Aeromonas salmonicida AI-1 and AI-2 quorum sensing pathways are differentially regulated by rainbow trout mucins and during in vivo colonization.

Aeromonas salmonicida is an opportunistic pathogen with relevance for aquaculture. Fish epithelia are covered by a mucus layer, composed mainly by highly glycosylated mucins, which are the first point of contact between fish and pathogens. Quorum sensing (QS), a bacterial communication mechanism through secreted autoinducer signals that governs gene expression, influences bacterial growth and virulence. The main A. salmonicida autoinducers are mediated by the luxS and asaI genes, corresponding to inter- and intraspecies communication, respectively. The aim of this study was to determine the effect of the mucins that pathogens encounter during colonization of the gill and skin on A. salmonicida QS. We found that expression of A. salmonicida asaI, but not luxS, was increased after culture at 20 °C compared to 10 °C. Rainbow trout gill and skin mucins up-regulated asaI expression 2-fold but down-regulated luxS 10-fold. The downregulation of luxS was reflected by a reduction in autoinducer-2 secretion. Mucins isolated from skin had a stronger inhibitory effect than mucins isolated from gills on both luxS expression and A1-2 secretion, consistent with a higher relative abundance of N-Acetylneuraminic acid on skin mucins than on gill mucins. Reduction of AI-2 production by mucins or luxS-deletion lead to a reduced A. salmonicida auto-aggregation. Furthermore, after colonization of the gill, luxS was down regulated whereas asaI expression was upregulated. Both in vivo and in vitro, the expression of luxS and asaI were thus differentially regulated, frequently in an inverse manner. The strong AI-2 inhibiting effect of the skin mucins is likely part of the mucin-based defense against pathogens.

[Effect of Quorum Sensing Systems on Biofilm Formation and Virulence in Salmonella]. / Salmonella'da Çogunlugu Algilama Sistemlerinin Biyofilm Olusumu ve Virülans Üzerine Etkisi.

In recent years, as the paradigm of communication between cells has been clarified, the ability of bacteria to change their gene expression patterns in response to various extracellular signals has attracted great interest. In particular, intracellular and intercellular communication between bacterial populations, called quorum sensing (QS), is essential for coordinating physiological and genetic activities. QS studies are critical, particularly in elucidating the regulatory mechanisms of infectious processes in food-borne pathogens. Elucidating the QS mechanisms in Salmonella is effective in silencing the virulence factors in the fight against this bacterium. The aims of this study were; to create luxS gene mutants that play a vital role in the QS activity of Salmonella and to determine the effect of this mutation on the expression of virulence genes in the bacteria and to determine the impact of synthetic N-hexanoyl-homoserine lactone (C6HSL) on biofilm formation and AI-2 signaling pathway of Salmonella wild strain and luxS gene mutants. luxS gene mutants were constructed by recombining the gene region with the chloramphenicol gene cassette based on homologous region recombination. In the luxS mutants obtained in this way, the expression of eight different virulence genes (hilA, invA, inv, glgC, fimF, fliF, lpfA, gyrA), which have essential roles in Salmonella pathogenicity, was determined by quantitative real-time reverse transcriptase polymerase chain reaction (rRT-qPCR) method and compared with natural strains. As a result of these studies, it was determined that the expression of each gene examined was significantly reduced in luxS mutant strains. The relative AI-2 activities of Salmonella strains were analyzed depending on time. It was determined that the highest activity occurred at the fourth hour and the AI-2 activities of luxS mutants were reduced compared to the wild strain. Finally, it was determined that C6HSL increased the biofilm activity of Salmonella Typhimurium DMC4, SL1344 wild strains, and mutants, mainly at the 72nd hour. In conclusion, our results proved that C6HSL stimulated QS communication in all strains and increased biofilm of Salmonella formation and autoinducer activity. This situation determines that Salmonella responds to external signals by using QS systems. In addition, this research contributed to provide additional information on interspecies communication mechanisms to develop strategies to prevent biofilm formation of this pathogen.

TusA influences Fe-S cluster assembly and iron homeostasis in E. coli by reducing the translation efficiency of Fur.

