Engineering Escherichia coli for enhanced sensitivity to the autoinducer-2 quorum sensing signal

The autoinducer-2 (AI-2) quorum sensing system is involved in a range of population-based bacterial behaviors and has been engineered for cell–cell communication in synthetic biology systems. Investigation into the cellular mechanisms of AI-2 processing has determined that overexpression of uptake genes increases AI-2 uptake rate, and genomic deletions of degradation genes lowers the AI-2 level required for activation of reporter genes. Here, we combine these two strategies to engineer an Escherichia coli strain with enhanced ability to detect and respond to AI-2. In an E. coli strain that does not produce AI-2, we monitored AI-2 uptake and reporter protein expression in a strain that overproduced the AI-2 uptake or phosphorylation units LsrACDB or LsrK, a strain with the deletion of AI-2 degradation units LsrF and LsrG, and an “enhanced” strain with both overproduction of AI-2 uptake and deletion of AI-2 degradation elements. By adding up to 40 μM AI-2 to growing cell cultures, we determine that this “enhanced” AI-2 sensitive strain both uptakes AI-2 more rapidly and responds with increased reporter protein expression than the others. This work expands the toolbox for manipulating AI-2 quorum sensing processes both in native environments and for synthetic biology applications.     

Symbiosis-stage associated alterations in quorum sensing autoinducer molecules biosynthesis in Sinorhizobium meliloti

Sinorhizobium meliloti strains use several N-acylhomoserine lactone (AHL) autoinducers to sense the population density during saprophytic growth. Using a sensitive gfp-based AHL biosensor system, we show that synthesis of short-chain AHL molecules is inhibited (or significantly diminished) during the symbiotic phase of growth and is undetectable in the nitrogen fixing bacteroids. In the saprophytic zone V of nodules occupied by intensively proliferating vegetative forms of rhizobia, AHL production recommenced and bright-green fluorescence was observed concomitantly with increasing population density.     

Stereochemical diversity of AI-2 analogs modulates quorum sensing in Vibrio harveyi and Escherichia coli

Bacteria coordinate population-dependent behaviors such as virulence by intra- and inter-species communication (quorum sensing). Autoinducer-2 (AI-2) regulates inter-species quorum sensing. AI-2 derives from the spontaneous cyclisation of linear (S)-4,5-dihydroxypentanedione (DPD) into two isomeric forms in dynamic equilibrium. Different species of bacteria have different classes of AI-2 receptors (LsrB and LuxP) which bind to different cyclic forms. In the present work, DPD analogs with a new stereocenter at C-5 (4,5-dihydroxyhexanediones (DHDs)) have been synthesized and their biological activity tested in two bacteria. (4S,5R)-DHD is a synergistic agonist in Escherichia coli (which contains the LsrB receptor), while it is an agonist in Vibrio harveyi (LuxP), displaying the strongest agonistic activity reported so far (EC(50)=0.65μM) in this organism. Thus, modification at C-5 opens the way to novel methods to manipulate quorum sensing as a method for controlling bacteria.     

Gravity sensing by Escherichia coli

We investigated the growth and protein profile of Escherichia coli under various gravity strengths to determine the effects of hypergravity on biochemical reactions. E. coli grows at less than 7,500 g without inhibition. Hypergravity induced OmpW and Antigen 43. Changes in gravity strength altered the expression levels of these proteins. This suggests that hypergravity regulates gene expression in bacteria.     

含自杀基因回路的大肠杆菌的生长和变异规律

具有鲁棒性的基因回路构建是合成生物学的基础工作.基于群体感应的自杀基因回路转化大肠杆菌后赋予宿主菌在一定菌群密度时启动自杀的特性.为了使基因回路更具鲁棒性,在转化后以不同浓度的IPTG为诱导剂,观察宿主菌的表型特征以及在自杀过程中突变菌产生的规律,并通过基因测序确定变异位点.结果表明:IPTG浓度与细菌自杀率呈正相关,自杀强度愈大,突变菌株出现得越早,蔓延的速度也越快.基因测序结果表明,在基因回路质粒上,luxR基因中间有转座子插入,从而破坏了群体感应系统.实验也表明:仅需该突变,就足以使宿主菌逃避自杀.结果为下一步优化基因回路设计,实现细菌密度的连续振荡提供了思路.     

Conditioned medium from Listeria innocua stimulates emergence from a resting state: not a response to E. coli quorum sensing autoinducer AI-2

The lag phase of the bacterial growth curve is an important determinant in speeding the detection of pathogens. It is affected by many factors including the prevailing growth environment and inoculum size, as well as specific signal molecules. The elucidation of growth-regulating signal molecules is further facilitated by culturing cells in defined growth media. In this study, a defined medium capable of supporting growth of Listeria innocua at similar levels as obtained using a complex brain heart infusion (BHI) media was developed. Further, the effects of conditioned medium (CM) on population lag time of L. innocua was investigated using a rapid parallel approach (with an automated microtiter plate reader). Importantly, the lag phase was shortened by up to approximately 50% by the addition of CM from L. innocua cultures obtained late in the exponential phase. Finally, while L. innocua were found to secrete bacterial signaling autoinducer, AI-2, tests using Escherichia coli based CM having a 90-fold difference in AI-2 level suggested that the observed decrease in lag phase was not due to E. coli-derived AI-2 and was instead due to elements specific to L. innocua. These findings indicate secreted signal molecules may be found in CM that speed detection of L. innocua.     

Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp

Background

Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N-acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out if the biofilm formation among clinical isolates of Acinetobacter spp. is under the control of autoinducing quorum sensing molecules.

Methodology/Principal Findings

Biofilm formation among clinical isolates of Acinetobacter spp. was assessed and the production of signal molecules were detected with Chromobacterium violaceum CV026 biosensor system. Characterisation of autoinducers was carried out by mass spectrometric analysis. We have also reported the identification of an autoinducer synthase gene, abaΙ among the isolates that produce quorum sensing signal molecules and have reported that the mutation in the abaI gene influences their biofilm forming capabilities. Using a microtitre-plate assay it was shown that 60% of the 50 Acinetobacter spp. isolates significantly formed biofilms. Further detection with the biosensor strain showed that some of these isolates produced long chain signal molecules. Mass spectrometric analysis revealed that five of these isolates produced N-decanoyl homoserine lactone and two isolates produced acyl-homoserine lactone with a chain length equal to C₁₂. The abaΙ gene was identified and a tetracycline mutant of the abaΙ gene was created and the inhibition in biofilm formation in the mutant was shown.

Conclusions/Significance

These data are of great significance as the signal molecules aid in biofilm formation which in turn confer various properties of pathogenicity to the clinical isolates including drug resistance. The use of quorum sensing signal blockers to attenuate bacterial pathogenicity is therefore highly attractive, particularly with respect to the emergence of multi antibiotic resistant bacteria.     

Effects of quorum sensing autoinducer degradation gene on virulence and biofilm formation of Pseudomonas aeruginosa

The aiiA gene from Bacillus thuringiensis was cloned into the Pseudomonas/E. coli shuttle vector and transformed into Pseudomonas aeruginosa strain PAO1. Western blotting showed that the AiiA protein was expressed in PAO1. After induction by IPTG for 6 h and 18 h, expression of the aiiA gene in PAO1 completely degraded the quorum sensing autoinducers N-acylhomoserine lactones (AHLs): N-oxododecanoyl-L-homoserine lactone (OdDHL) and N-butyryl-L-homoserine lactone (BHL). The reduced amount of AHLs in PAO1 was also correlated with decreased expression and production of several virulence factors such as elastase and pyocyanin. AiiA expression also influenced bacterial swarming motility. Most importantly, our studies indicated that aiiA played significant roles in P. aeruginosa biofilm formation and dispersion, as observed by the differences of the biofilm formation on liquid and solid surfaces, and biofilm structures under a scanning electron microscope. These authors contributed equally to this work Supported by the National Natural Science Foundation of China (Grant No. 30570020) and Natural Science Foundation of Hubei Province of China (Grant No. 2004ABA120)     

Engineering motility as a phenotypic response to LuxI/R-dependent quorum sensing in Escherichia coli

The repertoire of functional outputs interfaced with the LuxI/LuxR quorum sensing system in engineered Escherichia coli has been expanded to include motility via inducible expression of motB. Appropriate choice of ribosome binding site controlling MotB translation was crucial to achieving control over motility.     

Effects of quorum sensing autoinducer degradation gene on virulence and biofilm formation of Pseudomonas aeruginosa

The aiiA gene from Bacillus thuringiensis was cloned into the Pseudomonas/E. coli shuttle vector and transformed into Pseudomonas aeruginosa strain PAO1. Western blotting showed that the AiiA protein was expressed in PAO1. After induction by IPTG for 6 h and 18 h, expression of the aiiA gene in PAO1 completely degraded the quorum sensing autoinducers N-acylhomoserine lactones (AHLs): N-oxododecanoyl-L-homoserine lactone (OdDHL) and N-butyryl-L-homoserine lactone (BHL). The re- duced amount of AHLs in PAO1 was also correlated with decreased expression and production of several virulence factors such as elastase and pyocyanin. AiiA expression also influenced bacterial swarming motility. Most importantly, our studies indicated that aiiA played significant roles in P. aeruginosa biofilm formation and dispersion, as observed by the differences of the biofilm formation on liquid and solid surfaces, and biofilm structures under a scanning electron microscope.     

DNA microarray-based identification of serogroups and virulence gene patterns of Escherichia coli isolates associated with porcine postweaning diarrhea and edema disease

Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 10(3) CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.     

