N-acyl-l-homoserine lactones (AHLs) affect microbial community composition and function in activated sludge

The role of intercellular signalling in the regulation of genes and phenotypes in a broad range of bacterial species is now firmly established. In contrast, the impact of intercellular signalling on microbial community parameters, such as species diversity and function, is less well understood. In this study the role of N-acyl-l-homoserine lactones (AHLs) in microbial community dynamics in an industrial wastewater treatment system is addressed. Seven proteobacterial strains producing compounds with AHL-like activity were isolated from the treatment plant. Three of these belong to genera with no previously identified AHL producing species. Addition of AHLs at 2 micro M to sludge samples generated changes in both community function (phenol degradation) and composition as determined by length heterogeneity PCR and denaturing gradient gel electrophoresis. Phenol degradation was more stable as a result of the AHL augmentation. A dominant functional member of the Thauera genus was transiently supplanted by a member of the Comomonas genus in response to AHL addition. This suggests that AHLs can play a role in mediating microbial community parameters and has implications for ecosystem function and industrial wastewater treatment.     

Nonbioluminescent strains of Photobacterium phosphoreum produce the cell-to-cell communication signal N-(3-Hydroxyoctanoyl)homoserine lactone

Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (Rf value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated.     

N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.     

Quorum Sensing in <Emphasis Type="Italic">Aeromonas</Emphasis> Species Isolated from Patients in Malaysia

Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C₆. Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.     

Investigation of N-acyl homoserine lactone (AHL) molecule production in Gram-negative bacteria isolated from cooling tower water and biofilm samples

In this study, 99 Gram-negative rod bacteria were isolated from cooling tower water, and biofilm samples were examined for cell-to-cell signaling systems, N-acyl homoserine lactone (AHL) signal molecule types, and biofilm formation capacity. Four of 39 (10 %) strains isolated from water samples and 14 of 60 (23 %) strains isolated from biofilm samples were found to be producing a variety of AHL signal molecules. It was determined that the AHL signal molecule production ability and the biofilm formation capacity of sessile bacteria is higher than planktonic bacteria, and there was a statistically significant difference between the AHL signal molecule production of these two groups (p?<?0.05). In addition, it was found that bacteria belonging to the same species isolated from cooling tower water and biofilm samples produced different types of AHL signal molecules and that there were different types of AHL signal molecules in an AHL extract of bacteria. In the present study, it was observed that different isolates of the same strains did not produce the same AHLs or did not produce AHL molecules, and bacteria known as AHL producers did not produce AHL. These findings suggest that detection of signal molecules in bacteria isolated from cooling towers may contribute to prevention of biofilm formation, elimination of communication among bacteria in water systems, and blockage of quorum-sensing controlled virulence of these bacteria.     

食源假单胞菌群体感应信号分子的研究

从市售鲜鱼中分离的3株革兰氏阴性菌,经16S rDNA鉴定为假单胞菌属,该菌是一种导致食品腐败的重要腐败细菌。N-酰基-高丝氨酸内酯(AHLs)是革兰氏阴性菌群体感应(QS)系统中一类重要的信号分子,以密度依赖的方式调控某些生理性状的表达。利用AHLs检测菌株对3株假单胞菌进行检测发现,均产生AHLs类信号分子,且FML05-1和FML05-2至少产生两种AHLs,主要的信号分子是N-3-氧代-辛酰基-高丝氨酸内酯(N- 3-oxo-C_8-HSL)。同时对菌株FML05-2在生长过程中所产生的AHLs的活性变化进行研究,发现AHLs活性在菌体生长至12h时达到最大。首次对食源假单胞菌所产生的AHLs进行了研究,为以干扰腐败细菌群体感应为靶点的食品防腐保鲜策略提供研究基础。     

The presence, type and role of N-acyl homoserine lactone quorum sensing in fluorescent Pseudomonas originally isolated from rice rhizospheres are unpredictable

In Gram-negative bacteria, a typical quorum-sensing (QS) system involves the production and response to N-acyl homoserine lactones (AHLs). It still remains unclear as to how pivotal and conserved AHL QS is in root-colonizing rhizosphere Pseudomonas. We, therefore, performed a systematic study of AHL QS on a set of 50 rice rhizosphere Pseudomonas isolates. We also isolated the AHL QS genes in two representative strains and analyzed the role of AHL QS regulation of various phenotypes. Our results are discussed with the current knowledge of AHL QS of rhizosphere Pseudomonas, implicating a lack of conservation and an unpredictable role played by AHL QS in this group of bacteria.     

