Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene.

Myriad proteobacteria use N-acyl homoserine lactone (AHL) molecules as quorum sensing (QS) signals to regulate different physiological functions, including virulence, antibiotic production, and biofilm formation. Many of these proteobacteria possess LuxI/LuxR system as the QS mechanism. Recently, we reported the 3.89 Mb genome of Acinetobacter sp. strain GG2. In this work, the genome of this long chain AHL-producing bacterium was unravelled which led to the molecular characterization of luxI homologue, designated as aciI. This 552 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was ∼20.5 kDa and is highly similar to several autoinducer proteins of LuxI family among Acinetobacter species. To verify the AHL synthesis activity of this protein, high-resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-dodecanoyl-homoserine lactone and 3-hydroxy-dodecanoyl-homoserine lactone from induced E. coli harboring the recombinant AciI. Our data show for the first time, the cloning and characterization of the luxI homologue from Acinetobacter sp. strain GG2, and confirmation of its AHLs production. These data are of great significance as the annotated genome of strain GG2 has provided a valuable insight in the study of autoinducer molecules and its roles in QS mechanism of the bacterium.

Pseudomonas cremoricolorata strain ND07 produces N-acyl homoserine lactones as quorum sensing molecules.

Quorum sensing (QS) is a bacterial cell-to-cell communication system controlling QS-mediated genes which is synchronized with the population density. The regulation of specific gene activity is dependent on the signaling molecules produced, namely N-acyl homoserine lactones (AHLs). We report here the identification and characterization of AHLs produced by bacterial strain ND07 isolated from a Malaysian fresh water sample. Molecular identification showed that strain ND07 is clustered closely to Pseudomonas cremoricolorata. Spent culture supernatant extract of P. cremoricolorata strain ND07 activated the AHL biosensor Chromobacterium violaceum CV026. Using high resolution triple quadrupole liquid chromatography-mass spectrometry, it was confirmed that P. cremoricolorata strain ND07 produced N-octanoyl-L-homoserine lactone (C8-HSL) and N-decanoyl-L-homoserine lactone (C10-HSL). To the best of our knowledge, this is the first documentation on the production of C10-HSL in P. cremoricolorata strain ND07.

Short chain N-acyl homoserine lactone production by soil isolate Burkholderia sp. strain A9.

In the bacteria kingdom, quorum sensing (QS) is a cell-to-cell communication that relies on the production of and response to specific signaling molecules. In proteobacteria, N-acylhomoserine lactones (AHLs) are the well-studied signaling molecules. The present study aimed to characterize the production of AHL of a bacterial strain A9 isolated from a Malaysian tropical soil. Strain A9 was identified as Burkholderia sp. using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and 16S rDNA nucleotide sequence analysis. AHL production by A9 was detected with two biosensors, namely Chromobacterium violaceum CV026 and Escherichia coli [pSB401]. Thin layer chromatography results showed N-hexanoylhomoserine lactone (C6-HSL) and N-octanoylhomoserine lactone (C8-HSL) production. Unequivocal identification of C6-HSL and C8-HSL was achieved by high resolution triple quadrupole liquid chromatography-mass spectrometry analysis. We have demonstrated that Burkholderia sp. strain A9 produces AHLs that are known to be produced by other Burkholderia spp. with CepI/CepR homologs.

Degradation of bacterial quorum sensing signaling molecules by the microscopic yeast Trichosporon loubieri isolated from tropical wetland waters.

Proteobacteria produce N-acylhomoserine lactones as signaling molecules, which will bind to their cognate receptor and activate quorum sensing-mediated phenotypes in a population-dependent manner. Although quorum sensing signaling molecules can be degraded by bacteria or fungi, there is no reported work on the degradation of such molecules by basidiomycetous yeast. By using a minimal growth medium containing N-3-oxohexanoylhomoserine lactone as the sole source of carbon, a wetland water sample from Malaysia was enriched for microbial strains that can degrade N-acylhomoserine lactones, and consequently, a basidiomycetous yeast strain WW1C was isolated. Morphological phenotype and molecular analyses confirmed that WW1C was a strain of Trichosporon loubieri. We showed that WW1C degraded AHLs with N-acyl side chains ranging from 4 to 10 carbons in length, with or without oxo group substitutions at the C3 position. Re-lactonisation bioassays revealed that WW1C degraded AHLs via a lactonase activity. To the best of our knowledge, this is the first report of degradation of N-acyl-homoserine lactones and utilization of N-3-oxohexanoylhomoserine as carbon and nitrogen source for growth by basidiomycetous yeast from tropical wetland water; and the degradation of bacterial quorum sensing molecules by an eukaryotic yeast.

