Distribution and Diversity of Acyl Homoserine Lactone Producing Bacteria from Four Different Soils

The distribution and diversity of acyl homoserine lactone (AHL) producing bacteria in black, brown, red, and meadow soils were investigated using culture-dependent method. One out of seventy six and five out of fifty isolates from the black and the brown soil were AHL-biosensor active, respectively. They were affiliated to the genera Ensifer and Pseudomonas and the family Enterobacteriaceae. Thin layer chromatography showed that the most of them produced AHLs with acyl side chain of 6 or 8 carbons. No AHL-producer was found in red and meadow soils. A potential novel AHL-based quorum sensing and quenching system was identified by sequencing in the black soil isolate Ensifer adhaerens X097 that harbored two AHL synthetase-like proteins and one amidohydrolase-like protein. This is the first report of comparison of AHL-producers among different soils. Our data showed that composition of AHL-producers were niche specific and were not in proportion with community population.     

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.     

Synthesis of Signaling N-Acyl-Homoserine-Lactones Participating in Quorum Sensing Regulation in Rhizospheric and Soil-Borne 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 Pseudomonasand 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 these compounds. In at least a half of the isolated P. aureofaciens andP. aeruginosa strains, 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. putidastrains. None of the 41 Xanthomonas maltophilia strains examined synthesized AHL.     

Methods for detecting acylated homoserine lactones produced by Gram-negative bacteria and their application in studies of AHL-production kinetics

In the process of evaluating the role of acylated homoserine lactones (AHLs) in food-spoiling Gram-negative bacteria, we have combined a range of bacterial AHL monitor systems to determine the AHL-profile and the kinetics of AHL-production. AHL production from 148 strains of Enterobacteriaceae isolated from foods was tested using Escherichia coli pSB403 (LuxR), Agrobacterium tumefaciens A136 (TraR) and both induction and inhibition of Chromobacterium violaceum CV026 (CviR). All strains except one was found to produce AHL(s). In no case could a single monitor system identify more than 64% of the Enterobacteriaceae as AHL-producers, showing that the simultaneous use of monitor strains is required in the process of screening bacterial populations for AHL-production. AHLs from 20 selected strains were profiled by thin layer chromatography. Most strains produced more than one AHL with 3-N-oxo-hexanoyl homoserine lactone being the most prominent. It was found that the simultaneous use of monitor strains in the top-layer was necessary for the detection of (presumably) all the AHLs. An agar well-diffusion assay based on A. tumefaciens pDZLR4 was used for quantifying AHLs from bacterial supernatants and enabled an assessment of the kinetics of AHL-production of 3 strains (Serratia proteamaculans strain B5a, Erwinia carotovora ATCC 39048 and V. fischeri strain MJ-1). As expected, the production of AHL (OHHL) and luminescence in Vibrio fischeri strain MJ-1 increased faster than growth indicating up-regulation of the AHL regulated phenotype and auto-induction of AHL production. In contrast, production kinetics of AHL (OHHL) in the two Enterobacteriaceae indicated lack of auto-induction.     

Over-expression of an AT-hook gene, AHL22, delays flowering and inhibits the elongation of the hypocotyl in Arabidopsis thaliana

The Arabidopsis genome encodes 29 AHL (AT-hook motif nuclear localized) proteins, but the function for most of them remains unknown. We report here a study of the AHL22 gene, which was originally identified as a gain-of-function allele that enhanced the phenotype of the cry1 cry2 mutant. AHL22 is a nuclear protein with the binding activity for an AT-rich DNA sequence. AHL22 overexpression delayed flowering and caused a constitutive photomorphogenic phenotype. The loss-of-function AHL22 mutant showed no clear phenotype on flowering, but slightly longer hypocotyls. However, silencing four AHL genes (AHL22, AHL18, AHL27, and AHL29) resulted in early flowering and enhanced ahl22-1 mutant phenotype on the growth of hypocotyls, suggesting genetic redundancy of AHL22 with other AHL genes on these plant developmental events. Further analysis showed that AHL22 controlled flowering and hypocotyl elongation might result from primarily the regulation of FT and PIF4 expression, respectively.     

