosyl borate diester–play important regulatory roles in the synthesis of 2,3-butanediol
and prodigiosin. In the hope of understanding the effect of quorum sensing on physiologic metabolism, we established two molecular
strategies, one to express acyl-homoserine lactone hydrolase to inactivate AI-1 signaling molecule using an expression vector
with lactose as the inducer and the other to mutate luxS gene with a suicide plasmid pUTKm2 to inhibit the synthesis of AI-2 signaling molecule. 相似文献
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Minor Pilins of the Type IV Pilus System Participate in the Negative Regulation of Swarming Motility
Pseudomonas aeruginosa exhibits distinct surface-associated behaviors, including biofilm formation, flagellum-mediated swarming motility, and type IV pilus-driven twitching. Here, we report a role for the minor pilins, PilW and PilX, components of the type IV pilus assembly machinery, in the repression of swarming motility. Mutating either the pilW or pilX gene alleviates the inhibition of swarming motility observed for strains with elevated levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP) due to loss of BifA, a c-di-GMP-degrading phosphodiesterase. Blocking PilD peptidase-mediated processing of PilW and PilX renders the unprocessed proteins defective for pilus assembly but still functional in c-di-GMP-mediated swarming repression, indicating our ability to separate these functions. Strains with mutations in pilW or pilX also fail to exhibit the increase in c-di-GMP levels observed when wild-type (WT) or bifA mutant cells are grown on a surface. We also provide data showing that c-di-GMP levels are increased upon PilY1 overexpression in surface-grown cells and that this c-di-GMP increase does not occur in the absence of the SadC diguanylate cyclase. Increased levels of endogenous PilY1, PilX, and PilA are observed when cells are grown on a surface compared to liquid growth, linking surface growth and enhanced signaling via SadC. Our data support a model wherein PilW, PilX, and PilY1, in addition to their role(s) in type IV pilus biogenesis, function to repress swarming via modulation of intracellular c-di-GMP levels. By doing so, these pilus assembly proteins contribute to P. aeruginosa's ability to coordinately regulate biofilm formation with its two surface motility systems. 相似文献
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Veselova MA Lipasova VA Zaĭtseva IuV Koksharova OA Chernukha MIu Romanova IuM Khmel' IA 《Genetika》2012,48(5):608-616
By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice. 相似文献
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We redemonstrate that SwrA is essential for swarming motility in Bacillus subtilis, and we reassert that laboratory strains of B. subtilis do not swarm. Additionally, we find that a number of other genes, previously reported to be required for swarming in laboratory strains, are dispensable for robust swarming motility in an undomesticated strain. We attribute discrepancies in the literature to a lack of reproducible standard experimental conditions, selection for spontaneous swarming suppressors, inadvertent genetic linkage to swarming mutations, and auxotrophy.Many species of bacteria are capable of flagellum-mediated swimming motility in liquid broth. Of those species, a subset is also capable of a related, but genetically separable, form of flagellum-mediated surface movement called swarming motility (17). Examples of swarming-proficient species include Proteus mirabilis, Vibrio parahaemolyticus, Serratia marcescens, Escherichia coli, Salmonella enterica, and Bacillus subtilis (1, 15, 16, 20, 28). In general, swarming requires a surfactant or wetting agent to reduce surface tension, an increase in flagellar number per cell, and other genetic features that are distinct from swimming (7, 14).There is confusion in the literature concerning the genetic requirements of the swarming phenotype of B. subtilis. It is generally accepted that the ancestral undomesticated strain B. subtilis 3610 exhibits robust swarming motility (18, 20, 33). Swarming motility of strain 3610 requires the production of a secreted surfactant, called surfactin (6, 20), to reduce surface tension and permit surface spreading, and it also requires the protein SwrA to activate flagellar biosynthesis gene expression and increase the number of flagella on the cell surface (5, 20). Some reports claim that domesticated derivatives of 3610, such as the commonly used laboratory strain 168, are also swarming proficient (10, 18, 19, 24). Strain 168, however, is defective in both surfactin production (9, 25) and SwrA (5, 21, 31), and thus, swarming 168 strains challenge the genetic definition of swarming motility. Our lab has never observed swarming in laboratory strains, and here we investigated swarming motility in a reportedly swarming-proficient 168 strain.We obtained a reportedly swarming-proficient 168 strain (13) (generous gift of Simone Séror, Orsay University, Paris-Sud, France) (Table (Table1)1) and compared its swarming phenotype to that of 3610 under our standard conditions (20). Swarm plates were prepared one day prior to use with 25 ml of LB