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.     

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.     

Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1

The relevance of the acyl homoserine lactone (acyl-HSL) quorum signals N-3-oxododecanoyl-homoserine lactone (3OC12HSL) and N-butanoyl-homoserine lactone to the biology and virulence of Pseudomonas aeruginosa is well investigated. Previously, P. aeruginosa was shown to degrade long-chain, but not short-chain, acyl-HSLs as sole carbon and energy sources (J. J. Huang, J.-I. Han, L.-H. Zhang, and J. R. Leadbetter, Appl. Environ. Microbiol. 69:5941-5949, 2003). A gene encoding an enzyme with acyl-HSL acylase activity, pvdQ (PA2385), was identified, but it was not required for acyl-HSL utilization. This indicated that P. aeruginosa encodes another acyl-HSL acylase, which we identify here. A comparison of total cell proteins of cultures grown with long-acyl acyl-HSLs versus other substrates implicated the involvement of a homolog of PvdQ, the product of gene PA1032, for which we propose the name QuiP. Transposon mutants of quiP were defective for growth when P. aeruginosa was cultured in medium containing decanoyl-HSL as a sole carbon and energy source. Complementation with a functional copy of quiP rescued this growth defect. When P. aeruginosa was grown in buffered lysogeny broth, constitutive expression of QuiP in P. aeruginosa led to decreased accumulations of the quorum signal 3OC12HSL, relative to the wild type. Heterologous expression of QuiP was sufficient to confer long-chain acyl-HSL acylase activity upon Escherichia coli. Examination of gene expression patterns during acyl-HSL-dependent growth of P. aeruginosa further supported the involvement of quiP in signal decay and revealed other genes also possibly involved. It is not yet known under which "natural" conditions quiP is expressed or how P. aeruginosa balances the expression of its quorum-sensing systems with the expression of its acyl-HSL acylase activities.     

Identification of QuiP, the Product of Gene PA1032, as the Second Acyl-Homoserine Lactone Acylase of Pseudomonas aeruginosa PAO1

The relevance of the acyl homoserine lactone (acyl-HSL) quorum signals N-3-oxododecanoyl-homoserine lactone (3OC12HSL) and N-butanoyl-homoserine lactone to the biology and virulence of Pseudomonas aeruginosa is well investigated. Previously, P. aeruginosa was shown to degrade long-chain, but not short-chain, acyl-HSLs as sole carbon and energy sources (J. J. Huang, J.-I. Han, L.-H. Zhang, and J. R. Leadbetter, Appl. Environ. Microbiol. 69:5941-5949, 2003). A gene encoding an enzyme with acyl-HSL acylase activity, pvdQ (PA2385), was identified, but it was not required for acyl-HSL utilization. This indicated that P. aeruginosa encodes another acyl-HSL acylase, which we identify here. A comparison of total cell proteins of cultures grown with long-acyl acyl-HSLs versus other substrates implicated the involvement of a homolog of PvdQ, the product of gene PA1032, for which we propose the name QuiP. Transposon mutants of quiP were defective for growth when P. aeruginosa was cultured in medium containing decanoyl-HSL as a sole carbon and energy source. Complementation with a functional copy of quiP rescued this growth defect. When P. aeruginosa was grown in buffered lysogeny broth, constitutive expression of QuiP in P. aeruginosa led to decreased accumulations of the quorum signal 3OC12HSL, relative to the wild type. Heterologous expression of QuiP was sufficient to confer long-chain acyl-HSL acylase activity upon Escherichia coli. Examination of gene expression patterns during acyl-HSL-dependent growth of P. aeruginosa further supported the involvement of quiP in signal decay and revealed other genes also possibly involved. It is not yet known under which “natural” conditions quiP is expressed or how P. aeruginosa balances the expression of its quorum-sensing systems with the expression of its acyl-HSL acylase activities.     

Methylobacterium extorquens AM1 produces a novel type of acyl-homoserine lactone with a double unsaturated side chain under methylotrophic growth conditions

Acyl-homoserine lactones (acyl-HSLs) have emerged as important regulatory molecules for many gram-negative bacteria. We have found that Methylobacterium extorquens AM1, a member of the pink-pigmented facultative methylotrophs commonly present on plant surfaces, produces several acyl-HSLs depending upon the carbon source. A novel HSL was discovered with a double unsaturated carbon chain (N-(tetradecenoyl)) (C14:2) and characterized by MS and proton NMR. This long-chain acyl-HSL is synthesized by MlaI that also directs synthesis of C14:1-HSL. The Alphaproteobacterium also produces N-hexanoyl-HSL (C6-HSL) and N-octanoyl-HSL (C8-HSL) via MsaI.     