All sulfur transfer pathways have generally a l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of numerous sulfur-containing biomolecules in the cell. In Escherichia coli, the housekeeping l-cysteine desulfurase IscS has several interaction partners, which bind at different sites of the protein. So far, the interaction sites of IscU, Fdx, CyaY, and IscX involved in iron-sulfur (Fe-S) cluster assembly have been mapped, in addition to TusA, which is required for molybdenum cofactor biosynthesis and mnm5s2U34 tRNA modifications, and ThiI, which is involved in thiamine biosynthesis and s4U8 tRNA modifications. Previous studies predicted that the sulfur acceptor proteins bind to IscS one at a time. E. coli TusA has, however, been suggested to be involved in Fe-S cluster assembly, as fewer Fe-S clusters were detected in a ∆tusA mutant. The basis for this reduction in Fe-S cluster content is unknown. In this work, we investigated the role of TusA in iron-sulfur cluster assembly and iron homeostasis. We show that the absence of TusA reduces the translation of fur, thereby leading to pleiotropic cellular effects, which we dissect in detail in this study.IMPORTANCEIron-sulfur clusters are evolutionarily ancient prosthetic groups. The ferric uptake regulator plays a major role in controlling the expression of iron homeostasis genes in bacteria. We show that a ∆tusA mutant is impaired in the assembly of Fe-S clusters and accumulates iron. TusA, therefore, reduces fur mRNA translation leading to pleiotropic cellular effects.

Effects of the quorum sensing related luxS gene and lsr operon on Klebsiella michiganensis resisting copper stress.

Industrial wastewater is a major environmental concern due to its high copper content, which poses significant toxicity to microbial life. Autoinducer-2 (AI-2) can participate in the inter- and intra-species communication and regulate the physiological functions of different bacterial species by producing AI-2 signal molecules. However, there are few research reports on the luxS gene and lsr operon functions for AI-2 in bacteria with a certain tolerance to copper. This study delves into the potential of quorum sensing mechanisms, particularly the AI-2 system, for enhancing microbial resistance to copper toxicity in Klebsiella michiganensis (KM). We detail the critical roles of the luxS gene in AI-2 synthesis and the lsr operon in AI-2 uptake, demonstrating their collective impact on enhancing copper resistance. Our findings show that mutations in the lsr operon, alongside the knockout of the luxS gene in KM strain (KMΔluxSΔlsr), significantly impair the strain's motility (p < 0.0001) and biofilm formation (p < 0.01), underscoring the operon's role in AI-2 transport. These genetic insights are pivotal for developing bioremediation strategies aimed at mitigating copper pollution in wastewater. By elucidating the mechanisms through which KM modulates copper resistance, this study highlights the broader ecological significance of leveraging microbial quorum sensing pathways for sustainable wastewater management.

Identification and Characterization of Bacterial Alliinase: Resource and Substrate Stereospecificity.

Limited alliinase resources cause difficulties in the biosynthesis of thiosulfinates (e.g., allicin), restricting their applications in the agricultural and food industries. To effectively biosynthesize thiosulfinates, this study aimed to excavate bacterial alliinase resources and elucidate their catalytic properties. Two bacterial cystathionine ß-lyases (MetCs) possessing high alliinase activity (>60 U mg -1) toward L-(-)-alliin were identified from Allium sativum rhizosphere isolates. Metagenomic exploration revealed that cystathionine ß-lyase from Bacillus cereus (BcPatB) possessed high activity toward both L-(±)-alliin and L-(+)-alliin (208.6 and 225.1 U mg -1), respectively. Although these enzymes all preferred l-cysteine S-conjugate sulfoxides as substrates, BcPatB had a closer phylogenetic relationship with Allium alliinases and shared several similar features with A. sativum alliinase. Interestingly, the Trp30Ile31Ala32Asp33 Met34 motif in a cuspate loop of BcPatB, especially sites 31 and 32 at the top of the motif, was modeled to locate near the sulfoxide of L-(+)-alliin and is important for substrate stereospecificity. Moreover, the stereoselectivity and activity of mutants I31V and A32G were higher toward L-(+)-alliin than those of mutant I31L/D33E toward L-(-)-alliin. Using bacterial alliinases and chemically synthesized substrates, we obtained thiosulfinates with high antimicrobial and antinematode activities that could provide insights into the protection of crops and food.

Shared functions of Fe-S cluster assembly and Moco biosynthesis.

Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In the recent years it has become evident that the availability of Fe-S clusters play an important role for the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is an enzyme involved in the transfer of sulfur to various acceptor proteins with a main role in the assembly of Fe-S clusters. In this review, we dissect the dependence of the production of active molybdoenzymes in detail, starting from the regulation of gene expression and further explaining sulfur delivery and Fe-S cluster insertion into target enzymes. Further, Fe-S cluster assembly is also linked to iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, we explain that the expression of the genes is dependent on an active FNR protein. FNR is a very important transcription factor that represents the master-switch for the expression of target genes in response to anaerobiosis. Moco biosynthesis is further directly dependent on the presence of ArcA and also on an active Fur protein.

Baicalin Weakens the Virulence of Porcine Extraintestinal Pathogenic Escherichia coli by Inhibiting the LuxS/AI-2 Quorum-Sensing System.

Porcine extraintestinal pathogenic Escherichia coli (ExPEC) is a pathogenic bacterium that causes huge economic losses to the pig farming industry and considerably threatens human health. The quorum sensing (QS) system plays a crucial role in the survival and pathogenesis of pathogenic bacteria. Hence, it is a viable approach to prevent ExPEC infection by compromising the QS system, particularly the LuxS/AI-2 system. In this study, we investigated the effects of baicalin on the LuxS/AI-2 system of ExPEC. Baicalin at concentrations of 25, 50, and 100 µg/mL significantly diminished the survival ability of ExPEC in hostile environments and could inhibit the biofilm formation and autoagglutination ability in ExPEC. Moreover, baicalin dose-dependently decreased the production of AI-2 and down-regulated the expression level of luxS in PCN033. These results suggest that baicalin can weaken the virulence of PCN033 by inhibiting the LuxS/AI-2 system. After the gene luxS was deleted, AI-2 production in PCN033 was almost completely eliminated, similar to the effect of baicalin on the production of AI-2 in PCN033. This indicates that baicalin reduced the production of AI-2 by inhibiting the expression level of luxS in ExPEC. In addition, the animal experiment further showed the potential of baicalin as a LuxS/AI-2 system inhibitor to prevent ExPEC infection. This study highlights the potential of baicalin as a natural quorum-sensing inhibitor for therapeutic applications in preventing ExPEC infection by targeting the LuxS/AI-2 system.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

PLA inhibits TNF-α-induced PANoptosis of prostate cancer cells through metabolic reprogramming.

Previous studies have shown that phenyllactic acid (alpha-Hydroxyhydrocinnamic acid, 2-Hydroxy-3-phenylpropionic acid, PLA), a type of organic acid metabolite, has excellent diagnostic efficacy when used to differentiate between prostate cancer, benign prostatic hyperplasia, and prostatitis. This research aims to explore the molecular mechanism by which PLA influences the PANoptosis of prostate cancer (PCa) cell lines. First, we found that PLA was detected in all prostate cancer cell lines (PC-3, PC-3 M, DU145, LNCAP). Further experiments showed that the addition of PLA to prostate cancer cells could promote ATP generation, enhance cysteine desulfurase (NFS1) expression, and reduce tumor necrosis factor alpha (TNF-α) levels, thereby inhibiting apoptosis in prostate cancer cells. Notably, overexpression of NFS1 can inhibit the binding of TNF-α to serpin mRNA binding protein 1 (SERBP1), suggesting that NFS1 competes with TNF-α for binding to SERBP1. Knockdown of SERBP1 significantly reduced the level of small ubiquity-related modifier (SUMO) modification of TNF-α. This suggests that NFS1 reduces the SUMO modification of TNF-α by competing with SERBP1, thereby reducing the expression and stability of TNF-α and ultimately inhibiting apoptosis in prostate cancer cell lines. In conclusion, PLA inhibits TNF-α induced panapoptosis of prostate cancer cells through metabolic reprogramming, providing a new idea for targeted treatment of prostate cancer.

Impact of LuxS on virulence and pathogenicity in Klebsiella pneumoniae exhibiting varied mucoid phenotypes.