medium (10 g Bacto tryptone, 5 g Bacto yeast extract, 5 g NaCl per liter) fortified with 0.7% Bacto agar. To minimize water on the agar surface and thus minimize the potentially confounding influence of swimming motility, plates were dried 20 min prior to inoculation and 10 min postinoculation open-faced in a laminar flow hood. For qualitative swarm assays, plates were centrally inoculated with cells from a freshly grown overnight colony using a sterile stick. For quantitative swarm expansion assays, 1 ml of cells grown to mid-exponential phase (optical density at 600 nm [OD600], 0.5) was resuspended in PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 per liter, pH 7.0) containing 0.5% India ink (Higgins) to an OD600 of 10 and centrally spotted (10 μl). Swarm expansion was measured at 0.5-h intervals along a transect on the plate. Plates were incubated at 37°C in 20 to 30% humidity. Whereas strain 3610 was swarming proficient, strain 168 (Orsay) was swarming deficient (Fig. (Fig.1A).1A). Thus, strain 168 (Orsay) appeared to behave similarly to all other laboratory strains we have tested previously (20, 21).Open in a separate windowFIG. 1.Swarming motility on LB and B media. In qualitative plate images, colonized agar appears white and uncolonized agar appears black on LB and B media, as indicated. Swarming cells colonize a larger surface area than nonswarming cells. All strains are derivatives of strain 3610 unless otherwise indicated. Bar, 2 cm. (A) Quantitative swarm expansion assays on solid medium and growth in liquid medium of the indicated strains on LB medium (closed symbols) and on B medium (open symbols). To indicate variability in a particular experiment, we have reproduced the quantitative swarm expansion assay of strain 3610 on LB and B media with error bars in Fig. S5 in the supplemental material. (B) Quantitative swarm expansion assays on LB (closed symbols) and B (open symbols) media. The following strains were used: DS3337 (sfp), DS2415 (swrA), DS5106 (168 swrA+), DS5758 (168 sfp+), and DS5759 (168 swrA+ sfp+). In all assays, B medium was made according to reference 2 except for strain DS5759, for which B medium was supplemented with 780 μM threonine to compensate for thrC auxotrophy. (C) Swarm plates of the indicated strains on LB medium made with equal parts peptone instead of tryptone. (D) Quantitative swarm expansion assays of the indicated 3610-derived mutant strains on LB medium (closed symbols) and on B medium (open symbols). The following strains were used: DS72 (yvzB), DS2268 (epr), DS3903 (phrC), DS4978 (rapC), DS4979 (oppD), DS2509 (swrB), and DS3649 (degU). All points are averages for three replicates.
Open in a separate windowaAll strains are in the 3610 genetic background unless otherwise indicated.We next explored the genetic basis for the swarming defect we observed in strain 168 (Orsay). As with other laboratory strains, colonies of strain 168 (Orsay) failed to produce the transparent ring normally indicative of surfactin production, due to a mutation of the gene sfp (25). Complementation with the wild-type sfp gene in 168 was sufficient to restore surfactin production but was insufficient to restore swarming motility (Fig. (Fig.1B)1B) (20). Laboratory strains also fail to swarm because of a loss-of-function frameshift mutation in the gene encoding SwrA (5, 21). Sequencing of the swrA gene confirmed that strain 168 (Orsay) contained the frameshift mutation, but introduction of a swrA complementation construct at an ectopic site in the chromosome (amyE::PswrA-swrA) was also insufficient to restore swarming motility (Fig. (Fig.1B).1B). Swarming motility was fully rescued, however, when sfp and swrA were simultaneously complemented in the 168 strain (Fig. (Fig.1B)1B) or when the swrA frameshift mutation was repaired in spontaneous suppressors isolated from 168 complemented with sfp alone (see Fig. S1 in the supplemental material). Furthermore, mutation of either sfp or swrA in the 3610 genetic background abolished swarming (Fig. (Fig.1B).1B). We conclude that Sfp and SwrA are necessary for swarming. We further conclude that, with respect to swarming motility, strain 168 (Orsay) is genetically no different from any other laboratory strain we have tested, as it fails to swarm due to simultaneous defects in Sfp and SwrA (21). We infer that the apparent swarming observed in some laboratory strains is not due to genetic differences but rather due to differences in experimental conditions.In our swarming assays, we take steps to minimize surface water. In some cases of the reported swarming of strain 168, plates were poured 1 h before use, dried for 5 min, and incubated at 60 to 70% humidity (13). When 0.7% agar LB plates were freshly poured and not dried, we noticed that toothpick inoculation of the cells disturbed the agar surface and caused a pool of water to well forth from the agar (see Fig. S2 in the supplemental material). Pools of water emerged even when the plates were dried for 5 or 10 min prior to inoculation, but water did not emerge when the plates were dried for 15 min or longer (see Fig. S2 in the supplemental material). The colony size of strain 168 was proportional to the amount