Plant-derived natural products as sources of anti-quorum sensing compounds.

Quorum sensing is a system of stimuli and responses in relation to bacterial cell population density that regulates gene expression, including virulence determinants. Consequently, quorum sensing has been an attractive target for the development of novel anti-infective measures that do not rely on the use of antibiotics. Anti-quorum sensing has been a promising strategy to combat bacterial infections as it is unlikely to develop multidrug resistant pathogens since it does not impose any selection pressure. A number of anti-quorum sensing approaches have been documented and plant-based natural products have been extensively studied in this context. Plant matter is one of the major sources of chemicals in use today in various industries, ranging from the pharmaceutical, cosmetic, and food biotechnology to the textile industries. Just like animals and humans, plants are constantly exposed to bacterial infections, it is therefore logical to expect that plants have developed sophisticated of chemical mechanisms to combat pathogens. In this review, we have surveyed the various types of plant-based natural products that exhibit anti-quorum sensing properties and their anti-quorum sensing mechanisms.

Complete genome sequence of Burkholderia sp. Strain GG4, a betaproteobacterium that reduces 3-oxo-N-acylhomoserine lactones and produces different N-acylhomoserine lactones.

Burkholderia sp. strain GG4, isolated from the ginger rhizosphere, possesses a unique N-acylhomoserine lactone (AHL)-modifying activity that reduces 3-oxo-AHLs to 3-hydroxy-AHLs. To the best of our knowledge, this is the first sequenced genome from a bacterium of the genus Burkholderia that shows both quorum-sensing and signaling confusion activities.

Whole-genome sequence of N-acylhomoserine lactone-synthesizing and -degrading Acinetobacter sp. strain GG2.

Acinetobacter sp. strain GG2 is a quorum-sensing and quorum-quenching bacterium isolated from the ginger rhizosphere. It degrades a broad range of N-acylhomoserine lactone molecules via lactonase. The genome sequence of strain GG2 may provide insights on the regulation of quorum-sensing and quorum-quenching mechanisms in this bacterium.

Unusual long-chain N-acyl homoserine lactone production by and presence of quorum quenching activity in bacterial isolates from diseased tilapia fish.

Growth-dependent cell-cell communication termed quorum sensing is a key regulatory system in bacteria for controlling gene expression including virulence factors. In this study five potential bacterial pathogens including Bacillus sp. W2.2, Klebsiella sp. W4.2, Pseudomonas sp. W3 and W3.1 and Serratia sp. W2.3 were isolated from diseased Tilapia fish in Malaysia, supplied by the leading global fish supplier. Proteolytic activity assays confirmed that with the exception of Klebsiella sp. W4.2, all isolates showed distinct proteolytic activity. Furthermore Bacillus sp. W2.2 and Pseudomonas sp. strains W3 and W3.1 also displayed haemolytic activity. By using high resolution liquid chromatography mass spectrometry, we revealed the presence of unusually long-chain N-(3-oxohexadecanoyl)-homoserine lactone (3-oxo-C16-HSL) from Pseudomonas sp. W3.1 and N-dodecanoyl-homoserine lactone (C12-HSL) from Serratia sp. W2.3, respectively. Interestingly, Pseudomonas sp. W3.1 also produced a wide range of Pseudomonas quinolone signalling (PQS) molecules. Pseudomonas sp. W3 did not show any quorum sensing properties but possessed quorum quenching activity that inactivated AHLs. This study is the first documentation that shows unusual long-chain AHLs production in Serratia sp. and Pseudomonas sp. isolated from diseased fish and the latter also produce a wide range of PQS molecules.

Coexistence of quorum-quenching and quorum-sensing in tropical marine Pseudomonas aeruginosa strain MW3A.

