Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus

Acyl-homoserine lactones (acyl-HSLs) serve as dedicated cell-to-cell signaling molecules in many species of the class Proteobacteria. We have addressed the question of whether these compounds can be degraded biologically. A motile, rod-shaped bacterium was isolated from soil based upon its ability to utilize N-(3-oxohexanoyl)-L-homoserine lactone as the sole source of energy and nitrogen. The bacterium was classified as a strain of Variovorax paradoxus. The V. paradoxus isolate was capable of growth on all of the acyl-HSLs tested. The molar growth yields correlated with the length of the acyl group. HSL, a product of acyl-HSL metabolism, was used as a nitrogen source, but not as an energy source. Cleavage and partial mineralization of the HSL ring were demonstrated by using radiolabeled substrate. This study indicates that some strains of V. paradoxus degrade and grow on acyl-HSL signals as the sole energy and nitrogen sources. This study provides clues about the metabolic pathway of acyl-HSL degradation by V. paradoxus.     

Quorum-sensing signal synthesis by the Yersinia pestis acyl-homoserine lactone synthase YspI

The acyl-homoserine lactone molecular species (AHLs) produced by the Yersinia pestis AHL synthase YspI were identified by biochemical and physical/chemical techniques. Bioassays of extracts from culture supernatants of the recombinant YspI and wild-type Yersinia pestis showed similar profiles of AHLs. Analysis by liquid chromatography-mass spectrometry revealed that the predominant AHLs were N-3-oxooctanoyl-L-homoserine lactone and N-3-oxo-hexanoyl-L-homoserine lactone.     

Listening in on bacteria: acyl-homoserine lactone signalling

Bacterial cell-to-cell signalling has emerged as a new area in microbiology. Individual bacterial cells communicate with each other and co-ordinate group activities. Although a lot of detail is known about the mechanisms of a few well-characterized bacterial communication systems, other systems have been discovered only recently. Bacterial intercellular communication has become a target for the development of new anti-virulence drugs.     

Degradation of acyl-homoserine lactone molecules by Acinetobacter sp. strain C1010

A bacterium C1010, isolated from the rhizospheres of cucumbers in fields in Korea, degraded the microbial quorum-sensing molecules, hexanoyl homoserine lactone (HHSL), and octadecanoyl homoserine lactone (OHSL). Morphological characteristics and 16S rRNA sequence analysis identified C1010 as Acinetobacter sp. strain C1010. This strain was able to degrade the acyl-homoserine lactones (AHLs) produced by the biocontrol bacterium, Pseudomonas chlororaphis O6, and a phytopathogenic bacterium, Burkholderia glumae. Co-cultivation studies showed that the inactivation of AHLs by C1010 inhibited production of phenazines by P. chlororaphis O6. In virulence tests, the C1010 strain attenuated soft rot symptom caused by Erwinia carotovora ssp. carotovora. We suggest Acinetobacter sp. strain C1010 could be a useful bacterium to manipulate biological functions that are regulated by AHLs in various Gram-negative bacteria.     

Identification of acyl-homoserine lactone signal molecules produced by Nitrosomonas europaea strain Schmidt

Nitrosomonas europaea strain Schmidt produces at least three acyl homoserine lactone (AHL) signal molecules: C(6)-homoserine lactone (HSL), C(8)-HSL, and C(10)-HSL. These compounds were identified in extracts of chemostat culture effluent by three independent methods. The concentrations of AHL in effluent were low (0.4 to 2.2 nM) but within the range known to induce AHL-responsive systems. The absence of LuxI and LuxM homologs from the genome of N. europaea strain Schmidt suggested that AHL synthesis occurs by an alternate pathway, possibly mediated by an HdtS homolog. To the best of our knowledge, the present report is the first to document the types and levels of AHLs produced by N. europaea.     

