细菌中群体感应调节系统

细菌根据特定信号分子的浓度可以监测周围环境中自身或其它细菌的数量变化,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,这一调控系统被称为细菌的群体感应调节系统(QuorumSensing系统)。本文系统介绍了细菌感知种内与种间数量的群体感应调节系统,并阐述了植物针对病原菌这一信号系统的抗病策略。     

细菌生物被膜分散及分子调控机制研究进展

生物被膜分散(Biofilm Dispersal)是生物被膜发展后期细菌响应营养物、低浓度的一氧化氮、D-氨基酸、自诱导肽(Autoinducing Peptide,AIP)、酰基高丝氨酸内酯(Acyl Homoserine Lactones,AHL)、腺苷三磷酸(Adenosine Triphosphate,ATP)等信号变化而做出的一种程序性反应,有利于细菌从恶劣的生物被膜内部环境中脱离出来寻找新的定殖位点。此外,由生物被膜引起的细菌短暂的抗生素耐受性在分散过程中会恢复正常水平,这有助于治疗由致病菌引起的难治愈的生物被膜相关疾病。目前生物被膜分散的相关研究正处于起步阶段,本文希望通过综述生物被膜分散现象、信号分子及调控机制,可以更好地了解细菌生物被膜分散对于防控病原微生物和应用有益微生物的重要意义。     

A single mutation in P450BM-3 enhances acyl homoserine lactone: acyl homoserine substrate binding selectivity nearly 250-fold

Quorum sensing is the process by which bacteria alter gene regulation in response to their population density. The enzymatic inactivation of quorum signals has shown promise for use in genetically modified organisms resistant to pathogens. We recently characterized the ability of a cytochrome P450, P450BM-3, to oxidize the quorum sensing signals known as acyl homoserine lactones. The oxidation of the acyl homoserine lactones reduced their activity as quorum signals. The enzyme also oxidized the inactive lactonolysis products, acyl homoserines. The enzyme showed similar binding affinity for the acyl homoserine lactones and acyl homoserines. The latter reaction may lead to problems when lactonases and the P450-dependent system are used in tandem, as oxidation of the acyl homoserines produced by lactonolysis in vivo may compete with acyl homoserine lactone oxidation by the cytochrome P450. We report here that a single mutation (R47S) in P450BM-3 is capable of increasing the acyl homoserine lactone: acyl homoserine substrate binding selectivity of the enzyme nearly 250-fold, reducing the potential for competition by acyl homoserines and significantly enhancing the potential for use of P450BM-3 as part of a pathogen resistance system in genetically modified crops.     

Inhibition of quorum sensing in Serratia marcescens AS-1 by synthetic analogs of N-acylhomoserine lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C(6)-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C(9)-CPA), had a strong inhibitory effect on prodigiosin production. C(9)-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C(9)-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C(6)-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C(9)-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Quorum-sensing in Gram-negative bacteria

It has become increasingly and widely recognised that bacteria do not exist as solitary cells, but are colonial organisms that exploit elaborate systems of intercellular communication to facilitate their adaptation to changing environmental conditions. The languages by which bacteria communicate take the form of chemical signals, excreted from the cells, which can elicit profound physiological changes. Many types of signalling molecules, which regulate diverse phenotypes across distant genera, have been described. The most common signalling molecules found in Gram-negative bacteria are N-acyl derivatives of homoserine lactone (acyl HSLs). Modulation of the physiological processes controlled by acyl HSLs (and, indeed, many of the non-acyl HSL-mediated systems) occurs in a cell density- and growth phase-dependent manner. Therefore, the term 'quorum-sensing' has been coined to describe this ability of bacteria to monitor cell density before expressing a phenotype. In this paper, we review the current state of research concerning acyl HSL-mediated quorum-sensing. We also describe two non-acyl HSL-based systems utilised by the phytopathogens Ralstonia solanacearum and Xanthomonas campestris.     

In Vivo Evidence that S-Adenosylmethionine and Fatty Acid Synthesis Intermediates Are the Substrates for the LuxI Family of Autoinducer Synthases

Many gram-negative bacteria synthesize N-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In Escherichia coli, synthesis of N-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a fadD mutant blocked in β-oxidative fatty acid degradation. Also, conditions known to induce the fad regulon did not increase autoinducer synthesis. In contrast, cerulenin and diazoborine, specific inhibitors of fatty acid synthesis, both blocked autoinducer synthesis even in a strain dependent on β-oxidative fatty acid degradation for growth. These data provide the first in vivo evidence that the acyl chains in autoinducers synthesized by LuxI-family synthases are derived from acyl-acyl carrier protein substrates rather than acyl coenzyme A substrates. Also, we show that decreased levels of intracellular S-adenosylmethionine caused by expression of bacteriophage T3 S-adenosylmethionine hydrolase result in a marked reduction in autoinducer synthesis, thus providing direct in vivo evidence that the homoserine lactone ring of LuxI-family autoinducers is derived from S-adenosylmethionine.     