A Novel Degenerated Primer Pair Detects Diverse Genes of Acyl Homoserine Lactone Synthetase in Rhizobiaceae Family

A novel degenerated primer set was designed to amplify acyl homoserine lactone (AHL) synthetase genes from members of the family Rhizobiaceae. The primer set successfully amplified AHL synthetase genes from pure cultures of AHL producers from Rhizobiaceae, but not from AHL producers out of the Rhizobiaceae family, indicating the specificity of this primer set to the Rhizobiaceae family. An inoculation experiment showed that the minimal detectable concentration of AHL producers from the soil was around 2.5 × 10⁷ CFU/g soil. When applying to environmental samples, 7 and 14 different genotypes of AHL synthetase genes were identified in the rhizosphere of Glycine max and Vigna unguiculata, respectively, which revealed complicated and unknown AHL-based quorum-sensing networks in the rhizosphere. This is the first primer set that covers diverse AHL synthetase genes from different genera. It will be a useful culture-independent approach for better understanding of the ecological significance of QS in natural habitats.     

Diversity of the bacterial community in the rice rhizosphere managed under conventional and no-tillage practices

Bacterial diversity in the rice rhizosphere at different rice growth stages, managed under conventional and no-tillage practices, was explored using a culture-based approach. Actinobacteria are among the bacterial phyla abundant in the rice rhizosphere. Their diversity was further examined by constructing metagenomic libraries based on the 16S rRNA gene, using actinobacterial- and streptomycete-specific polymerase chain reaction (PCR) primers. The study included 132 culturable strains and 125 clones from the 16S rRNA gene libraries. In conventional tillage, there were 38% Proteobacteria, 22% Actinobacteria, 33% Firmicutes, 5% Bacteroidetes, and 2% Acidobacteria, whereas with no-tillage management there were 63% Proteobacteria, 24% Actinobacteria, 6% Firmicutes, and 8% Bacteroidetes as estimated using the culture-dependent method during the four stages of rice cultivation. Principal coordinates analysis was used to cluster the bacterial communities along axes of maximal variance. The different growth stages of rice appeared to influence the rhizosphere bacterial profile for both cultivation practices. Novel clones with low similarities (89–97%) to Actinobacteria and Streptomyces were retrieved from both rice fields by screening the 16S rRNA gene libraries using actinobacterial- and streptomycete-specific primers. By comparing the actinobacterial community retrieved by culture-dependent and molecular methods, it was clear that a more comprehensive assessment of microbial diversity in the rice rhizosphere can be obtained using a combination of both techniques than by using either method alone. We also succeeded in culturing a number of bacteria that were previously described as unculturable. These were in a phylogenetically deep lineage when compared with related cultivable genera.     

Quorum-sensing regulation in soil pseudomonads

228 strains of soil and rhizosphere pseudomonads isolated in different geographic zones were screened, with the use of two tester systems, for the capacity to produce N-acyl-homoserine lactones (AHLs), which are autoinducers involved in quorum-sensing (QS) regulation. AHL production was found in 11.4% of the strains investigated. In five Pseudomonas chlororaphis strains shown to be active AHL producers and chosen for further study, PCR identified two QS systems that involved the phzI, phzR, csaI, and csaR genes; this finding suggests the conservative nature of these regulation systems in P. chloroaphis. Strain P. chlororaphis 449, chosen as a model object and studied in greater detail, produced three AHL species including N-butanoyl-homoserine lactone and N-hexanoyl-homoserine lactone. This strain produced three types of phenazine antibiotics, as well as siderophores and cyanide; it also exhibited antagonistic properties toward a wide spectrum of phytopathogenic fungi. The phzI and csaI genes, coding for synthases of AHLs of two types, were cloned and sequenced; mutants with knocked-out phzI and csaI genes were obtained. With the use of transposon mutagenesis and the gene substitution method, mutations were obtained in the global expression regulator genes gacS, coding for the GacA-GacS regulation system kinase, and rpoS, coding for the sigma S subunit of RNA polymerase. The effect of these mutations on the AHL synthesis and on the regulation of various metabolic processes in P. chlororaphis was studied.     