碳源及温度对食源假单胞菌群体感应信号分子产生的影响

许多革兰氏阴性菌通过产生N-酰基-高丝氨酸内酯（AHLs）类信号分子来调控某些性状的表达,即群体感应（quorum sensing）。假单胞菌是一种导致食品腐败的重要腐败细菌,也产生AHLs。本文研究了不同温度及碳源对食源假单胞菌AHLs产生的影响。结果表明,该假单胞菌在25℃条件下,产生两种AHL信号分子,而在4℃条件下,所产生的短链AHL分子消失,主要产生长链AHL分子。而且在不同碳源（葡萄糖,果糖,木糖,麦芽糖等）的培养基中生长,所产生的AHLs分子种类也不同。同时发现当pH>7.5时,AHLs的稳定性下降。由此得出,在不同的环境条件(碳源及温度)下假单胞菌所产生的AHLs种类不同。为进一步研究群体感应现象在食品腐败中的作用以及开发基于干扰腐败菌群体感应的新型食品防腐技术提供研究基础。     

N-butanoyl-l-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.     

Detection of acylated homoserine lactones produced by Vibrio spp. and related species isolated from water and aquatic organisms

Aims: To assess the diversity in production of acylated homoserine lactones (AHLs) among Vibrio spp and related species. Methods and Results: A total of 106 isolates, with representatives of 28 Vibrio spp and related species, were investigated for the production of AHLs. For this, a rapid method for the screening of AHLs was developed based on the use of bacterial biosensors using a double‐layer microplate assay. At least one bacterial biosensor was activated in 20 species, Agrobacterium tumefaciens being the most frequently activated biosensor. One isolate of Vibrio anguillarum, Vibrio rotiferianus and Vibrio metschnikovii activated the Chromobacterium violaceum biosensor, which is not common among the Vibrionaceae family. For those species with more than one isolate, the biosensor activation profile was the same except for two species, V. anguillarum and V. metschnikovii, which varied among the different isolates. Conclusions: AHL production was observed in the majority of the studied species, with a diverse biosensor activation profile. Significance and Impact of the Study: The high diversity in AHL production is in consistence with the high diversity in ecological niches of the Vibrionaceae family. The absence of AHL detection in eight species warrants further work on their quorum‐sensing systems.     

Production of N-acyl Homoserine Lactones and Virulence Factors of Waterborne Aeromonas hydrophila

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. The purpose of this study was to investigate the production of N-acyl-homoserine lactone (AHL) signal molecules and some virulence factors, including hemolysins, proteases, extracellular nucleases production and cytotoxicity by waterborne Aeromonas hydrophila. A total of 24 strains isolated from fresh-water or diseased fish were used in the study. The majority A.hydrophila strains produce two AHL molecules (21/24), one is N-butanoyl homoserine lactone (BHL), and the other is N-hexanoyl homoserine lactone (HHL) according to thin-layer chromatography analysis. Among the virulence factors tested, more than 83 % of the isolates produced β haemolysin when inoculated on sheep blood agar, only 50 % of the isolates displayed DNase activity, 75 % of the isolates shown proteolytic activity on skimmed milk plate, and cytotoxic activity was detected in 20 of 24 of the isolates. The strains producing AHLs possessed one or more virulence factors. In conclusion, the production of quorum sensing signal molecules is common among the strains that we examined, and there seems to some relationships between quorum sensing signal production and virulence factors in A. hydrophila.     

Isolation and characterization of acyl homoserine lactone–producing bacteria during an urban river biofilm formation

The presence and diversity of acyl homoserine lactone (AHL)-producers in an urban river biofilm were investigated during 60-day biofilm formation. AHL biosensors detected the presence of AHL-producers in 1–60-day river biofilms. Screening for AHL-producers resulted in 17 Aeromonas spp., 3 Pseudomonas spp., 3 Ensifer spp., and 1 Acinetobacter sp. Among these isolates, six of them were closely related to Acinetobacter tjernbergiae, Aeromonas allosaccharophila, Aeromonas aquariorum, Aeromonas jandaei, Pseudomonas panipatensis, and Ensifer adhaerens and represented novel AHL-producing species. Thin layer chromatography revealed that C4-homoserine lactone was prevailing in Aeromonas spp., whereas C6- and C8-homoserine lactones and their derivatives were prevailing in other strains. Using degenerate primers, novel AHL synthetase genes from the three Ensifer spp. were successfully amplified. This study reports for the first time the diversity of AHL-producers from a river biofilm and the variety of novel AHL synthetase genes in Ensifer group.     