Rapid acyl-homoserine lactone quorum signal biodegradation in diverse soils

Signal degradation impacts all communications. Although acyl-homoserine lactone (acyl-HSL) quorum-sensing signals are known to be degraded by defined laboratory cultures, little is known about their stability in nature. Here, we show that acyl-HSLs are biodegraded in soils sampled from diverse U.S. sites and by termite hindgut contents. When amended to samples at physiologically relevant concentrations, 14C-labeled acyl-HSLs were mineralized to 14CO2 rapidly and, at most sites examined, without lag. A lag-free turf soil activity was characterized in further detail. Heating or irradiation of the soil prior to the addition of radiolabel abolished mineralization, whereas protein synthesis inhibitors did not. Mineralization exhibited an apparent Km of 1.5 microM acyl-HSL, ca. 1,000-fold lower than that reported for a purified acyl-HSL lactonase. Under optimal conditions, acyl-HSL degradation proceeded at a rate of 13.4 nmol x h(-1) x g of fresh weight soil(-1). Bioassays established that the final extent of signal inactivation was greater than for its full conversion to CO2 but that the two processes were well coupled kinetically. A most probable number of 4.6 x 10(5) cells . g of turf soil(-1) degraded physiologically relevant amounts of hexanoyl-[1-14C]HSL to 14CO2. It would take chemical lactonolysis months to match the level of signal decay achieved in days by the observed biological activity. Rapid decay might serve either to quiet signal cross talk that might otherwise occur between spatially separated microbial aggregates or as a full system reset. Depending on the context, biological signal decay might either promote or complicate cellular communications and the accuracy of population density-based controls on gene expression in species-rich ecosystems.     

一种新的乙酰高丝氨酸内酯水解酶基因的克隆和表达

目的 获得新的降解革兰阴性细菌数量阈值感应信号分子乙酰高丝氨酸内酯类化合物(AHL)的水解酶基因。方法 选择性富集和培养土壤中耐热细菌,抽取细菌总DNA作为模板,特异性聚合酶链反应扩增乙酰高丝氨酸内酯水解酶基因,进行克隆和DNA序列分析及原核表达。结果 得到1个新的AHL水解酶基因,该基因与已知基因的核苷酸序列和对应的氨基酸序列同源性最高分别为87％和94％。该基因在原核表达系统中表达,得到了与预期相对分子质鲢(Mr)一致的蛋白质。结论 证实乙酰高丝氨酸内酯水解酶广泛存在于环境微生物中。为进一步研究提供条件。     

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has proven to be a useful tool for examining noncovalent complexes between proteins and a variety of ligands. It has also been used to distinguish between denatured and refolded forms of proteins. Surfactants are frequently employed to enhance solubilization or to modify the tertiary or quaternary structure of proteins, but are usually considered incompatible with mass spectrometry. A broad range of ionic, nonionic, and zwitterionic surfactants was examined to characterize their effects on ESI-MS and on protein structure under ESI-MS conditions. Solution conditions studied include 4% acetic acid/50% acetonitrile/46% H2O and 100% aqueous. Of the surfactants examined, the nonionic saccharides, such as n-dodecyl-beta-D-glucopyranoside, at 0.1% to 0.01% (w/v) concentrations, performed best, with limited interference from chemical background and adduct formation. Under the experimental conditions used, ESI-MS performance in the presence of surfactants was found to be unrelated to critical micelle concentration. It is demonstrated that surfactants can affect both the tertiary and quaternary structures of proteins under conditions used for ESI-MS. However, several of the surfactants caused significant shifts in the charge-state distributions, which appeared to be independent of conformational effects. These observations suggest that surfactants, used in conjunction with ESI-MS, can be useful for protein structure studies, if care is used in the interpretation of the results.     