of water extracted from the agar, but the cells did not exhibit swarming motility (see Fig. S2 in the supplemental material). We conclude that excess water was not sufficient to promote swarming of the laboratory strain. Nonetheless, we recommend drying plates for 20 min prior to inoculation to minimize any contribution of swimming motility to apparent surface migration.Another difference in experimental conditions may concern the nutritional content of the medium. Some labs have tested swarming motility on LB medium in which tryptone was replaced by an equal amount of peptone (13). We reproduced the “LB” medium containing peptone and found that whereas strain 3610 was swarming proficient, strain 168 was swarming deficient (Fig. (Fig.1C).1C). Thus, the peptone substitution did not promote swarming in lab strains.Some labs have also reported swarming of laboratory strains on a defined medium called B medium [15 mM (NH4)2SO4, 8 mM MgSO4·7H2O, 27 mM KCl, 7 mM sodium citrate·H2O, 50 mM Tris·HCl (pH 7.5), 2 mM CaCl2·2H2O, 1 μM FeSO4·7H2O, 10 μM MnSO4·4H2O, 0.6 mM KH2PO4, 4.5 mM glutamic acid, 860 μM lysine, 780 μM tryptophan, and 0.5% glucose) (2, 13, 18, 19). In our hands, 3610 was swarming proficient on B medium, but strain 168 was swarming deficient (Fig. (Fig.1A).1A). We conclude that altering medium composition was insufficient to promote swarming of laboratory strains. Furthermore, mutation of either sfp or swrA rendered strain 3610 nonswarming on B medium, and complementation of sfp and swrA restored B medium swarming to strain 168 (Fig. (Fig.1B).1B). We conclude that the genetic requirements for swarming are the same for both LB and B medium.On undefined rich LB medium, strain 3610 swarmed rapidly as a featureless monolayer, whereas on defined B medium, it swarmed in a branched dendritic pattern (18, 20) (Fig. (Fig.1A).1A). In addition, the growth rate of 3610 in liquid B medium and swarm rate on solid B medium were both reduced fivefold relative to comparable assays with LB (Table (Table2),2), suggesting that the rate of swarming and the rate of growth were related. To further explore the connection between growth rate and swarming rate, we performed swarm expansion assays at lower temperatures. At 30°C, the growth rate in LB broth was reduced 2.5-fold relative to 37°C, and the swarming rate on LB agar was reduced 2.5-fold as well (Table (Table2;2; also, see Fig. S3 in the supplemental material). We conclude that swarming rate is correlated with growth rate. We infer that differences in growth may account for differences in swarm patterns (11). We note that regardless of the medium composition or the growth rate, the duration of the lag prior to swarming initiation was relatively constant.
Open in a separate windowaStrain 3610 was used to generate all data.bRelative to cells cultured in LB at 37°C (standard conditions).Ultimately we were unable to reproduce swarming in laboratory strains, and we reassert that laboratory strains are defective for swarming-motility. It is difficult to explain reports of swarming-proficient laboratory strains, because these cells are defective for both surfactin and swrA. Thus, the apparent swarming of strain 168 must be due to poorly reproducible environmental factors and/or selection for genetic revertants. 相似文献
TABLE 1.
Strains| Strain | Genotypea |
|---|---|
| 168 | trpC2 swrA sfp (13) |
| 3610 | Wild type |
| DS72 | yvzB::tet (21) |
| DS2268 | epr::kan |
| DS2415 | ΔswrA |
| DS2509 | ΔswrB |
| DS3337 | sfp::mls |
| DS3649 | ΔdegU |
| DS3903 | phrC::spec |
| DS4978 | rapC::spec |
| DS4979 | oppD::kan |
| DS5106 | 168 trpC2 swrA sfp amyE::PswrA-swrA cat |
| DS5758 | 168 trpC2 swrA sfp amyE::sfp+ cat |
| DS5759 | 168 trpC2 swrA sfp amyE::PswrA-swrA cat thrC::sfp+ mls |
TABLE 2.
Growth rates and swarm ratesa| Medium | Temp (°C) | Swarm rate (mm/h) | Growth rate (generations/h) | Reduction inb: | |
|---|---|---|---|---|---|
| Swarm rate | Growth rate | ||||
| LB | 37 | 15 | 3.5 | 1 | 1 |
| LB | 30 | 6 | 1.4 | 2.5 | 2.5 |
| B | 37 | 3 | 0.8 | 5 | 5 |
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Tim Eiseler Angelika Hausser Line De Kimpe Johan Van Lint Klaus Pfizenmaier 《The Journal of biological chemistry》2010,285(24):18672-18683
We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein cortactin and the Arp actin polymerization machinery. PKD phosphorylates cortactin in vitro and in vivo at serine 298 thereby generating a 14-3-3 binding motif. In vitro, a phosphorylation-deficient cortactin-S298A protein accelerated VCA-Arp-cortactin-mediated synergistic actin polymerization and showed reduced F-actin binding, indicative of enhanced turnover of nucleation complexes. In vivo, cortactin co-localized with the nucleation promoting factor WAVE2, essential for lamellipodia extension, in the actin polymerization zone in Heregulin-treated MCF-7 cells. Using a 3-dye FRET-based approach we further demonstrate that WAVE2-Arp and cortactin prominently interact at these structures. Accordingly, cortactin-S298A significantly enhanced lamellipodia extension and directed cell migration. Our data thus unravel a previously unrecognized mechanism by which PKD controls cancer cell motility. 相似文献