Utilization of homoserine lactone as a sole source of carbon and energy by soil Arthrobacter and Burkholderia species

Homoserine lactone (HSL) is a ubiquitous product of metabolism. It is generated by all known biota during the editing of certain mischarged aminoacyl-tRNA reactions, and is also released as a product of quorum signal degradation by bacterial species expressing acyl-HSL acylases. Little is known about its environmental fate over long or short periods of time. The mammalian enzyme paraoxonase, which has no known homologs in bacteria, has been reported to degrade HSL via a lactonase mechanism. Certain strains of Variovorax and Arthrobacter utilize HSL as a sole source of nitrogen, but not as a sole source of carbon or energy. In this study, the enrichment and isolation of four strains of soil bacteria capable of utilizing HSL as a carbon and energy source are described. Phylogenetic analysis of these isolates indicates that three are distinct members of the genus Arthrobacter, whereas the fourth clusters within the non-clinical Burkholderia. The optimal pH for growth of the isolates ranged from 6.0 to 6.5, at which their HSL-dependent doubling times ranged from 1.4 to 4 h. The biodegradation of HSL by these 4 isolates far outpaced its chemical decay. HSL degradation by soil bacteria has implications for the consortial mineralization of acyl-homoserine lactones by bacteria associated with quorum sensing populations.     

The Burkholderia mallei BmaR3-BmaI3 quorum-sensing system produces and responds to N-3-hydroxy-octanoyl homoserine lactone

Burkholderia mallei has two acyl-homoserine lactone (acyl-HSL) signal generator-receptor pairs and two additional signal receptors, all of which contribute to virulence. We show that B. mallei produces N-3-hydroxy-octanoyl HSL (3OHC8-HSL) but a bmaI3 mutant does not. Recombinant Escherichia coli expressing BmaI3 produces hydroxylated acyl-HSLs, with 3OHC8-HSL being the most abundant compound. In recombinant E. coli, BmaR3 responds to 3OHC8-HSL but not to other acyl-HSLs. These data indicate that the signal for BmaR3-BmaI3 quorum sensing is 3OHC8-HSL.     

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.     

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.     

Aerobic degradation of mercaptosuccinate by the gram-negative bacterium Variovorax paradoxus strain B4

The Gram-negative bacterium Variovorax paradoxus strain B4 was isolated from soil under mesophilic and aerobic conditions to elucidate the so far unknown catabolism of mercaptosuccinate (MS). During growth with MS this strain released significant amounts of sulfate into the medium. Tn5::mob-induced mutagenesis was successfully employed and yielded nine independent mutants incapable of using MS as a carbon source. In six of these mutants, Tn5::mob insertions were mapped in a putative gene encoding a molybdenum (Mo) cofactor biosynthesis protein (moeA). In two further mutants the Tn5::mob insertion was mapped in the gene coding for a putative molybdopterin (MPT) oxidoreductase. In contrast to the wild type, these eight mutants also showed no growth on taurine. In another mutant a gene putatively encoding a 3-hydroxyacyl-coenzyme A dehydrogenase (paaH2) was disrupted by transposon insertion. Upon subcellular fractionation of wild-type cells cultivated with MS as sole carbon and sulfur source, MPT oxidoreductase activity was detected in only the cytoplasmic fraction. Cells grown with succinate, taurine, or gluconate as a sole carbon source exhibited no activity or much lower activity. MPT oxidoreductase activity in the cytoplasmic fraction of the Tn5::mob-induced mutant Icr6 was 3-fold lower in comparison to the wild type. Therefore, a new pathway for MS catabolism in V. paradoxus strain B4 is proposed: (i) MPT oxidoreductase catalyzes the conversion of MS first into sulfinosuccinate (a putative organo-sulfur compound composed of succinate and a sulfino group) and then into sulfosuccinate by successive transfer of oxygen atoms, (ii) sulfosuccinate is cleaved into oxaloacetate and sulfite, and (iii) sulfite is oxidized to sulfate.     

Long- and short-chain plant-produced bacterial N-acyl-homoserine lactones become components of phyllosphere, rhizosphere, and soil

Two N-acyl-homoserine lactone (acyl-HSL) synthase genes, lasI from Pseudomonas aeruginosa and yenI from Yersinia enterocolitica, were introduced into tobacco, individually and in combination. Liquid chromatograph-tandem mass spectrometry and thin-layer chromatography confirmed products of lasI and yenI activity in single and cotransformants. Cotransformants expressing plastid-localized LasI and YenI synthases produced the major acyl-HSLs for each synthase in all tissues tested. Total acyl-HSL signals accumulated in leaf tissue up to 3 pmol/mg of fresh weight, half as much in stem tissue, and approximately 10-fold less in root tissues. Acyl-HSLs were present in aqueous leaf washes from greenhouse-grown transgenic plants. Transgenic lines grown for 14 days under axenic conditions produced detectable levels of acyl-HSLs in root exudates. Ethyl acetate extractions of rhizosphere and nonrhizosphere soil from transgenically grown plants contained active acyl-HSLs, whereas plant-free soil or rhizosphere and nonrhizosphere soil from wild-type plants lacked detectable amounts of acyl-HSLs. This work shows that bioactive acyl-HSLs are exuded from leaves and roots and accumulate in the phytosphere of plants engineered to produce acyl-HSLs. These data further suggest that plants that are bioengineered to synthesize acyl-HSLs can foster beneficial plant-bacteria communications or deter deleterious interactions. Therefore, it is feasible to use bioengineered plants to supplement soils with specific acyl-HSLs to modulate bacterial phenotypes and plant-associated bacterial community structures.     