Production of acyl-homoserine lactone quorum-sensing signals is widespread in gram-negative Methylobacterium

Members of Methylobacterium, referred as pink-pigmented facultative methylotrophic bacteria, are frequently associated with terrestrial and aquatic plants, tending to form aggregates on the phyllosphere. We report here that the production of autoinducer molecules involved in the cell-to-cell signaling process, which is known as quorum sensing, is common among Methylobacterium species. Several strains of Methylobacterium were tested for their ability to produce N-acyl-homoserine lactone (AHL) signal molecules using different indicators. Most strains of Methylobacterium tested could elicit a positive response in Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. The synthesis of these compounds was cell-density dependent, and the maximal activity was reached during the late exponential to stationary phases. The bacterial extracts were separated by thin-layer chromatography and bioassayed with A. tumefaciens NT1 (traR, tra::lacZ749). They revealed the production of various patterns of the signal molecules, which are strain dependent. At least two signal molecules could be detected in most of the strains tested, and comparison of their relative mobilities suggested that they are homologs of N-octanoyl-DL-homoserine lactone (C8-HSL) and N-decanoyl-DL-homoserine lactone (C10-HSL).     

Metabolic changes during development of Phytophthora palmivora examined by gas chromatography/mass spectrometry

Metabolic profiles from four stages of differentiation of the fungus Phytophthora palmivora were obtained by gas chromatography/mass spectrometry. The profiles showed the presence of sterols in the asexual reproduction stage of the organism, and confirmed their virtual absence from the mycelial stages. The zoospore stage was characterized by the presence of polyunsaturated fatty acids of C20 and C22 chain length. The transition from zoospore to cyst was also marked by the appearance of disaccharides and by a decrease in the amount of phosphate present. There were also distinctive shifts in the proportions and the total amounts of amino acids present, with gamma-aminobutyrate and alanine increasing as germination took place. These distinctive profiles identify some of the metabolic changes which accompany differentiation in this fungus.     

Arthrobacter strain VAI-A utilizes acyl-homoserine lactone inactivation products and stimulates quorum signal biodegradation by Variovorax paradoxus

Many Proteobacteria produce acyl-homoserine lactones (acyl-HSLs) and employ them as dedicated cell-to-cell signals in a process known as quorum sensing. Previously, Variovorax paradoxus VAI-C was shown to utilize diverse acyl-HSLs as sole sources of energy and nitrogen. We describe here the properties of a second isolate, Arthrobacter strain VAI-A, obtained from the same enrichment culture that yielded V. paradoxus VAI-C. Although strain VAI-A grew rapidly and exponentially on a number of substrates, it grew only slowly and aberrantly (i.e., linearly) in media amended with oxohexanoyl-HSL as the sole energy source. Increasing the culture pH markedly improved the growth rate in media containing this substrate but did not abolish the aberrant kinetics. The observed growth was remarkably similar to the known kinetics of the pH-influenced half-life of acyl-HSLs, which decay chemically to yield the corresponding acyl-homoserines. Strain VAI-A grew rapidly and exponentially when provided with an acyl-homoserine as the sole energy or nitrogen source. The isolate was also able to utilize HSL as a sole source of nitrogen but not as energy for growth. V. paradoxus, known to release HSL as a product of quorum signal degradation, was examined for the ability to support the growth of Arthrobacter strain VAI-A in defined cocultures. It did. Moreover, the acyl-HSL-dependent growth rate and yield of the coculture were dramatically superior to those of the monocultures. This suggested that the original coenrichment of these two organisms from the same soil sample was not coincidental and that consortia may play a role in quorum signal turnover and mineralization. The fact that Arthrobacter strain VAI-A utilizes the two known nitrogenous degradation products of acyl-HSLs, acyl-homoserine and HSL, begins to explain why none of the three compounds are known to accumulate in the environment.     

Long-chain acyl-homoserine lactone quorum-sensing regulation of Rhodobacter capsulatus gene transfer agent production

Many proteobacteria use acyl-homoserine lactones as quorum-sensing signals. Traditionally, biological detection systems have been used to identify bacteria that produce acyl-homoserine lactones, although the specificities of these detection systems can limit discovery. We used a sensitive approach that did not require a bioassay to detect production of long-acyl-chain homoserine lactone production by Rhodobacter capsulatus and Paracoccus denitrificans. These long-chain acyl-homoserine lactones are not readily detected by standard bioassays. The most abundant acyl-homoserine lactone was N-hexadecanoyl-homoserine lactone. The long-chain acyl-homoserine lactones were concentrated in cells but were also found in the culture fluid. An R. capsulatus gene responsible for long-chain acyl-homoserine lactone synthesis was identified. A mutation in this gene, which we named gtaI, resulted in decreased production of the R. capsulatus gene transfer agent, and gene transfer agent production was restored by exogenous addition of N-hexadecanoyl-homoserine lactone. Thus, long-chain acyl-homoserine lactones serve as quorum-sensing signals to enhance genetic exchange in R. capsulatus.     