Synthesis of multiple N-acylhomoserine lactones is wide-spread among the members of the Burkholderia cepacia complex

Seventy strains of the Burkholderia cepacia complex, which currently comprises six genomic species, were tested for their ability to produce N-acylhomoserine lactone (AHL) signal molecules. Using thin layer chromatography in conjunction with a range of AHL biosensors, we show that most strains primarily produce two AHLs, namely N-octanoylhomoserine lactone (C8-HSL) and N-hexanoylhomoserine lactone (C6-HSL). Furthermore, some strains belonging to B. vietnamiensis (genomovar V) produce additional long chain AHL molecules with acyl chains ranging from C10 to C14. For B. vietnamiensis R-921 the structure of the most abundant long chain AHL was confirmed as N-decanoylhomoserine lactone (C10-HSL) by liquid chromatography-mass spectrometry (LC-MS) in combination with total chemical synthesis. Interestingly, a number of strains, most notably all representatives of B. multivorans (genomovar II), did not produce AHLs at least under the growth conditions used in this study. All strains were also screened for the production of extracellular lipase, chitinase, protease, and siderophores. However, no correlation between the AHL production and the synthesis of these exoproducts was apparent. Southern blot analysis showed that all the B. cepacia complex strains investigated, including the AHL-negative strains, possess genes homologous to the C8-HSL synthase cepI and to cepR, which encodes the cognate receptor protein. The nucleotide sequence of the cepI and cepR genes from one representative strain from each of the six genomovars was determined. Furthermore, the cepI genes from the different genomovars were expressed in Escherichia coli and it is demonstrated that all genes encode functional proteins that direct the synthesis of C8-HSL and C6-HSL. Given that cepI from the B. multivorans strain encodes a functional AHL synthase, yet detectable levels of AHLs were not produced by the wild-type, this suggests that additional regulatory functions may be present in members of this genomovar that negatively affect expression of cepI.     

Altering substrate chain length specificity of an acylhomoserine lactone synthase in bacterial communication

Quorum sensing mediated by specific signal compounds (autoinducers) allows bacteria to monitor their cell density and enables a synchronized regulation of target gene sets. The best studied group of autoinducers are the acylhomoserine lactones (AHSLs), which are central to the regulation of virulence in many plant and animal pathogens. Variation of the acyl side chain of the AHSLs underlies the observed species specificity of this communication system. Here we show that even different strains of the plant pathogen Erwinia carotovora employ different dialects of this language and demonstrate the molecular basis for the acyl chain length specificity of distinct AHSL synthases. Under physiological concentrations, only the cognate AHSL with the "right" acyl chain is recognized as a signal that will switch on virulence genes. Mutagenesis of the AHSL synthase gene expI(SCC1) identified the changes M127T and F69L as sufficient to effectively alter ExpI(SCC1) (an N-3-oxohexanoyl-l-homoserine lactone producer) substrate specificity to that of an N-3-oxooctanoyl-l-homoserine lactone producer. Our data identify critical residues that define the size of the substrate-binding pocket of the AHSL synthase and will help in understanding and manipulating this bacterial language.     

Acylhomoserine lactone synthase activity of the Vibrio fischeri AinS protein.

Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone from an acyl-acyl carrier protein and S-adenosylmethionine. Another V. fischeri gene, ainS, directs the synthesis of N-octanoylhomoserine lactone. The AinS protein shows no significant sequence similarity with LuxI family members, but it does show sequence similarity with the Vibrio harveyi LuxM protein. The luxM gene is required for the synthesis of N-(3-hydroxybutyryl)-L-homoserine lactone. To gain insights about whether AinS and LuxM represent a second family of acylhomoserine lactone synthases, we have purified AinS as a maltose-binding protein (MBP) fusion protein. The purified MBP-AinS fusion protein catalyzed the synthesis of N-octanoylhomoserine lactone from S-adenosylmethionine and either octanoyl-acyl carrier protein or, to a lesser extent, octanoyl coenzyme A. With the exception that octanoyl coenzyme A served as an acyl substrate for the MBP-AinS fusion protein, the substrates for and reaction kinetics of the MBP-AinS fusion protein were similar to those of the several LuxI family members previously studied. We conclude that AinS is an acylhomoserine lactone synthase and that it represents a second family of such enzymes.     