How the LuxS/AI-2 quorum sensing (QS) system influences the pathogenicity of K. pneumoniae is complicated by the heterogeneity of the bacterial mucoid phenotypes. This study aims to explore the LuxS-mediated regulation of the pathogenicity of K. pneumoniae with diverse mucoid phenotypes, including hypermucoid, regular-mucoid, and nonmucoid. The wild-type, luxS knockout, and complemented strains of three K. pneumoniae clinical isolates with distinct mucoid phenotypes were constructed. The results revealed the downregulation of virulence genes of regular-mucoid, and nonmucoid but not hypermucoid strains. The deletion of luxS reduced the pathogenicity of the regular-mucoid, and nonmucoid strains in mice; while in hypermucoid strain, luxS knockout reduced virulence in late growth but enhanced virulence in the early growth phase. Furthermore, the absence of luxS led the regular-mucoid and nonmucoid strains to be more sensitive to the host cell defense, and less biofilm-productive than the wild-type at both the low and high-density growth state. Nevertheless, luxS knockout enhanced the resistances to adhesion and phagocytosis by macrophage as well as serum-killing, of hypermucoid K. pneumoniae at its early low-density growth state, while it was opposite to those in its late high-density growth phase. Collectively, our results suggested that LuxS plays a crucial role in the pathogenicity of K. pneumoniae, and it is highly relevant to the mucoid phenotypes and growth phases of the strains. LuxS probably depresses the capsule in the early low-density phase and promotes the capsule, biofilm, and pathogenicity during the late high-density phase, but inhibits lipopolysaccharide throughout the growth phase, in K. pneumoniae.IMPORTANCECharacterizing the regulation of physiological functions by the LuxS/AI-2 quorum sensing (QS) system in Klebsiella pneumoniae strains will improve our understanding of this important pathogen. The genetic heterogeneity of K. pneumoniae isolates complicates our understanding of its pathogenicity, and the association of LuxS with bacterial pathogenicity has remained poorly addressed in K. pneumoniae. Our results demonstrated strain and growth phase-dependent variation in the contributions of LuxS to the virulence and pathogenicity of K. pneumoniae. Our findings provide new insights into the important contribution of the LuxS/AI-2 QS system to the networks that regulate the pathogenicity of K. pneumoniae. Our study will facilitate our understanding of the regulatory mechanisms of LuxS/AI-2 QS on the pathogenicity of K. pneumoniae under the background of their genetic heterogeneity and help develop new strategies for diminished bacterial virulence within the clinical K. pneumoniae population.

Characterization of AI-2/LuxS quorum sensing system in biofilm formation, pathogenesis of Streptococcus equi subsp. zooepidemicus.

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of both humans and animals. Quorum sensing (QS) plays an important role in the regulation of bacterial group behaviors. The aim of this study was to characterize the LuxS in SEZ and evaluate its impact on biofilm formation, pathogenesis and gene expression. The wild-type SEZ and its LuxS mutant (ΔluxS) were examined for growth, biofilm formation, virulence factors, and transcriptomic profiles. Our results showed that LuxS deficiency did not affect SEZ hemolytic activity, adhesion or capsule production. For biofilm assay demonstrated that mutation in the luxS gene significantly enhances biofilm formation, produced a denser biofilm and attached to a glass surface. RAW264.7 cell infection indicated that ΔluxS promoted macrophage apoptosis and pro-inflammatory responses. In mice infection, there was no significant difference in mortality between SEZ and ΔluxS. However, the bacterial load in the spleen of mice infected with ΔluxS was significantly higher than in those infected with SEZ. And the pathological analysis further indicated that spleen damage was more severe in the ΔluxS group. Moreover, transcriptomics analysis revealed significant alterations in carbon metabolism, RNA binding and stress response genes in ΔluxS. In summary, this study provides the first evidence of AI-2/LuxS QS system in SEZ and reveals its regulatory effects on biofilm formation, pathogenicity and gene expression.

Roles of luxS in regulation of probiotic characteristics and inhibition of pathogens in Lacticaseibacillus paracasei S-NB.