Post-translational processing of preprosomatostatin-II examined using fast atom bombardment mass spectrometry

The products and an intermediate of preprosomatostatin-II processing in the anglerfish islet were purified and subjected to structural analysis. The peptides isolated identify the site of signal cleavage (between Ser-24 and Gln-25). The prohormone is further processed at Arg-97 and, to a lesser extent, at the two adjacent basic amino acid residues Lys-61 and Arg-62. A 28-residue somatostatin is also generated which can be hydroxylated at Lys-23. A proteolytic processing site which would form the 14-residue somatostatin does not appear to be used to a significant degree. Fast atom bombardment mass spectrometry (FABMS) was used to demonstrate that the amino-terminal residues of peptides 25-60, and 25-90 are pyroglutamic acid, a modification which precludes Edman degradation of these peptides. Analysis of the peptides and tryptic peptide maps by FABMS allowed confirmation of the sites of prohormone conversion and indicated that terminal basic residues were removed during processing. Three amino acid residues were also found to differ from the amino acid sequence deduced from the cDNA and were localized to specific regions by FABMS analysis. Residues found to differ from the cDNA (cDNA in parentheses) were: Asp-77 (Thr), Val-78 (Phe), and Gly-90 (Glu). Mass assignments were confirmed by running a single cycle of Edman degradation prior to FABMS. The peptides noted above were also examined by Edman sequence analysis. The sequence of a cDNA clone to preprosomatostatin-II was re-examined in light of the observed differences at the protein level. This study emphasizes the utility of FABMS in prohormone processing studies and in identification of post-translational processing events.     

Utilization of acyl-homoserine lactone quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42 degrees C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other gamma-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.     

Bronchoalveolar lavage examined by solid phase microextraction, gas chromatography--mass spectrometry and selected ion flow tube mass spectrometry

Samples (210 in total) of broncholaveolar lavages (BALs), obtained from patients hospitalized with pneumonia in various departments of two hospitals, were analysed using the method of solid phase microextraction-gas chromatography (SPME-GC) with FID detection. Up to 20% (9% unequivocally, 11% probably) of these samples was found to contain volatile fatty acids (VFAs) in the series from acetic acid to heptanoic acid. Importantly, the presence of these acids indicates the presence of fermenting anaerobic bacteria, which were not detected by the conventional microbiological examination. Other compounds, namely the heptanol and cyclohexanone, were also detected by this method in some samples. Cyclohexanone occurred almost exclusively in samples from patients receiving intensive care with mechanical ventilation, and is suspected to originate from plastic parts of ventilators. Selected representative samples were also analysed using further methods, namely gas chromatography-mass spectrometry (GC-MS) of native and silylated samples, and selected ion flow tube mass spectrometry (SIFT-MS). These methods confirmed the identities of above mentioned compounds, and detected numerous other compounds tentatively identified as various alcohols, aldehydes, ketones, esters and hydrogen cyanide, HCN. Most of these compounds occurred in small amounts and their origin and diagnostic significance remains uncertain, except, that is, for the HCN, which indicates the presence of Pseudomonas aeruginosa.     

Functional sites and evolutionary connections of acylhomoserine lactone synthases

Acylhomoserine lactone (AHL) synthases act as chemical communication signals or pheromones in Gram-negative bacteria and regulate diverse physiological events in a cell density-dependent manner. The recent crystal structure determination of EsaI, a key enzyme in this pathway, shows that the AHL synthase superfamily members adopt the fold of the N-acetyltransferase superfamily. We suggest, by the identification of intermediate sequences, that the two superfamilies are evolutionarily related. Evolutionary trace analyses of aligned sequences and docking studies have been used to discuss functionally important residues of EsaI homologues.     

Structural basis and specificity of acyl-homoserine lactone signal production in bacterial quorum sensing

Synthesis and detection of acyl-homoserine lactones (AHLs) enables many gram-negative bacteria to engage in quorum sensing, an intercellular signaling mechanism that activates differentiation to virulent and biofilm lifestyles. The AHL synthases catalyze acylation of S-adenosyl-L-methionine by acyl-acyl carrier protein and lactonization of the methionine moiety to give AHLs. The crystal structure of the AHL synthase, EsaI, determined at 1.8 A resolution, reveals a remarkable structural similarity to the N-acetyltransferases and defines a common phosphopantetheine binding fold as the catalytic core. Critical residues responsible for catalysis and acyl chain specificity have been identified from a modeled substrate complex and verified through functional analysis in vivo. A mechanism for the N-acylation of S-adenosyl-L-methionine by 3-oxo-hexanoyl-acyl carrier protein is proposed.     

Protein structural dynamics at the gas/water interface examined by hydrogen exchange mass spectrometry

Gas/water interfaces (such as air bubbles or foam) are detrimental to the stability of proteins, often causing aggregation. This represents a potential problem for industrial processes, for example, the production and handling of protein drugs. Proteins possess surfactant-like properties, resulting in a high affinity for gas/water interfaces. The tendency of previously buried nonpolar residues to maximize contact with the gas phase can cause significant structural distortion. Most earlier studies in this area employed spectroscopic tools that could only provide limited information. Here we use hydrogen/deuterium exchange (HDX) mass spectrometry (MS) for probing the conformational dynamics of the model protein myoglobin (Mb) in the presence of N₂ bubbles. HDX/MS relies on the principle that unfolded and/or highly dynamic regions undergo faster deuteration than tightly folded segments. In bubble-free solution Mb displays EX2 behavior, reflecting the occurrence of short-lived excursions to partially unfolded conformers. A dramatically different behavior is seen in the presence of N₂ bubbles; EX2 dynamics still take place, but in addition the protein shows EX1 behavior. The latter results from interconversion of the native state with conformers that are globally unfolded and long-lived. These unfolded species likely correspond to Mb that is adsorbed to the surface of gas bubbles. N₂ sparging also induces aggregation. To explain the observed behavior we propose a simple model, that is, “semi-unfolded” ↔ “native” ↔ “globally unfolded” → “aggregated”. This model quantitatively reproduces the experimentally observed kinetics. To the best of our knowledge, the current study marks the first exploration of surface denaturation phenomena by HDX/MS.     

Kinetic folding mechanism of an integral membrane protein examined by pulsed oxidative labeling and mass spectrometry

We report the application of pulsed oxidative labeling for deciphering the folding mechanism of a membrane protein. SDS-denatured bacteriorhodopsin (BR) was refolded by mixing with bicelles in the presence of free retinal. At various time points (20 ms to 1 day), the protein was exposed to a microsecond ·OH pulse that induces oxidative modifications at solvent-accessible methionine side chains. The extent of labeling was determined by mass spectrometry. These measurements were complemented by stopped-flow spectroscopy. Major time-dependent changes in solvent accessibility were detected for M20 (helix A) and M118 (helix D). Our kinetic data indicate a sequential folding mechanism, consistent with models previously suggested by others on the basis of optical data. Yet, ·OH labeling provides additional structural insights. An initial folding intermediate I(1) gets populated within 20 ms, concomitantly with formation of helix A. Subsequent structural consolidation leads to a transient species I(2). Noncovalent retinal binding to I(2) induces folding of helix D, thereby generating an intermediate I(R). In the absence of retinal, the latter transition does not take place. Hence, formation of helix D depends on retinal binding, whereas this is not the case for helix A. As the cofactor settles deeper into its binding pocket, a final transient species I(R) is generated. This intermediate converts into native BR within minutes by formation of the retinal-K216 Schiff base linkage. The combination of pulsed covalent labeling and optical spectroscopy employed here should also be suitable for exploring the folding mechanisms of other membrane proteins.