Design of a noncovalently linked bifunctional enzyme for whole-cell biotransformation

Optical pure d-p-hydroxyphenylglycine (d-HPG) is a precursor for semi-synthetic antibiotics. It can be synthesized from d,l-hydroxyphenyl hydantoin (HPH) by a two-step reaction mediated by d-hydantoinase (HDT) and amidohydrolase (AHL). In this study, a bifunctional enzyme was originally created by in-frame fusion of AHL with HDT genes (AHL-HDT). However, the AHL-HDT fusion protein expressed in Escherichia coli was prone to aggregates, recognized as a frequently encountered problem for this conventional method. To address this issue, small interacting motifs, cohesin (Coh) and dockerin (Doc) domains of cellulosomes, were explored and illustrated to interact in vivo. Accordingly, Coh and Doc were fused with AHL and HDT, respectively. After co-expression in E. coli, Coh-tagged AHL and Doc-tagged HDT assembled into a soluble protein complex via the high-affinity interaction of Coh and Doc. Consequently, the protein assembly exhibited both AHL and HDT activities and a higher reaction rate than free counterparts. Whole cells expressing the protein assembly were more stable than ones with free proteins for d-HPG production, and they could be recycled six times with a conversion yield of d-HPG exceeding 90%.     

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.     

Biochemical detection of N-Acyl homoserine lactone from biofilm-forming uropathogenic Escherichia coli isolated from urinary tract infection samples

Background:

N-Acyl homoserine lactone (AHL) is found to be the main component of quorum sensing (QS) in Gram-negative bacteria and plays an important role in biofilm formation. Little information is available regarding the role of AHL in biofilm formation in Escherichia coli (E. coli). The purpose of this investigation was to biochemically detect and characterize AHL activity in biofilm-forming uropathogenic E. coli (UPEC) isolated from urine samples of the patients with urinary tract infections (UTIs) in Kerman, Iran.

Methods:

Thirty-five UPEC isolates were obtained from urine samples of the patients with UTIs referred to the Afzalipoor hospital. The isolates were identified by biochemical tests. Biofilm analyses of all the isolates were performed using the microtiter plate method at OD 490nm. N-Acyl homoserine lactone was separated from cell mass supernatants by liquid-liquid extraction (LLE) and analyzed by a colorimetric method. N-Acyl homoserine lactone functional groups were identified by Fourier Transform-Infrared Spectroscopy (FT-IR).

Results:

The biofilm formation assay identified 10 (28.57%) isolates with strong, 16 (45.71%) with moderate, and 9 (25.71%) with weak biofilm activities. The UPEC isolates with strong and weak biofilm activities were subjected to AHL analyses. It was found that isolates with the highest AHL activities also exhibited strong adherence to microplate wells (P≤0.05). Two E. coli isolates with the highest AHL activities were selected for FT-IR spectroscopy. Peaks at 1764.33, 1377.99, and 1242.90 cm^-1 correspond to the C=O bond of the lactone ring, and the N=H and C-O bonds of the acyl chain, respectively.

Conclusion:

We found that many UPEC isolates exhibited strong biofilm formation. The control of this property by AHL may contribute to the pathogenesis of the organism in UTI’s.Key Words: Biofilm, FT-IR, N-acylhomoserine lactone, Uropathogenic Escherichia coli     

Genes Encoding the N-Acyl Homoserine Lactone-Degrading Enzyme Are Widespread in Many Subspecies of Bacillus thuringiensis

Gram-negative bacteria can communicate with each other by N-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers. Recently, the aiiA gene (encoding an enzyme catalyzing the degradation of AHL) has been cloned from Bacillus sp. strain 240B1. During investigations in the course of the ongoing Bacillus thuringiensis subsp. morrisoni genome project, an aiiA homologue gene in the genome sequence was found. These results led to consideration of the possibility of the widespread existence of the gene in B. thuringiensis. aiiA homologue genes were found in 16 subspecies of B. thuringiensis, and their sequences were determined. Comparison of the Bacillus sp. strain 240B1 aiiA gene with the B. thuringiensis aiiA homologue genes showed high homologies of 89 to 95% and 90 to 96% in the nucleotide sequence and deduced amino acid sequence, respectively. Among the subspecies of B. thuringiensis having an aiiA gene, the subspecies aizawai, galleriae, kurstaki, kyushuensis, ostriniae, and subtoxicus were shown to degrade AHL. It was observed that recombinant Escherichia coli producing AiiA proteins also had AHL-degrading activity and could also attenuate the plant pathogenicity of Erwinia carotovora. These results indicate that insecticidal B. thuringiensis strains might have potential to compete with gram-negative bacteria in natural ecosystems by autoinducer-degrading activity.     