Lactic acid bacteria (LAB) have excellent tolerance to the gastrointestinal environment and high adhesion ability to intestinal epithelial cells, which could be closely related to the LuxS/AI-2 Quorum sensing (QS) system. Here, the crucial enzymes involved in the synthesis of AI-2 was analyzed in Lacticaseibacillus paracasei S-NB, and the luxS deletion mutant was constructed by homologous recombination based on the Cre-lox system. Afterwards, the effect of luxS gene on the probiotic activities in L. paracasei S-NB was investigated. Notably, the tolerance of simulated gastrointestinal digestion, AI-2 production, ability of auto-aggregation and biofilm formation significantly decreased (p < 0.05 for all) in the S-NB△luxS mutant. Compared to the wild-type S-NB, the degree of reduction in the relative transcriptional level of the biofilm -related genes in Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 was diminished when co-cultured with S-NB△luxS. Furthermore, the inhibitory effect of S-NB△luxS on the adhesion (competition, exclusion and displacement) of E. coli ATCC 25922 and S. aureus ATCC 25923 to Caco-2 cells markedly decreased. Therefore, comprehensive analysis of the role by luxS provides an insight into the LuxS/AI-2 QS system of L. paracasei S-NB in the regulation of strain characteristics and inhibition of pathogens.

Effects of luxS gene on growth characteristics, biofilm formation, and antimicrobial resistance of multi-antimicrobial-resistant Vibrio parahaemolyticus Vp2015094 isolated from shellfish.

AIMS: Vibrio parahaemolyticus is an important foodborne pathogen worldwide, which can cause gastroenteritis. This study aimed to investigate the effect of quorum sensing system LuxS/AI-2-related gene luxS on the biological characteristics and antimicrobial resistance of V. parahaemolyticus Vp2015094 from shellfish, which carried a multi-antimicrobial-resistant plasmid. METHODS AND RESULTS: The critical gene luxS related to the synthesis of AI-2 in V. parahaemolyticus Vp2015094 was knocked out by homologous recombination with suicide plasmid. The effect of luxS on the biological characteristics of V. parahaemolyticus was determined by comparing the growth, AI-2 activity, motility, biofilm formation ability, and antibiotic resistance between the wildtype strain and the luxS deletion mutant. Compared with wildtype strain, the production of AI-2, the motility and biofilm formation ability, antimicrobial resistance, and conjugation frequency of luxS deletion mutant strain were decreased. The transcriptome sequencing showed that the transcriptional levels of many genes related to motility, biofilm formation, antimicrobial resistance, and conjugation were significantly downregulated after luxS deletion. CONCLUSIONS: Quorum sensing system LuxS/AI-2-related gene luxS in V. parahaemolyticus Vp2015094 played an important role in growth characteristics, biofilm formation, antimicrobial resistance, and resistance genes' transfer.

Effects of metformin on Streptococcus suis LuxS/AI-2 quorum sensing system and biofilm formation.

Streptococcus suis (S. suis) regulates biofilm formation through LuxS/AI-2 quorum sensing system, increasing drug resistance and exacerbating infection. The anti-hyperglycaemic agent metformin has anti-bacterial and anti-biofilm activities. This study aimed to investigate the anti-biofilm and anti-quorum sensing activity of metformin in S. suis. We first determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of metformin on S. suis. The results indicated that metformin showed no obvious inhibitory or bactericidal effect. Crystal violet staining showed that metformin significantly inhibited the formation of S. suis biofilm at sub-MIC concentration, which was also confirmed by scanning electron microscopy. Then, we quantified the AI-2 signal molecules in S. suis, and the results showed that metformin had a significant inhibitory effect on the production of AI-2 signal in S. suis. Inhibition of enzyme activity and molecular docking experiments showed that metformin has a significant binding activity to LuxS protein. In addition, qRT-PCR results showed that metformin significantly down-regulated the expression of AI-2 synthesis-related genes luxS and pfs, and adhesion-related genes luxS, pfs, gapdh, sly, fbps, and ef. Western blotting also showed that metformin significantly reduced the expression of LuxS protein. Our study suggests that metformin seems to be a suitable candidate for the inhibition of S. suis LuxS/AI-2 QS system and prevention of biofilm formation, which provided a new idea for the prevention and control of S. suis.

Structural and Biochemical Characterization of Mycobacterium tuberculosis Zinc SufU-SufS Complex.