Diversity and quorum-sensing signal production of Proteobacteria associated with marine sponges

Marine sponges are hosts to diverse and dense bacterial communities and thus provide a potential environment for quorum sensing. Quorum sensing, a key factor in cell–cell communication and bacterial colonization of higher animals, might be involved in the symbiotic interactions between bacteria and their sponge hosts. Given that marine Proteobacteria are known to produce N -acyl homoserine lactone (AHL) signal molecules, we tested the production of AHLs by Alpha - and Gammaproteobacteria isolated from marine sponges Mycale laxissima and Ircinia strobilina and the surrounding water column. We used three different AHL biodetection systems in diffusion assays: Chromobacterium violaceum , Agrobacterium tumefaciens and Sinorhizobium meliloti with optimal sensitivity to short-chain (C4–C6), moderate-chain (C8–C12) and long-chain (≥ C14) AHLs respectively. Thirteen of 23 isolates from M. laxissima and five of 25 isolates from I. strobilina were found to produce AHLs. Signals were detected from two of eight proteobacterial strains from the water column. Thin-layer chromatographic assays based on the A. tumefaciens reporter system were utilized to determine the AHL profiles of the positive isolates. The types and amounts of AHLs synthesized varied considerably among the strains. Small ribosomal rRNA gene sequencing revealed that the AHL-producing alphaproteobacterial isolates were mainly from the Silicibacter–Ruegeria subgroup of the Roseobacter clade. Two-dimensional gel electrophoresis (2DGE)-based proteomic analyses were congruent with phylogenetic relationships but provided higher resolution to differentiate these closely related AHL-producing strains.     

Detection of Homoserine Lactone-Like Quorum Sensing Molecules in Bradyrhizobium Strains

One hundred and forty-two Bradyrhizobium strains were screened for their ability to produce N-acyl homoserine lactone-like molecules (AHLs) by using an Agrobacterium tumefaciens biosensor strain containing a traI-lacZ fusion. Approximately 22% (31 of 142) of the tested strains produced AHLs that induced moderate to elevated β-galactosidase activity levels in the biosensor strain. Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were both shown to produce AHLs. Age of culture, and media composition were each shown to influence production of AHL(s), with greater production occurring in 2 day-old cultures grown in rich media. Reverse-phase high-performance liquid chromatography and thin-layer chromatography analyses indicated that the B. japonicum strain USDA 290 produced at least two types of AHLs. Our results indicate that the production AHL-like autoinducers is widespread among both B. japonicum and B. elkanii strains.     

Occurrence of N-acyl-L-homoserine lactones in extracts of some Gram-negative bacteria evaluated by gas chromatography-mass spectrometry

Acylated homoserine lactones (AHLs) are self-generated signal molecules that mediate population density-dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria. These signal molecules diffuse from bacterial cells and accumulate in the medium as a function of cell growth. In selected foods AHLs contribute to product spoilage. As different bacterial species produce AHL analogs that differ in length of the N-acyl chain, ranging from 4 to 14 carbons and in the substitution at the C-3 position of the side chain (i.e., oxo or hydroxyl group), the suitability and applicability of a gas chromatography-mass spectrometry direct method for characterizing trace amounts of AHLs was evaluated using N-heptanoyl-homoserine lactone as internal standard. Crude cell-free supernatants of bacterial cultures of Aeromonas hydrophila, Aeromonas salmonicida, Pseudomonas aeruginosa, Pseudomonas fluorescens, Yersinia enterocolitica, and Serratia liquefaciens were screened for AHL production in selected ion monitoring mode, using the prominent fragment at m/z 143. The observed profiles of distinguishable N-acyl-homoserine lactones occurring in bacterial extracts were compared and discussed. The presence of a labile 3-oxo-hexanoylhomoserine lactone was evidenced but serious difficulties arose in estimating its concentration as thermal degradation occurs during the gas chromatographic separation. Its electron impact mass spectra was, however, given and interpreted.     

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.     