A chemically defined medium called KGm medium was used to isolate from a sample of sea water a bacterial strain, MW3A, capable of using N-3-oxohexanoyl-L: -homoserine lactone as the sole carbon source. MW3A was clustered closely to Pseudomonas aeruginosa by 16S ribosomal DNA sequence analysis. It degraded both N-acylhomoserine lactones (AHLs) with a 3-oxo group substitution and, less preferably, AHLs with unsubstituted groups at C3 position in the acyl side chain, as determined by Rapid Resolution Liquid Chromatography. Its quiP and pvdQ homologue gene sequences showed high similarities to those of known acylases. Spent supernatant of MW3A harvested at 8-h post inoculation was shown to contain long-chain AHLs when assayed with the biosensor Escherichia coli [pSB1075], and specifically N-dodecanoyl-L: -homoserine lactone and N-3-oxotetradecanoyl-L: -homoserine lactone by high resolution mass spectrometry. Hence, we report here a novel marine P. aeruginosa strain MW3A possessing both quorum-quenching and quorum-sensing properties.

N-acyl homoserine lactone production by Klebsiella pneumoniae isolated from human tongue surface.

Bacteria communicate by producing quorum sensing molecules called autoinducers, which include autoinducer-1, an N-hexanoyl homoserine lactone (AHL), and autoinducer-2. Bacteria present in the human oral cavity have been shown to produce autoinducer-2, but not AHL. Here, we report the isolation of two AHL-producing Klebsiella pneumoniae strains from the posterior dorsal surface of the tongue of a healthy individual. Spent culture supernatant extracts from K. pneumoniae activated the biosensors Agrobacterium tumefaciens NTL4(pZLR4) and Escherichia coli [pSB401], suggesting the presence of both long and short chain AHLs. High resolution mass spectrometry analyses of these extracts confirmed that both K. pneumoniae isolates produced N-octanoylhomoserine lactone and N-3-dodecanoyl-L-homoserine lactone. To the best of our knowledge, this is the first report of the isolation of K. pneumoniae from the posterior dorsal surface of the human tongue and the production of these AHLs by this bacterium.

Quorum quenching Bacillus sonorensis isolated from soya sauce fermentation brine.

An N-acylhomoserine lactone (AHL)-degrading bacterial strain, L62, was isolated from a sample of fermentation brine of Chinese soya sauce by using rich medium agar supplemented with soya sauce (10% v/v). L62, a rod-shaped Gram positive bacterium with amylolytic activity, was phylogentically related to Bacillus sonorensis by 16S ribosomal DNA and rpoB sequence analyses. B. sonorensis L62 efficiently degraded N-3-oxohexanoyl homoserine lactone and N-octanoylhomoserine lactone. However, the aiiA homologue, encoding an autoinducer inactivation enzyme catalyzing the degradation of AHLs, was not detected in L62, suggesting the presence of a different AHL-degrading gene in L62. To the best of our knowledge, this is the first report of AHL-degrading B. sonorensis from soya sauce liquid state fermentation.

Quorum quenching revisited--from signal decays to signalling confusion.

In a polymicrobial community, while some bacteria are communicating with neighboring cells (quorum sensing), others are interrupting the communication (quorum quenching), thus creating a constant arms race between intercellular communication. In the past decade, numerous quorum quenching enzymes have been found and initially thought to inactivate the signalling molecules. Though this is widely accepted, the actual roles of these quorum quenching enzymes are now being uncovered. Recent evidence extends the role of quorum quenching to detoxification or metabolism of signalling molecules as food and energy source; this includes "signalling confusion", a term coined in this paper to refer to the phenomenon of non-destructive modification of signalling molecules. While quorum quenching has been explored as a novel anti-infective therapy targeting, quorum sensing evidence begins to show the development of resistance against quorum quenching.

Characterization of quorum sensing and quorum quenching soil bacteria isolated from Malaysian tropical montane forest.

We report the production and degradation of quorum sensing N-acyl-homoserine lactones by bacteria isolated from Malaysian montane forest soil. Phylogenetic analysis indicated that these isolates clustered closely to the genera of Arthrobacter, Bacillus and Pseudomonas. Quorum quenching activity was detected in six isolates of these three genera by using a series of bioassays and rapid resolution liquid chromatography analysis. Biosensor screening and high resolution liquid chromatography-mass spectrometry analysis revealed the production of N-dodecanoyl-L-homoserine lactone (C12-HSL) by Pseudomonas frederiksbergensis (isolate BT9). In addition to degradation of a wide range of N-acyl-homoserine lactones, Arthrobacter and Pseudomonas spp. also degraded p-coumaroyl-homoserine lactone. To the best of our knowledge, this is the first documentation of Arthrobacter and Pseudomonas spp. capable of degrading p-coumaroyl-homoserine lactone and the production of C12-HSL by P. frederiksbergensis.