Iron-sulfur (Fe-S) clusters are inorganic prosthetic groups in proteins composed exclusively of iron and inorganic sulfide. These cofactors are required in a wide range of critical cellular pathways. Iron-sulfur clusters do not form spontaneously in vivo; several proteins are required to mobilize sulfur and iron, assemble and traffic-nascent clusters. Bacteria have developed several Fe-S assembly systems, such as the ISC, NIF, and SUF systems. Interestingly, in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), the SUF machinery is the primary Fe-S biogenesis system. This operon is essential for the viability of Mtb under normal growth conditions, and the genes it contains are known to be vulnerable, revealing the Mtb SUF system as an interesting target in the fight against tuberculosis. In the present study, two proteins of the Mtb SUF system were characterized for the first time: Rv1464(sufS) and Rv1465(sufU). The results presented reveal how these two proteins work together and thus provide insights into Fe-S biogenesis/metabolism by this pathogen. Combining biochemistry and structural approaches, we showed that Rv1464 is a type II cysteine-desulfurase enzyme and that Rv1465 is a zinc-dependent protein interacting with Rv1464. Endowed with a sulfurtransferase activity, Rv1465 significantly enhances the cysteine-desulfurase activity of Rv1464 by transferring the sulfur atom from persulfide on Rv1464 to its conserved Cys40 residue. The zinc ion is important for the sulfur transfer reaction between SufS and SufU, and His354 in SufS plays an essential role in this reaction. Finally, we showed that Mtb SufS-SufU is more resistant to oxidative stress than E. coli SufS-SufE and that the presence of zinc in SufU is likely responsible for this improved resistance. This study on Rv1464 and Rv1465 will help guide the design of future anti-tuberculosis agents.

Enhancing the AI-2/LuxS quorum sensing system in Lactiplantibacillus plantarum: Effect on the elimination of biofilms grown on seafoods.

The biofilm clustered with putrefying microorganisms and seafood pathogens could cover the surface of aquatic products that pose a risk to cross-contaminating food products or even human health. Fighting biofilms triggers synchronous communication associated with microbial consortia to regulate their developmental processes, and the enhancement of the quorum sensing system in Lactiplantibacillus plantarum can serve as an updated starting point for antibiofilm-forming strategies. Our results showed that the exogenous 25 mM L-cysteine induced a significant strengthening in the AI-2/LuxS system of Lactiplantibacillus plantarum SS-128 along with a stronger bacteriostatic ability, resulting in an effective inhibition of biofilms formed by the simplified microbial consortia constructed by Vibrio parahaemolyticus and Shewanella putrefaciens grown on shrimp and squid surfaces. The accumulation of AI-2 allowed the suppression of the expression of biofilm-related genes in V. parahaemolyticus under the premise of L. plantarum SS-128 treatment, contributing to the inhibition effect. In addition, strengthening the AI-2/LuxS system is also conducive to eliminating preexisting biofilms by L. plantarum SS-128. This study suggests that the enhancement of the AI-2/LuxS system of lactic acid bacteria enables the regulation of interspecific communication within biofilms to be a viable tool to efficiently reduce and eradicate potentially harmful biofilms from aquatic product sources, opening new horizons for combating biofilms.

The ß-latch structural element of the SufS cysteine desulfurase mediates active site accessibility and SufE transpersulfurase positioning.

Under oxidative stress and iron starvation conditions, Escherichia coli uses the Suf pathway to assemble iron-sulfur clusters. The Suf pathway mobilizes sulfur via SufS, a type II cysteine desulfurase. SufS is a pyridoxal-5'-phosphate-dependent enzyme that uses cysteine to generate alanine and an active-site persulfide (C364-S-S-). The SufS persulfide is protected from external oxidants/reductants and requires the transpersulfurase, SufE, to accept the persulfide to complete the SufS catalytic cycle. Recent reports on SufS identified a conserved "ß-latch" structural element that includes the α6 helix, a glycine-rich loop, a ß-hairpin, and a cis-proline residue. To identify a functional role for the ß-latch, we used site-directed mutagenesis to obtain the N99D and N99A SufS variants. N99 is a conserved residue that connects the α6 helix to the backbone of the glycine-rich loop via hydrogen bonds. Our x-ray crystal structures for N99A and N99D SufS show a distorted beta-hairpin and glycine-rich loop, respectively, along with changes in the dimer geometry. The structural disruption of the N99 variants allowed the external reductant TCEP to react with the active-site C364-persulfide intermediate to complete the SufS catalytic cycle in the absence of SufE. The substitutions also appear to disrupt formation of a high-affinity, close approach SufS-SufE complex as measured with fluorescence polarization. Collectively, these findings demonstrate that the ß-latch does not affect the chemistry of persulfide formation but does protect it from undesired reductants. The data also indicate the ß-latch plays an unexpected role in forming a close approach SufS-SufE complex to promote persulfide transfer.