AmiE,a Novel N-Acylhomoserine Lactone Acylase Belonging to the Amidase Family,from the Activated-Sludge Isolate Acinetobacter sp. Strain Ooi24

Many Gram-negative bacteria use N-acyl-l-homoserine lactones (AHLs) as quorum-sensing signal molecules. We have reported that Acinetobacter strains isolated from activated sludge have AHL-degrading activity. In this study, we cloned the amiE gene as an AHL-degradative gene from the genomic library of Acinetobacter sp. strain Ooi24. High-performance liquid chromatography analysis revealed that AmiE functions as an AHL acylase, which hydrolyzes the amide bond of AHL. AmiE showed a high level of degrading activity against AHLs with long acyl chains but no activity against AHLs with acyl chains shorter than eight carbons. AmiE showed homology with a member of the amidases (EC 3.5.1.4) but not with any known AHL acylase enzymes. An amino acid sequence of AmiE from Ooi24 showed greater than 99% identities with uncharacterized proteins from Acinetobacter ursingii CIP 107286 and Acinetobacter sp. strain CIP 102129, but it was not found in the draft or complete genome sequences of other Acinetobacter strains. The presence of transposase-like genes around the amiE genes of these three Acinetobacter strains suggests that amiE is transferred by a putative transposon. Furthermore, the expression of AmiE in Pseudomonas aeruginosa PAO1 reduced AHL accumulation and elastase activity, which were regulated by AHL-mediated quorum sensing.     

Synthesis of signaling N-acyl-homoserine-lactones participating in quorum sensing in rhizosphere and soil bacteria Pseudomonas and Xanthomonas

Signaling molecules assigned to N-acyl-homoserine-lactones (AHL) serve as autoinducers for the genes controlling the quorum sensing regulatory system. In many gram-negative bacteria, AHL are the key factors responsible for density-dependent regulation of exoenzyme and secondary metabolite production; they also participate in interaction between bacteria and higher organisms. The soil and rhisosphere bacteria Pseudomonas and Xanthomonas from different geographical zones of Russia and the former USSR were analyzed for the presence of the AHL producers. Screening was conducted by using a test system based on the mutant strain Chromobacterium violaceum, which was unable to synthesize AHL but produced a pigment violacein in the presence of exogenous AHL. The AHL-like compounds proved to be formed by 9.7% of the studied bacteria. Various Pseudomonas species differed in the capacity to synthesize this compounds. In at least a half of the isolated P. aureofaciens and P. aeruginosa, an intense AHL production was observed, whereas the AHL-producers were far less frequent among the P. fluorescens, P. chlororaphis, P. lemonnieri, P. geniculata, and P. putida. None of the 41 Xanthomonas maltophilia strains examined synthesized AHL.     

Production and Degradation of N-Acylhomoserine Lactone Quorum Sensing Signal Molecules in Bacteria Isolated from Activated Sludge

N-Acylhomoserine lactones (AHLs) function as quorum-sensing signaling molecules in many Gram-negative bacteria. We isolated a total of 672 bacterial strains from activated sludge obtained from seven sewage treatment plants in Tochigi Prefecture, Japan, and screened for AHL-producing and degrading strains. Isolates (n=107) stimulated AHL-mediated purple pigment production in AHL reporter strains Chromobacterium violaceum CV026 and VIR07. Based on their 16S rRNA gene sequences, most of these AHL-producing isolates were assigned to the genus Aeromonas, and they were divided into six groups. Isolates (n=46) degraded N-decanoyl-L-homoserine lactone (C10-HSL) within 24 h. Based on their 16S rRNA gene sequences, the most dominant AHL-degrading isolates were assigned to the genus Acinetobacter and divided into six groups. Strains Ooi24, Omo91, and Uzu81, which showed higher C10-HSL-degrading activity, showed putative AHL-acylase activity.     

N-acylhomoserine lactonase producing Rhodococcus spp. with different AHL-degrading activities

N-acylhomoserine lactones (AHLs) are conserved signal molecules that control diverse biological activities in quorum sensing system of Gram-negative bacteria. Recently, several soil bacteria were found to degrade AHLs, thereby interfering with the quorum sensing system. Previously, Rhodococcus erythropolis W2 was reported to degrade AHLs by both oxido-reductase and AHL-acylase. In the present study, two AHL-utilizing bacteria, strains LS31 and PI33, were isolated and identified as the genus Rhodococcus. They exhibited different AHL-utilization abilities: Rhodococcus sp. strain LS31 rapidly degraded a wide range of AHLs, including N-3-oxo-hexanoyl-l-homoserine lactone (OHHL), whereas Rhodococcus sp. strain PI33 showed relatively less activity towards 3-oxo substituents. Coculture of strain LS31 with Erwinia carotovora effectively reduced the amount of OHHL and pectate lyase activity, compared with coculture of strain PI33 with E. carotovora. A mass spectrometry analysis indicated that both strains hydrolyzed the lactone ring of AHL to generate acylhomoserine, suggesting that AHL-lactonases (AHLases) from the two Rhodococcus strains are involved in the degradation of AHL, in contrast to R. erythropolis W2. To the best of our knowledge, this is the first report on AHLases of Rhodococcus spp.