The Ti plasmid of Agrobacterium tumefaciens harbors an attM-paralogous gene,aiiB, also encoding N-Acyl homoserine lactonase activity

The Agrobacterium tumefaciens C58 genome contains three putative N-acyl homoserine lactone (acyl-HSL) hydrolases, which are closely related to the lactonase AiiA of BACILLUS: When expressed in Escherichia coli, two of the putative acyl-HSL hydrolases, AttM and AiiB, conferred the ability to degrade acyl-HSLs on the host. In Erwinia strain 6276, the lactonases reduced the endogenous acyl-HSL level and the bacterial virulence in planta.     

Biodegradation of 3-nitrotyrosine by Burkholderia sp. strain JS165 and Variovorax paradoxus JS171

The cascade of reactive nitrogen species generated from nitric oxide causes modification of proteins, lipids, and nucleic acids in a wide range of organisms. 3-Nitrotyrosine is one of the most common products of the action of reactive nitrogen species on proteins. Although a great deal is known about the formation of 3-nitrotyrosine, the subsequent metabolism of this compound is a mystery. Variovorax paradoxus JS171 and Burkholderia sp. strain JS165 were isolated from soil slurries when 3-nitrotyrosine was provided as the sole carbon, nitrogen, and energy source. During growth on 3-nitrotyrosine stoichiometric amounts of nitrite were released along with approximately one-half of the theoretically available ammonia. The catabolic pathway involving oxidative denitration is distinct from the pathway for tyrosine metabolism. The facile isolation and the specific, regulated pathway for 3-nitrotyrosine degradation in natural ecosystems suggest that there is a significant flux of 3-nitrotyrosine in such environments.     

Degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Stenotrophomonas maltophilia PB1.

A mixed microbial culture capable of metabolizing the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A bacterium, Stenotrophomonas maltophilia PB1, isolated from the culture used RDX as a sole source of nitrogen for growth. Three moles of nitrogen was used per mole of RDX, yielding a metabolite identified by mass spectroscopy and 1H nuclear magnetic resonance analysis as methylene-N-(hydroxymethyl)-hydroxylamine-N'-(hydroxymethyl)nitroamin e. The bacterium also used s-triazine as a sole source of nitrogen but not the structurally similar compounds octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, cyanuric acid, and melamine. An inducible RDX-degrading activity was present in crude cell extracts.     

<Emphasis Type="Italic">Rhodococcus</Emphasis> sp. Q5, a novel agarolytic bacterium isolated from printing and dyeing wastewater

An agar-degrading bacterium, Rhodococcus sp. Q5, was isolated from printing and dyeing wastewater using a mineral salts agar plate containing agar as the sole carbon source. The bacterium grew from pH 4.0 to 9.0, from 15 to 35°C, and in NaCl concentrations of 0–5 %; optimal values were pH 6.0, 30°C, and 1 % NaCl. Maximal agarase production was observed at pH 6.0 and 30°C. The bacterium did not require NaCl for growth or agarase production. The agarase secreted by Q5 was inducible by agar and was repressed by all simple sugars tested except lactose. Strain Q5 could hydrolyze starch but not cellulose or carboxymethyl cellulose. Agarase activity could also be detected in the medium when lactose or starch was the sole source of carbon and energy. Strain Q5 could grow in nitrogen-free mineral media; an organic nitrogen source was more effective than inorganic carbon sources for growth and agarase production. Addition of more organic nitrogen (peptone) to the medium corresponded with reduced agarase activity.     

一株聚氨酯降解菌的分离及其降解特性解析

利用微生物对聚氨酯 (Polyurethane,PUR) 类污染物进行生物降解是目前的研究热点之一。寻找能高效降解聚氨酯的微生物是该类研究的重要前提。文中从塑料垃圾填埋场中分离培养了1株PUR高效降解菌株P10。基于菌落形态观察和16S rDNA系统发育分析,鉴定其为短芽孢杆菌属Brevibacillus的细菌。通过PUR的模式底物水性聚氨酯 (Impranil DLN) 比浊法,确定了该菌株能在6 d内降解71.4%的Impranil DLN。此外,菌株P10能够利用商业聚氨酯海绵作为唯一碳源进行生长;通过降解条件的优化,在5% (V/V) LB作为额外碳源的辅助下实现了6 d内对50 mg PUR泡沫的降解。以上结果表明Brevibacillus sp. P10在聚氨酯废弃物的生物降解过程中具有一定的应用潜力。