A new dimethylsulfoniopropionate lyase of the cupin superfamily in marine bacteria.

Dimethylsulfoniopropionate (DMSP) is a marine organosulfur compound with important roles in stress protection, marine biogeochemical cycling, chemical signalling and atmospheric chemistry. Diverse marine microorganisms catabolize DMSP via DMSP lyases to generate the climate-cooling gas and info-chemical dimethyl sulphide. Abundant marine heterotrophs of the Roseobacter group (MRG) are well known for their ability to catabolize DMSP via diverse DMSP lyases. Here, a new DMSP lyase DddU within the MRG strain Amylibacter cionae H-12 and other related bacteria was identified. DddU is a cupin superfamily DMSP lyase like DddL, DddQ, DddW, DddK and DddY, but shares <15% amino acid sequence identity with these enzymes. Moreover, DddU proteins forms a distinct clade from these other cupin-containing DMSP lyases. Structural prediction and mutational analyses suggested that a conserved tyrosine residue is the key catalytic amino acid residue in DddU. Bioinformatic analysis indicated that the dddU gene, mainly from Alphaproteobacteria, is widely distributed in the Atlantic, Pacific, Indian and polar oceans. For reference, dddU is less abundant than dddP, dddQ and dddK, but much more frequent than dddW, dddY and dddL in marine environments. This study broadens our knowledge on the diversity of DMSP lyases, and enhances our understanding of marine DMSP biotransformation.

Structural Basis of the Inhibition of L-Methionine γ-Lyase from Fusobacterium nucleatum.

Fusobacterium nucleatum is a lesion-associated obligate anaerobic pathogen of destructive periodontal disease; it is also implicated in the progression and severity of colorectal cancer. Four genes (FN0625, FN1055, FN1220, and FN1419) of F. nucleatum are involved in producing hydrogen sulfide (H2S), which plays an essential role against oxidative stress. The molecular functions of Fn1419 are known, but their mechanisms remain unclear. We determined the crystal structure of Fn1419 at 2.5 Å, showing the unique conformation of the PLP-binding site when compared with L-methionine γ-lyase (MGL) proteins. Inhibitor screening for Fn1419 with L-cysteine showed that two natural compounds, gallic acid and dihydromyricetin, selectively inhibit the H2S production of Fn1419. The chemicals of gallic acid, dihydromyricetin, and its analogs containing trihydroxybenzene, were potentially responsible for the enzyme-inhibiting activity on Fn1419. Molecular docking and mutational analyses suggested that Gly112, Pro159, Val337, and Arg373 are involved in gallic acid binding and positioned close to the substrate and pyridoxal-5'-phosphate-binding site. Gallic acid has little effect on the other H2S-producing enzymes (Fn1220 and Fn1055). Overall, we proposed a molecular mechanism underlying the action of Fn1419 from F. nucleatum and found a new lead compound for inhibitor development.

Metabolism of Cysteine Conjugates and Production of Flavor Sulfur Compounds by a Carbon-Sulfur Lyase from the Oral Anaerobe Fusobacterium nucleatum.

Flavor perception is a key factor in the acceptance or rejection of food. Aroma precursors such as cysteine conjugates are present in various plant-based foods and are metabolized into odorant thiols in the oral cavity. To date, the involved enzymes are unknown, despite previous studies pointing out the likely involvement of carbon-sulfur lyases (C-S lyases) from the oral microbiota. In this study, we show that saliva metabolizes allyl-cysteine into odorant thiol metabolites, with evidence suggesting that microbial pyridoxal phosphate-dependent C-S lyases are involved in the enzymatic process. A phylogenetic analysis of PatB C-S lyase sequences in four oral subspecies of Fusobacterium nucleatum was carried out and led to the identification of several putative targets. FnaPatB1 from F. nucleatum subspecies animalis, a putative C-S lyase, was characterized and showed high activity with a range of cysteine conjugates. Enzymatic and X-ray crystallographic data showed that FnaPatB1 metabolizes cysteine derivatives within a unique active site environment that enables the formation of flavor sulfur compounds. Using an enzymatic screen with a library of pure compounds, we identified several inhibitors able to reduce the C-S lyase activity of FnaPatB1 in vitro, which paves the way for controlling the release of odorant sulfur compounds from their cysteine precursors in the oral cavity.