Novel roles of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> to control plant diseases

Bacillus thuringiensis is well known as an effective bio-insecticidal bacterium. However, the roles of B. thuringiensis to control plant diseases are not paid great attention to. In recent years, many new functions in protecting plants from pathogen infection have been discovered. For example, acyl homoserine lactone lactonase produced by B. thuringiensis can open the lactone ring of N-acyl homoserine lactone, a signal molecule in the bacterial quorum-sensing system. This in turn, significantly silences bacterial virulence. This finding resulted in the development of a new strategy against plant bacterial diseases by quenching bacterial quorum sensing. Another new discovery about B. thuringiensis function is zwittermicin A, a linear aminopolyol antibiotic with high activity against the Oomycetes and their relatives, as well as some gram-negative bacteria. This paper summarized the relative progresses of B. thuringiensis in plant disease control and its favorable application prospects.     

"Quorum sensing", or how bacteria "talk" to each other

Recent advances in studying the quorum-sensing systems, which regulate gene expression depending on population density, are reviewed. Low-molecular-weight acyl derivatives of L-homoserine lactone (N-AHL) freely diffuse through cell membranes and determine cell-to-cell communications in bacteria. The quorum-sensing systems have first been found to regulate bioluminescence in marine bacteria Photobacterium (Vibrio) fischeri and Vibrio harveyi. Such systems are widespread and control expression of genes for virulence factors, proteases, antibiotics, etc., in various Gram-negative bacteria, including plant, animal, and human pathogens. Quorum sensing is a prominent example of social behavior in bacteria, as signal exchange among individual cells allows the entire population to choose an optimal way of interaction with the environment and with higher organisms.     

Quorum-sensing molecules: Sampling,identification and characterization of N-acyl-homoserine lactone in Vibrio sp

Quorum sensing (QS) is a mechanism by which gram-negative bacteria regulate their gene expression by making use of cell density. QS is triggered by a small molecule known as an autoinducer. Typically, gram-negative bacteria such as Vibrio produce signaling molecules called acyl homoserine lactones (AHLs). However, their levels are very low, making them difficult to detect. We used thin layer chromatography (TLC) to examine AHLs in different Vibrio species, such as Vibrio alginolyticus, Vibrio parahemolyticus, and Vibrio cholerae, against a standard- Chromobacterium violaceum. Further, AHLs were characterised by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC–MS). C4-HSL (N- butanoyl- L- homoserine lactone), C6-HSL (N- hexanoyl- L- homoserine lactone), 3-oxo-C8-HSL (N-(3-Oxooctanoyl)-DL-homoserine lactone), C8-HSL (N- octanoyl- L- homoserine lactone), C₁10-HSL (N- decanoyl- L- homoserine lactone), C12-HSL (N- dodecanoyl- L- homoserine lactone) and C14-HSL (N- tetradecanoyl- L- homoserine lactone) were identified from Vibrio. These results may provide a basis for blocking the AHL molecules of Vibrio, thereby reducing their pathogenicity and eliminating the need for antimicrobials.     

Inhibition of human leukocyte elastase, cathepsin G, chymotrypsin A alpha, and porcine pancreatic elastase with substituted isobenzofuranones and benzopyrandiones

Several 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones, 3-(1-haloalkylidene)-1(3H)-isobenzofuranones, and 3-bromomethyl-1H-2-benzopyran-1-ones containing masked halo ketone functional groups were synthesized and tested as inhibitors of several serine proteases including human leukocyte (HL) elastase and cathepsin G. While many of the 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones were quite potent inhibitors of the enzymes tested, the alkylideneisobenzofuranones and benzopyran-1-ones inhibited poorly or not at all. The 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones decomposed rapidly upon addition to buffer to give the corresponding 3-alkyl-1H-2-benzopyran-1,4(3H)-diones. The pure benzopyran-1,4-diones were extremely potent inhibitors of HL elastase and chymotrypsin A alpha but did not inactivate porcine pancreatic elastase or cathepsin G. Enzymes inhibited by the isobenzofuranones and benzopyran-1,4-diones regained activity slowly upon standing or after dialysis (t1/2 = 5-16 h) and more rapidly in the presence of 0.5 M hydroxylamine, which indicated the presence of labile acyl moieties in the inhibited enzyme. These results are consistent with a scheme in which the active site serine of the protease reacts with the lactone carbonyl of these inhibitors to give a stable acyl enzyme and alkylation of another active site residue by the unmasked halo ketone functional group does not occur.     