Isolation and identification of Fur binding genes in Escherichia coli

Fur (ferric uptake regulation) binding fragments were isolated by in vitro binding of purified Fur protein with Sau3AI-digested genomic DNA fragments. The Fur-bound DNA fragments were filtered on nitrocellulose paper, isolated, cloned, and sequenced. The protein binding was confirmed by gel retardation assay for five DNA fragments. The sequence data were used to identify the genes by comparison with the GenBank data. The proposed Fur binding regions lie on or near the putative promoter regions of marAB (multiple antibiotic resistance), pyrC (dihydroorotase), mreB (mecillinam resistance) and an unidentified gene (ecouw93) near argI and in the middle of the treBC (trehalose permease enzyme II) coding region. The proposed Fur binding sites of the known iron regulating operators including the genes of this work are AAT(pyrimidine) and A(purine)TT. The two conserved sequences are 10 bases apart and palindromic to each other, which might suggest the classical pattern of protein binding toward one side of the DNA in contrast to the concept of the Fur protein wrapping around the DNA.     

Structure of the Escherichia coli quorum sensing protein SdiA: activation of the folding switch by acyl homoserine lactones

The three-dimensional structure of a complex between the N-terminal domain of the quorum sensing protein SdiA of Escherichia coli and a candidate autoinducer N-octanoyl-L-homoserine lactone (C8-HSL) has been calculated in solution from NMR data. The SdiA-HSL system shows the "folding switch" behavior that has been seen for quorum-sensing factors produced by other bacterial species. In the presence of C8-HSL, a significant proportion of the SdiA protein is produced in a folded, soluble form in an E.coli expression system, whereas in the absence of acyl homoserine lactones, the protein is expressed into insoluble inclusion bodies. In the three-dimensional structure, the autoinducer molecule is sequestered in a deep pocket in the hydrophobic core, forming an integral part of the core packing of the folded SdiA. The NMR spectra of the complex show that the bound C8-HSL is conformationally heterogeneous, either due to motion within the pocket or to heterogeneity of the bound structure. The C8-HSL conformation is defined by NOEs to the protein only at the terminal methyl group of the octanoyl chain. Unlike other well-studied bacterial quorum sensing systems such as LuxR of Vibrio fischeri and TraR of Agrobacterium tumefaciens, there is no endogenous autoinducer for SdiA in E.coli: the E.coli genome does not contain a gene analogous to the LuxI and TraI autoinducer synthetases. We show that two other homoserine lactone derivatives are also capable of acting as a folding-switch autoinducers for SdiA. The observed structural heterogeneity of the bound C8-HSL in the complex, together with the variety of autoinducer-type molecules that can apparently act as folding switches in this system, are consistent with the postulated biological function of the SdiA protein as a detector of the presence of other species of bacteria.     

Phasmid vectors for identification of genes by complementation of Escherichia coli mutants.

A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from procaryotic organisms by complementation of Escherichia coli mutants. This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176, 1980). This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host. This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency. An E. coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified. A typical clone, lambda ant+, was shown to be readily mutagenized by a mini-Tn10 transposon. A general method for transferring cloned DNA segments onto bacteriophage lambda was developed. The method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4. Possible uses of these vectors and of the method for transferring cloned DNA onto phage lambda are discussed.     

Genome-scale identification of conditionally essential genes in E. coli by DNA microarrays

Identifying the genes required for the growth or viability of an organism under a given condition is an important step toward understanding the roles these genes play in the physiology of the organism. Currently, the combination of global transposon mutagenesis with PCR-based mapping of transposon insertion sites is the most common method for determining conditional gene essentiality. In order to accelerate the detection of essential gene products, here we test the utility and reliability of a DNA microarray technology-based method for the identification of conditionally essential genes of the bacterium, Escherichia coli, grown in rich medium under aerobic or anaerobic growth conditions using two different DNA microarray platforms. Identification and experimental verification of five hypothetical E. coli genes essential for anaerobic growth directly demonstrated the utility of the method. However, the two different DNA microarray platforms yielded largely non-overlapping results after a two standard deviations cutoff and were subjected to high false positive background levels. Thus, further methodological improvements are needed prior to the use of DNA microarrays to reliably identify conditionally essential genes on genome-scale.     

Influence of mutations at genes of global transcriptional regulators on production of autoinducer AI-2 Quorum Sensing in the system of Escherichia coli

The control of gene expression in response to an increase in the bacterial population density (Quorum Sensing) involves low-molecular-weight signal molecules (autoinducers, AI). AI-2 and synthase LuxS mediating its synthesis are widely distributed in Gram-negative and Gram-positive bacteria. In this work, the data were obtained on the role of global regulators of gene expression in AI-2 synthesis in Escherichia coli cells. The mutation inactivating gene rpoS (encodes sigma S subunit of RNA polymerase) was shown to drastically decrease an amount of active AI-2 in the culture medium. Mutations at gene rpoN that encodes sigma N subunit of RNA polymerase and also at gene lon, which encodes Lon proteinase, on the contrary, increase an amount of active AI-2 in supernatants of cultures. Mutant strains lacking histone-like proteins H-NS and StpA accumulate a slightly higher amount of AI-2 than the isogenic wild-type strain: however, an amount of AI-2 decreased in the culture medium of the double mutant devoid of both these proteins.