Characterization of wetland quorum quenching Pseudomonas aeruginosa strain 2SW8 and its 2-heptyl-3-hydroxy-4-quinolone production.

Most Proteobacteria produce N-acylhomoserine lactones for bacterial cell-to-cell communication, a process called quorum sensing. Interference of quorum sensing, commonly known as quorum quenching, represents an important way to control quorum sensing. This work reports the isolation of quorum quenching bacterium strain 2WS8 from Malaysia tropical wetland water (2°11'8"N, 102°15'2"E, in 2007) by using a modified version of a previously reported KG medium. Strain 2WS8 was isolated based on its ability to utilize N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) as the sole source of energy. This bacterium clustered closely to Pseudomonas aeruginosa PAO1. Strain 2SW8 possesses both quiP and pvdQ homologue acylase genes. Rapid Resolution Liquid Chromatography analysis confirmed that strain 2SW8 preferentially degraded N-acylhomoserine lactones with 3-oxo group substitution but not those with unsubstituted groups at C3 position in the acyl side chain. Strain 2SW8 also showed 2-heptyl-3-hydroxy-4-quinolone production.

A complex multilevel attack on Pseudomonas aeruginosa algT/U expression and algT/U activity results in the loss of alginate production.

Infection by the opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. This is mainly due to the genotypic and phenotypic changes of the bacteria that cause conversion from a typical nonmucoid to a mucoid form in the CF lung. Mucoid conversion is indicative of overproduction of a capsule-like polysaccharide called alginate. The alginate-overproducing (Alg(+)) mucoid phenotype seen in the CF isolates is extremely unstable. Low oxygen tension growth of mucoid variants readily selects for nonmucoid variants. The switching off mechanism has been mapped to the algT/U locus, and the molecular basis for this conversion was partially attributed to mutations in the algT/U gene itself. To further characterize molecular changes resulting in the unstable phenotype, an isogenic PAO1 derivative that is constitutively Alg(+) due to the replacement of the mucA with mucA22 (PDO300) was used. The mucA22 allele is common in mucoid CF isolates. Thirty-four spontaneous nonmucoid variants, or sap (suppressor of alginate production) mutants, of PDO300 were isolated under low oxygen tension. About 40% of the sap mutants were rescued by a plasmid carrying algT/U (Group A). The remaining sap mutants were not (Group B). The members of Group B fall into two subsets: one similar to PAO1, and another comparable to PDO300. Sequence analysis of the algT/U and mucA genes in Group A shows that mucA22 is intact, whereas algT/U contains mutations. Genetic complementation and sequencing of one Group B sap mutant, sap22, revealed that the nonmucoid phenotype was due to the presence of a mutation in PA3257. PA3257 encodes a putative periplasmic protease. Mutation of PA3257 resulted in decreased algT/U expression. Thus, inhibition of algT/U is a primary mechanism for alginate synthesis suppression.

Malabaricone C from Myristica cinnamomea exhibits anti-quorum sensing activity.

A methanol-soluble extract of the bark of Myristica cinnamomea was found to exhibit anti-quorum sensing activity, and subsequent bioassay-guided isolation led to the identification of the active compound malabaricone C (1). Compound 1 inhibited violacein production by Chromobacterium violaceum CV026 when grown in the presence of a cognate signaling molecule, N-3-oxohexanoyl-homoserine lactone. Furthermore, 1 inhibited the quorum sensing-regulated pyocyanin production and biofilm formation in Pseudomonas aeruginosa PAO1. These results suggest that the anti-quorum sensing activity of 1 and related molecules should be investigated further.

Characterization of N-acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale (ginger) rhizosphere: co-existence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia.

BACKGROUND: Cell-to-cell communication (quorum sensing (QS)) co-ordinates bacterial behaviour at a population level. Consequently the behaviour of a natural multi-species community is likely to depend at least in part on co-existing QS and quorum quenching (QQ) activities. Here we sought to discover novel N-acylhomoserine lactone (AHL)-dependent QS and QQ strains by investigating a bacterial community associated with the rhizosphere of ginger (Zingiber officinale) growing in the Malaysian rainforest. RESULTS: By using a basal growth medium containing N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) as the sole source of carbon and nitrogen, the ginger rhizosphere associated bacteria were enriched for strains with AHL-degrading capabilities. Three isolates belonging to the genera Acinetobacter (GG2), Burkholderia (GG4) and Klebsiella (Se14) were identified and selected for further study. Strains GG2 and Se14 exhibited the broadest spectrum of AHL-degrading activities via lactonolysis while GG4 reduced 3-oxo-AHLs to the corresponding 3-hydroxy compounds. In GG2 and GG4, QQ was found to co-exist with AHL-dependent QS and GG2 was shown to inactivate both self-generated and exogenously supplied AHLs. GG2, GG4 and Se14 were each able to attenuate virulence factor production in both human and plant pathogens. CONCLUSIONS: Collectively our data show that ginger rhizosphere bacteria which make and degrade a wide range of AHLs are likely to play a collective role in determining the QS-dependent phenotype of a polymicrobial community.

Phenotype and TCR-gamma gene rearrangements in a Malaysian cohort of T-cell leukaemia/lymphoma cases.

T cells undergo a series of complex phenotypic changes before achieving maturation. Discrete stages of T-cell differentiation are simplified to four stages (pro-, pre-, cortical and mature-T cell) and used in the classification of T-cell leukaemia. HLA-DR has been reported to be expressed in immature T-cell acute lymphoblastic leukemia (ALL) and also confer a poorer treatment outcome. Simultaneously, the genotype goes through distinct pattern changes due to rearrangement of T-cell receptor (TCR) genes. TCR gene rearrangement is important in the diagnosis of clonality and used as markers to detect minimal residual disease in lymphoproliferative disorders. We identified a subset within Pro-T and Pre-T cell cases distinguished by the expression of HLA-DR. These subgroups appeared to be more immature as rearrangement of the TCR-gamma gene was either at germline or involved only the first constant region (C1) unlike a more rearranged pattern in the HLA-DR-subgroups. We also observed a higher incidence of mediastinal mass (67%) in the HLA-DR-subgroup in the Pre-T stage. These characteristics may be useful as markers to further refine staging of T-cell ALL and determine prognosis.

Rapid degradation of N-3-oxo-acylhomoserine lactones by a Bacillus cereus isolate from Malaysian rainforest soil.

A bacterial strain, KM1S, was isolated from a Malaysian rainforest soil sample by using a defined enrichment medium that specifically facilitates selection of quorum quenching bacteria. KM1S was clustered closely to Bacillus cereus by 16S ribosomal DNA sequence analysis. It degraded N-3-oxo-hexanoyl homoserine lactone and N-3-oxo-octanoyl homoserine lactone in vitro rapidly at 4.98 and 6.56 microg AHL h(-1) per 10(9) CFU/ml, respectively, as determined by the Rapid Resolution Liquid Chromatography. The aiiA homologue, encoding an autoinducer inactivation enzyme catalyzing the degradation of N-acylhomoserine lactones, of KM1S was amplified and cloned. Sequence analysis indicated the presence of the motif (106)HXDH-59 amino acids-H(169)-21 amino acids-D(191) for N-acylhomoserine lactone lactonases.

A novel medium for the isolation of N-acylhomoserine lactone-degrading bacteria.

A novel chemically defined medium, named KG medium, supplemented with N-3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL), an acylhomoserine lactone (AHL) used as signalling molecules in Gram-negative bacterial cell-to-cell communication, as the sole source of carbon and nitrogen, was designed and successfully used for the enrichment and isolation of AHL-degrading bacteria. A 3-oxo-C6-HSL-degrading bacterium, 13sw7, was isolated from sewage after six enrichment transfers in the 3-oxo-C6-HSL-containing KG medium. On the basis of the almost complete 16S ribosomal DNA sequence, isolate 13sw7 was clustered with unculturable beta-proteobacteria. This study indicates that the AHL-containing KG medium is effective in isolating AHL-degrading bacteria, including those previously considered unculturable, from environmental sources. To the best of our knowledge, this is the first documentation of the isolation of an AHL-degrading proteobacterium from sewage.