Synthesis of Pseudomonas quorum-sensing autoinducer analogs and structural entities required for induction of apoptosis in macrophages

The synthesis of the analogs of N-3-oxododecanoyl-L-homoserine lactone (1) and their structure-activity relationship for the apoptotic induction in macrophages, P388D1 cells, are described. It was revealed that the position of the oxo group in the acyl side chain in addition to the presence of the L-homoserine lactone unit is crucial for the apoptosis-inducing activity. Furthermore, the long acyl side chains with hydrophobic distal ends are preferable for the activity.     

Inhibition of Quorum Sensing in Serratia marcescens AS-1 by Synthetic Analogs of N-Acylhomoserine Lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C₆-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C₉-CPA), had a strong inhibitory effect on prodigiosin production. C₉-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C₉-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C₆-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C₉-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Analysis of random and site-directed mutations in rhlI, a Pseudomonas aeruginosa gene encoding an acylhomoserine lactone synthase

The opportunistic human pathogen Pseudomonas aeruginosa possesses two cell density-dependent genetic regulatory systems that control expression of a number of secreted virulence factors. These two systems, the lasI–lasR and rhlI–rhlR gene pairs, are members of the luxI–luxR family of quorum-sensing signal generators and signal receptors. The rhlI gene in P. aeruginosa encodes a 201-amino-acid protein that catalyses the synthesis of an autoinducer, butyrylhomoserine lactone. Through a programme of random and site-specific mutagenesis of rhlI we have gained a better understanding of how its protein product functions. Eight residues critical to butyrylhomoserine lactone synthesis by RhlI were identified by random mutagenesis, and all mapped to a conserved region that spans residues 24–104. Seven of the eight residues were charged amino acids and the other was a glycine. By using site-specific mutagenesis we showed that an active-site cysteine or serine was not required for butyrylhomoserine lactone synthesis, and that two conserved aromatic amino acids in the postulated active site region could be altered without complete loss of RhlI activity. Furthermore, two residues towards the C-terminus that align with critical residues in LuxI can be altered in RhlI without loss of activity. These studies suggest that as opposed to the current models for acyl substrate binding to quorum-sensing signal generators, charged amino acid residues participate directly in the catalysis of butyrylhomoserine lactone synthesis rather than cysteines, serines or hydrophobic amino acids.     

Proline-valine pseudo peptide enol lactones. Effective and selective inhibitors of chymotrypsin and human leukocyte elastase

Pro-Val pseudo dipeptides incorporating protio and halo enol lactones were tested for inhibitory activity against the serine proteases human leukocyte elastase (HLE), porcine pancreatic elastase, alpha-chymotrypsin, trypsin, thrombin, and urokinase. The protio enol lactones 1a-c were found to be HLE substrates but were poor alternate substrate inhibitors. The bromo enol lactone trans isomer 2a was found to be a very effective inhibitor of HLE and chymotrypsin, as shown by the binding constants (KI), acylation rates (ka), inactivation rates, and partition ratios determined for each enzyme. This inhibitor shows better specificity toward its target enzyme HLE than monosubstituted halo enol lactones; we attribute this to a pseudo dipeptide acyl enzyme whose structure is similar to that adopted by good peptide substrates of HLE. Inactivation of chymotrypsin by the bromo enol lactone 2a is permanent, but inactivation of HLE is partially recoverable upon treatment with the nucleophile hydrazine, indicating that lactone 2a produces two species of inactivated HLE. The more stable of these species could be the result of alkylation of His-57 by the electrophilic bromomethyl ketone revealed in the acyl enzyme, and the less stable, hydrazine-reactivatable species could be the result of alkylation of Asp-102 or the hydrolysis of the bromomethyl ketone group in the initially formed acyl enzyme to form a new, more stable acyl enzyme.     

Quorum Sensing,or How Bacteria “Talk” to Each Other

Reviewed are recent advances in studying the quorum-sensing systems, which regulate gene expression depending on population density. Low-molecular-weight acyl derivatives of L-homoserine lactone (N-AHL) freely diffuse through cell membranes and determine cell-to-cell communication in bacteria. The quorum-sensing systems have first been found to regulate bioluminescence in marine bacteria Photobacterium(Vibrio) fischeriand Vibrio harveyi. Such systems are widespread and control expression of genes for virulence factors, proteases, antibiotics, etc., in various Gram-negative bacteria, including plant, animal, and human pathogens. Quorum sensing is a prominent example of social behavior in bacteria, as signal exchange among individual cells allows the entire population to choose an optimal way of interaction with the environment and with higher organisms.