In-vitro antioxidative,antiinflammatory properties of Aurea helianthus leaf extract a Korean traditional medicinal plant

The leaf of Aurea helianthus (A. helianthus Jinhuakui) is popularly used in China traditional medicine, however, scientific evidence on its antioxidant properties rarely studied. In this study, biological activities of A. helianthus leave’s 80% ethanol extract (AHL) were investigated. The measured total polyphenol and flavonoid content of AHL was 184.24 ± 5.01 mg GAE/g and 102.53 ± 0.98 mg NAR/g. AHL showed the highest α, α-diphenyl-β-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) radical scavenging activities of 98.30 ± 0.18% at 1000 µg/mL. DPPH and ABTS radical scavenging activities significantly increased in a AHL concentration-dependent manner. AHL treatment significantly suppressed the generation of pro-inflammatory mediators, including nitric oxide (NO), in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. AHL demonstrated strong anti-inflammatory activity that reduced NO production in LPS-stimulated RAW 264.7 cells. To test the potential protective effect of AHL, the antioxidant capacity, on the cell growth, viability of a human hepatoma cell (HepG2) and Raw 264.7 cell were investigated. AHL also enhanced cytotoxicity on the proliferation of HepG2 cells and was capable of inhibiting 56% against LPS at 400 µg/mL. The results of this study the potential of AHL as an excellent antioxidant substance for inhibiting inflammatory mediators. Therefore, AHL may be used as a therapeutic approach to various inflammatory diseases.     

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.     

Baseline Survey of Root-Associated Microbes of Taxus chinensis (Pilger) Rehd

Taxol (paclitaxel) a diterpenoid is one of the most effective anticancer drugs identified. Biosynthesis of taxol was considered restricted to the Taxus genera until Stierle et al. discovered that an endophytic fungus isolated from Taxus brevifolia could independently synthesize taxol. Little is known about the mechanism of taxol biosynthesis in microbes, but it has been speculated that its biosynthesis may differ from plants. The microbiome from the roots of Taxus chinensis have been extensively investigated with culture-dependent methods to identify taxol synthesizing microbes, but not using culture independent methods.,Using bar-coded high-throughput sequencing in combination with a metagenomics approach, we surveyed the microbial diversity and gene composition of the root-associated microbiomefrom Taxus chinensis (Pilger) Rehd. High-throughput amplicon sequencing revealed 187 fungal OTUs which is higher than any previously reported fungal number identified with the culture-dependent method, suggesting that T. chinensis roots harbor novel and diverse fungi. Some operational taxonomic units (OTU) identified were identical to reported microbe strains possessing the ability to synthesis taxol and several genes previously associated with taxol biosynthesis were identified through metagenomics analysis.     

The Gut Bacteria Associated with Camponotus japonicus Mayr with Culture-Dependent and DGGE Methods

The bacterial composition and distribution in the different gut regions of Camponotus japonicus were investigated using both culture-dependent method and culture-independent method of polymerase chain reaction and denaturing gradient gel electrophoresis (PCR–DGGE). Five different bacterial strains were isolated using culture-dependent method, and they all belong to the phylum Firmicutes, including three genera of bacteria Bacillus, Paenibacillus, and Enterococcus. Bacillus cereus and Enterococcus mundtii were found in the midgut; Paenibacillus sp. was isolated from the hindgut; and the other two Bacillus spp. were isolated from the crop. Twelve distinct DGGE bands were found using PCR–DGGE method, and their sequences blasting analysis shows that they are members of the Proteobacteria and the Firmicutes, respectively, including three genera (Pseudomonas, Candidatus Blochmannia, Fructobacillus) and one uncultured bacterium, in which Pseudomonas was the most dominant bacteria group in all the three gut regions. According to the DGGE profile, the three gut regions had very similar gut communities, and all the DGGE bands were presented in the midgut and hindgut, while just two bands representing Blochmannia were not present in the crop. The results of our study indicate that the gut of C. japonicus harbors several other bacteria besides the obligate endosymbionts Blochmannia, and more work should be carried on to verify if they are common in the guts of other Camponotus ants.     

Identification of Quorum-Quenching N-Acyl Homoserine Lactonases from Bacillus Species

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.     

MomL,a Novel Marine-Derived N-Acyl Homoserine Lactonase from Muricauda olearia

Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C₆-HSL is ∼2.9 × 10⁵ s⁻¹ M⁻¹. Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the “HXHXDH” motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent.