Use of acetate as substrate for sustainable production of homoserine and threonine by Escherichia coli W3110: A modular metabolic engineering approach.

Acetate, a promising yet underutilized carbon source for biological production, was explored for the efficient production of homoserine and threonine in Escherichia coli W. A modular metabolic engineering approach revealed the crucial roles of both acetate assimilation pathways (AckA/Pta and Acs), optimized TCA cycle flux and glyoxylate shunt activity, and enhanced CoA availability, mediated by increased pantothenate kinase activity, for efficient homoserine production. The engineered strain W-H22/pM2/pR1P exhibited a high acetate assimilation rate (5.47 mmol/g cell/h) and produced 44.1 g/L homoserine in 52 h with a 53% theoretical yield (0.18 mol/mol) in fed-batch fermentation. Similarly, strain W-H31/pM2/pR1P achieved 45.8 g/L threonine in 52 h with a 65% yield (0.22 mol/mol). These results represent the highest reported levels of amino acid production using acetate, highlighting its potential as a valuable and sustainable feedstock for biomanufacturing.

Quorum sensing and iron-dependent coordinated control of autoinducer-2 production via small RNA RyhB in Vibrio vulnificus.

Roles for the non-coding small RNA RyhB in quorum-sensing and iron-dependent gene modulation in the human pathogen V. vulnificus were assessed in this study. Both the quorum sensing master regulator SmcR and the Fur-iron complex were observed to bind to the region upstream of the non-coding small RNA RyhB gene to repress expression, which suggests that RyhB is associated with both quorum-sensing and iron-dependent signaling in this pathogen. We found that expression of LuxS, which is responsible for the biosynthesis of autoinducer-2 (AI-2), was higher in wild type than in a ryhB-deletion isotype. RyhB binds directly to the 5'-UTR (untranslated region) of the luxS transcript to form a heteroduplex, which not only stabilizes luxS mRNA but also disrupts the secondary structure that normally obscures the translational start codon and thereby allows translation of LuxS to begin. The binding of RyhB to luxS mRNA requires the chaperone protein Hfq, which stabilizes RyhB. These results demonstrate that the small RNA RyhB is a key element associated with feedback control of AI-2 production, and that it inhibits quorum-sensing signaling in an iron-dependent manner. This study, taken together with previous studies, shows that iron availability and cell density signals are funneled to SmcR and RyhB, and that these regulators coordinate cognate signal pathways that result in the proper balance of protein expression in response to environmental conditions.

Study of the Dynamics of Biofilm Formation and Elastase Activity of Pseudomonas aeruginosa in the Presence of Dodecanoyl-Homoserine Lactone.

We studied the effect of early accumulation of N-3-oxo-dodecanoyl-homoserine lactone on the suppression of Pseudomonas aeruginosa reproduction, biofilm formation, and elastase activity. N-3-oxo-dodecanoyl-homoserine lactone in various concentrations was added to the P. aeruginosa culture, and changes in the concentration of bacteria and the formation of biofilms were studied in dynamics. N-3-oxo-dodecanoyl-homoserine lactone in a concentration of 25 µM, decelerated proliferation of bacterial cells during the first 6 h of culturing (p<0.05) and stimulated biofilm formation after 18 h of culturing. Elastase activity of P. aeruginosa increased significantly after addition of N-3-oxo-dodecanoyl-homoserine lactone in a concentration of 0.75 µM.

5-Fluorouracil blocks quorum-sensing of biofilm-embedded methicillin-resistant Staphylococcus aureus in mice.

Antibiotic-resistant pathogens often escape antimicrobial treatment by forming protective biofilms in response to quorum-sensing communication via diffusible autoinducers. Biofilm formation by the nosocomial pathogen methicillin-resistant Staphylococcus aureus (MRSA) is triggered by the quorum-sensor autoinducer-2 (AI-2), whose biosynthesis is mediated by methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) and S-ribosylhomocysteine lyase (LuxS). Here, we present a high-throughput screening platform for small-molecular inhibitors of either enzyme. This platform employs a cell-based assay to report non-toxic, bioavailable and cell-penetrating inhibitors of AI-2 production, utilizing engineered human cells programmed to constitutively secrete AI-2 by tapping into the endogenous methylation cycle via ectopic expression of codon-optimized MTAN and LuxS. Screening of a library of over 5000 commercial compounds yielded 66 hits, including the FDA-licensed cytostatic anti-cancer drug 5-fluorouracil (5-FU). Secondary screening and validation studies showed that 5-FU is a potent quorum-quencher, inhibiting AI-2 production and release by MRSA, Staphylococcus epidermidis, Escherichia coli and Vibrio harveyi. 5-FU efficiently reduced adherence and blocked biofilm formation of MRSA in vitro at an order-of-magnitude-lower concentration than that clinically relevant for anti-cancer therapy. Furthermore, 5-FU reestablished antibiotic susceptibility and enabled daptomycin-mediated prevention and clearance of MRSA infection in a mouse model of human implant-associated infection.

Transglutaminase-mediated assembly of multi-enzyme pathway onto TMV brush surfaces for synthesis of bacterial autoinducer-2.

Bioelectronic microdevices, with spatially arranged biosynthetic machinery, can be programmed to convert raw materials to high-value products in a controlled manner. Generic methods for biofunctionalization that enable precise control over biocomponent assembly at the nano and meso scales are necessary to diversify the range and capabilities of these systems. Here, we used tobacco mosaic virus (TMV) derived virus like particles (VLPs) as 3D interfacial scaffolds for the assembly of biosynthetic enzymes onto gold electrodes. The TMV capsids are aligned in a vertical brush configuration by cysteine modifications to the capsid protein and by taking advantage of the well-known gold/cysteine affinity. This alignment enables high surface density and biosynthetic enzyme-enzyme proximity. Enzymes are covalently tethered to the capsid protein of TMV by the N- and C-terminal addition of lysine-rich assembly domains which react with surface exposed glutamine residues on the capsid surfaces; the lysine/glutamine linkages are mediated by a microbial transglutaminase (mTG). We demonstrate flexible mTG-mediated assembly of a three-enzyme biosynthetic pathway that converts S-adenosylmethionine (SAM) to autoinducer-2 (AI-2), a bacterial signal molecule that mediates quorum sensing behavior. We propose that our VLP and mTG based fabrication approach will help in the modular assembly of biological components onto microelectronic devices and that these will find utility in many applications including sensing and lab on chip devices.

The use of multifunctional yeast-lactobacilli starter cultures improves fermentation performance of Spanish-style green table olives.

In this work, Lactobacillus pentosus LPG1, Lactobacillus pentosus Lp13, Lactobacillus plantarum Lpl15, and Wickerhanomyces anomalous Y12, all of them previously isolated from fermented table olive biofilms, were used (alone or in combination) as multifunctional starters for Manzanilla Spanish-style green table olive fermentations. Their performances were evaluated through the changes in the key physico-chemical and microbiological parameters, correlation between AI-2 production and biofilm formation, inoculum imposition, metataxonomic analysis and sensory characteristics of the finished products. Inoculation only with lactic acid bacteria (LAB) strains led to higher titratable acidities and lower pH values than the spontaneous fermentation (non-inoculated control), mainly during the first steps of processing. However, the sequential inoculation of the yeast and then the combination of the 3 LAB strains showed the most favourable evolution. LPG1 strain and, particularly Lp13, were excellent biofilms former and showed the major imposition on the fruit epidermis, as corroborated by rep-PCR analysis. Production of AI-2 was lower in the treatment inoculated exclusively with yeast Y12 but had the highest presence in the sequential yeast-LAB inoculum, with its maximum concentration and maximum LAB population on fruits (19th days) strongly related. Metataxonomic analysis of the biofilms at the end of the fermentation revealed, in addition to Lactobacillus, high proportions of sequences from genera Marinilactobacillus, Alkalibacterium, Halolactobacillus, and low levels of Halomonas and Aerococcus. Compositional data analysis of the omics data revealed that Lpl15 was scarcely efficient for controlling the spontaneous microbiota since its treatment presented the highest proportions of Aerococcus genus. Finally, the sensory analysis showed similar characteristics for the treatment inoculated with LPG1 and the spontaneous process, with olives inoculated with the yeast (alone or in combination with Lactobacillus strains) showing attractive scores. Then, inoculation of Spanish-style table olive fermentations with a sequential yeast and LAB combination could be an advisable practice.

Increasing L-homoserine production in Escherichia coli by engineering the central metabolic pathways.

l-homoserine is an important functional amino acid. Based on the system metabolic engineering strategy, the key genes in the central metabolic pathway of Escherichia coli W3110 were engineered to construct the strain for l-homoserine production. To construct an engineered strain with high yield of l-homoserine, the work was carried out from the following aspects: (1) Disrupt the competitive and degradative pathways of l-homoserine, and the l-homoserine was initially accumulated with a titer of 0.2 g/L; (2) Exploring the effect of weakening TCA cycle, modification of the glyoxylate branch, and reduction of the pyruvate synthesis for l-homoserine synthesis. The concentration of l-homoserine in the final recombinant strain LJL12 reached a titer of 3.2 g/L at shake flask and 35.8 g/L in fed-batch fermentation, showing a high l-homoserine production capacity (0.82 g/L/h). The study provides a well research foundation for l-homoserine production with the capacity for industrial application.

The Marine Bacterium Shewanella woodyi Produces C8-HSL to Regulate Bioluminescence.

Quorum sensing (QS), a cell-to-cell communication system involved in the synchronization of bacterial behavior in a cell-density-dependent manner has been shown to control phenotypes such as luminescence, virulence, and biofilm formation. The marine strain, Shewanella woodyi MS32 has been identified as a luminous bacterium. Very little information is known on this bacterium, in particular if its luminescence and biofilm formation are controlled by QS. In this study, we have demonstrated that S. woodyi MS32 emits luminescence in planktonic and sessile conditions. The putative QS regulatory genes homologous to luxI and luxR identified in the S. woodyi MS32 genome, named swoI and swoR, are divergently transcribed and are not genetically linked to the lux operon in contrast with its closest parent Shewanella hanedai and with Aliivibrio fischeri. Interestingly, the phylogenetic analysis based on the SwoI and SwoR sequences shows that a separate horizontal gene transfer (HGT) occurred for the regulatory genes and for the lux operon. Functional analyses demonstrate that the swoI and swoR mutants were non-luminescent. Expression of lux genes was impaired in the QS regulatory mutants. N-octanoyl-L-homoserine lactone (C8-HSL) identified using liquid chromatography mass spectrometry in the wild-type strain (but not in ΔswoI) can induce S. woodyi luminescence. No significant difference has been detected between the wild-type and mutants on adhesion and biofilm formation in the conditions tested. Therefore, we have demonstrated that the luxCDABEG genes of S. woodyi MS32 are involved in luminescence emission and that the swoR/swoI genes, originated from a separate HGT, regulate luminescence through C8-HSL production.

Suppressive effect of Lactobacillus fermentum Lim2 on Clostridioides difficile 027 toxin production.

Clostridioides difficile is a spore-forming, Gram-positive, anaerobic pathogen that caused gastrointestinal illness. During dysbiosis, overgrowth of C. difficile resulting in higher levels of toxin production. Since Lactobacillus has been commonly used to alleviate gastrointestinal discomfort, this study aimed to investigate the effects of Lactobacillus isolated from kimchi on the quorum-sensing and virulence factors of C. difficile 027. Among the isolated Lactobacillus strains, the acid and bile tolerant L. fermentum Lim2 was only able to reduce C. difficile 027 growth by one log10 CFU per ml. In keeping with this finding, C. difficile 027 growth was unaffected by either untreated or heat-inactivated cell extracts from L. fermentum Lim2. Both untreated and heat-inactivated cell extracts did, however, significantly reduce the autoinducer-2 (AI-2) activity of C. difficile 027, with the most prominent suppression effect (654-fold) being found from 100 mg ml-1 of heat-inactivated cell extract. A gene expression analysis indicated that in the presence of 100 mg ml-1 heat-inactivated cell extract, the quorum-sensing (luxS) and the virulence factors (tcdA, tcdB and tcdE) were significantly suppressed, whereas the negative regulator gene (tcdC) was significantly up-regulated. Taken together, the significant anti-pathogenic effect from L. fermentum Lim2 could potentially be used to treat C. difficile-infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridioides difficile is a Gram-positive pathogenic bacteria that caused gastrointestinal illness via toxic production. The emergence of highly virulence and foodborne C. difficile strains has further increased the incident and severity of C. difficile-infections (CDIs). Numerous studies have reported the immunomodulatory activity of Lactobacillus, a member of healthy gut microbiota, to maintain gastrointestinal health. Here, we successfully isolated L. fermentum Lim2 from kimchi, and identified a promising anti-pathogenic effect against C. difficile 027, from the heat-inactivated L. fermentum cell extract via suppression on the C. difficile 027 quorum-sensing system and toxin production, which could potentially be used to treat and prevent CDIs.

In-Silico Prediction and Modeling of the Quorum Sensing LuxS Protein and Inhibition of AI-2 Biosynthesis in Aeromonas hydrophila.

luxS is conserved in several bacterial species, including A. hydrophila, which causes infections in prawn, fish, and shrimp, and is consequently a great risk to the aquaculture industry and public health. luxS plays a critical role in the biosynthesis of the autoinducer-2 (AI-2), which performs wide-ranging functions in bacterial communication, and especially in quorum sensing (QS). The prediction of a 3D structure of the QS-associated LuxS protein is thus essential to better understand and control A. hydrophila pathogenecity. Here, we predicted the structure of A. hydrophila LuxS and characterized it structurally and functionally with in silico methods. The predicted structure of LuxS provides a framework to develop more complete structural and functional insights and will aid the mitigation of A. hydrophila infection, and the development of novel drugs to control infections. In addition to modeling, the suitable inhibitor was identified by high through put screening (HTS) against drug like subset of ZINC database and inhibitor ((-)-Dimethyl 2,3-O-isopropylidene-l-tartrate) molecule was selected based on the best drug score. Molecular docking studies were performed to find out the best binding affinity between LuxS homologous or predicted model of LuxS protein for the ligand selection. Remarkably, this inhibitor molecule establishes agreeable interfaces with amino acid residues LYS 23, VAL 35, ILE76, and SER 90, which are found to play an essential role in inhibition mechanism. These predictions were suggesting that the proposed inhibitor molecule may be considered as drug candidates against AI-2 biosynthesis of A. hydrophila. Therefore, (-)-Dimethyl 2,3-O-isopropylidene-l-tartrate inhibitor molecule was studied to confirm its potency of AI-2 biosynthesis inhibition. The results shows that the inhibitor molecule had a better efficacy in AI-2 inhibition at 40 µM concentration, which was further validated using Western blotting at a protein expression level. The AI-2 bioluminescence assay showed that the decreased amount of AI-2 biosynthesis and downregulation of LuxS protein play an important role in the AI-2 inhibition. Lastly, these experiments were conducted with the supplementation of antibiotics via cocktail therapy of AI-2 inhibitor plus OXY antibiotics, in order to determine the possibility of novel cocktail drug treatments of A. hydrophila infection.

Production, detection and application perspectives of quorum sensing autoinducer-2 in bacteria.

Autoinducer-2 (AI-2) is a major signal molecule in bacterial quorum sensing (QS) besides N-acyl homoserine lactones (AHLs or AI-1). AI-2 mediated QS pathways have been proved to regulate gene expression and physiological behaviors of bacteria in either intraspecies or interspecies communication. Recent reviews have mainly summarized AI-2 structures, AI-2 mediated QS pathways and the role of AI-2 in gene regulation, etc. In this article, we present a comprehensive review of AI-2 production, detection and applications. Firstly, intracellular AI-2 synthetic routes were outlined and environmental influences on AI-2 production were focused. Furthermore, recent advances in AI-2 detection and quantification were elucidated from an overall perspective. An in-depth understanding of mechanisms and features of various detection methods may facilitate development of new technologies aimed at signal molecule detection. Finally, utilization of AI-2 mediated QS in health improvement, water treatment and drug production indicate promising and extensive application perspectives of QS strategies.

Streptococcus gordonii LuxS/autoinducer-2 quorum-sensing system modulates the dual-species biofilm formation with Streptococcus mutans.

Dental plaques are mixed-species biofilms that are related to the development of dental caries. Streptococcus mutans (S. mutans) is an important cariogenic bacterium that forms mixed-species biofilms with Streptococcus gordonii (S. gordonii), an early colonizer of the tooth surface. The LuxS/autoinducer-2(AI-2) quorum sensing system is involved in the regulation of mixed-species biofilms, and AI-2 is proposed as a universal signal for the interaction between bacterial species. In this work, a S. gordonii luxS deficient strain was constructed to investigate the effect of the S. gordonii luxS gene on dual-species biofilm formed by S. mutans and S. gordonii. In addition, AI-2 was synthesized in vitro by incubating recombinant LuxS and Pfs enzymes of S. gordonii together. The effect of AI-2 on S. mutans single-species biofilm formation and cariogenic virulence gene expression were also assessed. The results showed that luxS disruption in S. gordonii altered dual-species biofilm formation, architecture, and composition, as well as the susceptibility to chlorhexidine. And the in vitro synthesized AI-2 had a concentration-dependent effect on S. mutans biofilm formation and virulence gene expression. These findings indicate that LuxS/AI-2 quorum-sensing system of S. gordonii plays a role in regulating the dual-species biofilm formation with S. mutans.

Pichia pastoris Mut(S) strains are prone to misincorporation of O-methyl-L-homoserine at methionine residues when methanol is used as the sole carbon source.

BACKGROUND: Over the last few decades the methylotrophic yeast Pichia pastoris has become a popular host for a wide range of products such as vaccines and therapeutic proteins. Several P. pastoris engineered strains and mutants have been developed to improve the performance of the expression system. Yield and quality of a recombinant product are important parameters to monitor during the host selection and development process but little information is published regarding quality differences of a product produced by different P. pastoris strains. RESULTS: We compared titer and quality of several Nanobodies(®) produced in wild type and Mut(S) strains. Titer in fed-batch fermentation was comparable between all strains for each Nanobody but a significant difference in quality was observed. Nanobodies expressed in Mut(S) strains contained a product variant with a Δ-16 Da mass difference that was not observed in wild type strains. This variant showed substitution of methionine residues due to misincorporation of O-methyl-L-homoserine, also called methoxine. Methoxine is likely synthesized by the enzymatic action of O-acetyl homoserine sulfhydrylase and we confirmed that Nanobodies produced in the corresponding knock-out strain contained no methoxine variants. We could show the incorporation of methoxine during biosynthesis by its addition to the culture medium. CONCLUSION: We showed that misincorporation of methoxine occurs particularly in P. pastoris Mut(S) strains. This reduction in product quality could outweigh the advantages of using Mut strains, such as lower oxygen and methanol demand, heat formation and in some cases improved expression. Methoxine incorporation in recombinant proteins is likely to occur when an excess of methanol is present during fermentation but can be avoided when the methanol feed rate protocol is carefully designed.

PhoB activation in non-limiting phosphate condition by the maintenance of high polyphosphate levels in the stationary phase inhibits biofilm formation in Escherichia coli.

Polyphosphate (polyP) degradation in Escherichia coli stationary phase triggers biofilm formation via the LuxS quorum sensing system. In media containing excess of phosphate (Pi), high polyP levels are maintained in the stationary phase with the consequent inhibition of biofilm formation. The transcriptional-response regulator PhoB, which is activated under Pi limitation, is involved in the inhibition of biofilm formation in several bacterial species. In the current study, we report, for the first time, we believe that E. coli PhoB can be activated in non-limiting Pi conditions, leading to inhibition of biofilm formation. In fact, PhoB was activated when high polyP levels were maintained in the stationary phase, whereas it remained inactive when the polymer was degraded or absent. PhoB activation was mediated by acetyl phosphate with the consequent repression of biofilm formation owing to the downregulation of c-di-GMP synthesis and the inhibition of autoinducer-2 production. These results allowed us to propose a model showing that PhoB is a component in the signal cascade regulating biofilm formation triggered by fluctuations of polyP levels in E. coli cells during stationary phase.

Chania multitudinisentens gen. nov., sp. nov., an N-acyl-homoserine-lactone-producing bacterium in the family Enterobacteriaceae isolated from landfill site soil.

Phylogenetic and taxonomic characterization was performed for bacterium RB-25T, which was isolated from a soil sample collected in a former municipal landfill site in Puchong, Malaysia. Growth occurred at 20-37 °C at pH 5-8 but not in the presence of 9 % (w/v) NaCl or higher. The principal fatty acids were C16:0, C18:1ω7c and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH). Ubiquinone-8 was the only isoprenoid quinone detected. Polar lipid analysis revealed the presence of phospholipid, phosphoaminolipid, phosphatidylethanolamine, phosphatidylglycerol and one unidentified aminolipid. DNA G+C content was 50.9 mol% phylogenetic analysis based on 16S rRNA gene sequence showed that strain RB-25T formed a distinct lineage within the family Enterobacteriaceae of the class Gammaproteobacteria. It exhibited a low level of 16S rRNA gene sequence similarity with its phylogenetic neighbours Pantoea rwandensis LMG 26275T (96.6 %), Rahnella aquatilis CIP 78.65T (96.5 %), Pectobacterium betavasculorum ATCC 43762T (96.4 %), Pantoea rodasii LMG 26273T (96.3 %), Gibbsiella dentisursi NUM 1720T (96.3 %) and Serratia glossinae C1T (96.2 %). Multilocus sequence analyses based on fusA, pyrG, rplB, rpoB and sucA sequences showed a clear distinction of strain RB-25T from the most closely related genera. Isolate RB-25T could also be distinguished from members of these genera by a combination of the DNA G+C content, respiratory quinone system, fatty acid profile, polar lipid composition and other phenotypic features. Strain RB-25T represents a novel species of a new genus, for which the name Chaniamultitudinisentens gen. nov., sp. nov. is proposed. The type strain is RB-25T (=DSM 28811T=LMG 28304T).

The response of Serratia marcescens JG to environmental changes by quorum sensing system.

Many bacterial cells are known to regulate their cooperative behaviors and physiological processes through a molecular mechanism called quorum sensing. Quorum sensing in Serratia marcescens JG is mediated by the synthesis of autoinducer 2 (AI-2) which is a furanosyl borate diester. In this study, the response of quorum sensing in S. marcescens JG to environment changes such as the initial pH, carbon sources and boracic acid was investigated by a bioreporter and real-time PCR analysis. The results show that glucose can affect AI-2 synthesis to the greatest extent, and 2.0 % glucose can stimulate S. marcescens JG to produce more AI-2, with a 3.5-fold increase in activity compared with control culture. Furthermore, the response of quorum sensing to changes in glucose concentration was performed by changing the amount of luxS RNA transcripts. A maximum of luxS transcription appeared during the exponential growth phase when the glucose concentration was 20.0 g/L. AI-2 production was also slightly impacted by the low initial pH. It is significant for us that the addition of boracic acid at microdosage (0.1-0.2 g/L) can also induce AI-2 synthesis, which probably demonstrated the feasible fact that the 4,5-dihydroxy-2, 3-pentanedione cyclizes by the addition of borate and the loss of water, is hydrated and is converted to the final AI-2 in S. marcescens JG.

The role of autoinducer-2 in aerobic granulation using alternating feed loadings strategy.

Quorum sensing (QS) plays an important role in aerobic granulation while how QS system regulates the formation of aerobic granules needs further discussion. This study cultivated activated sludge in two identical sequencing batch reactors (R1 and R2) at different influent organic loading rate (OLR) strategies: R1 was operated using constant OLR (around 8.0kg/m(3)d), while R2 was operated at alternating OLR (4.0-17.0kg/m(3)d). Microbial aggregates appeared in R2 on day 19, while the morphology of sludge in R1 changed little compared with the initial sludge. The concentration of autoinducer-2 (AI-2) in R2 showed an ascending trend, along with the increase of cell adhesiveness. The total extracellular polymeric substances (EPS) amount and large molecular weight EPS of R2 rose steadily, which was different from R1. Some bacteria able to self-aggregate and promote EPS secretion were exclusive in R2. A mechanism about aerobic granulation at alternating OLR was proposed.

Quorum sensing signal production and microbial interactions in a polymicrobial disease of corals and the coral surface mucopolysaccharide layer.

Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome.

Pantoea sp. isolated from tropical fresh water exhibiting N-acyl homoserine lactone production.

N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry.

O-Succinyl-L-homoserine-based C4-chemical production: succinic acid, homoserine lactone, γ-butyrolactone, γ-butyrolactone derivatives, and 1,4-butanediol.

There has been a significant global interest to produce bulk chemicals from renewable resources using engineered microorganisms. Large research programs have been launched by academia and industry towards this goal. Particularly, C4 chemicals such as succinic acid (SA) and 1,4-butanediol have been leading the path towards the commercialization of biobased technology with the effort of replacing chemical production. Here we present O-Succinyl-L-homoserine (SH) as a new, potentially important platform biochemical and demonstrate its central role as an intermediate in the production of SA, homoserine lactone (HSL), γ-butyrolactone (GBL) and its derivatives, and 1,4-butanediol (BDO). This technology encompasses (1) the genetic manipulation of Escherichia coli to produce SH with high productivity, (2) hydrolysis into SA and homoserine (HS) or homoserine lactone hydrochloride, and (3) chemical conversion of either HS or homoserine lactone HCL (HSL·HCl) into drop-in chemicals in polymer industry. This production strategy with environmental benefits is discussed in the perspective of targeting of fermented product and a process direction compared to petroleum-based chemical conversion, which may reduce the overall manufacturing cost.