RESUMEN
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.
Asunto(s)
Acetatos , Escherichia coli , Homoserina , Ingeniería Metabólica , Treonina , Escherichia coli/genética , Escherichia coli/metabolismo , Treonina/biosíntesis , Treonina/metabolismo , Treonina/genética , Acetatos/metabolismo , Homoserina/metabolismo , Homoserina/análogos & derivados , Homoserina/genética , Homoserina/biosíntesis , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
O-acetyl-homoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis, which catalyzes the conversion of o-acetyl-homoserine (OAH) to homocysteine. In our previous study, we found that OAHS of Streptococcus suis serotype 2 (SS2) can interact with the porcine blood-brain barrier (BBB) model, but whether OAHS regulates the penetration of BBB during SS2 infection is still unclear. To explore the role of OAHS in SS2 infection, OAHS-deficient SS2 mutant strain (SC19-ΔOAHS) and gene complemental strain (SC19-cΔOAHS) were constructed. Compared to the parent strain, with the loss of oahs, the chain length of SC19-ΔOAHS was shortened, the virulence was significantly reduced, the survival rate of mice infected with SC19-ΔOAHS was obviously increased accompanied by the relieved clinical symptoms. And the survival ability of SC19-ΔOAHS in whole blood was also remarkably decreased. Interestingly, the adhesion of SC19-ΔOAHS to endothelial cells was markedly increased, but the deficiency of OAHS significantly inhibited the strain penetrating BBB both in vivo and in vitro. Most of these phenomena can be reversed by the complemental strain (SC19-cΔOAHS). Further study showed that the deficiency of OAHS severely reduced SC19-induced endothelial cell apoptosis, tight junctions (TJs) protein impairment and the expression of SS2 virulence factor Enolase (Eno), involved in the destruction of BBB. Additionally, SC19-ΔOAHS immunized mice were able to resist SC19 or JZLQ022 infection. In conclusion, we confirmed that OAHS promoted the pathogenicity by enhancing host's BBB permeability and immune escape, and SC19- ΔOAHS is a potential live vaccine.
Asunto(s)
Infecciones Estreptocócicas , Streptococcus suis , Enfermedades de los Porcinos , Animales , Ratones , Células Endoteliales , Homoserina/genética , Serogrupo , Infecciones Estreptocócicas/veterinaria , Porcinos , Enfermedades de los Porcinos/metabolismo , VirulenciaRESUMEN
L-Homoserine is an important amino acid as a precursor in synthesizing many valuable products. However, the low productivity caused by slow L-homoserine production during active cell growth in fermentation hinders its potential applications. In this study, strategies of engineering the synthetic pathway combined with regulating cell division were employed in an L-homoserine-producing Escherichia coli strain for efficiently biomanufacturing L-homoserine. First, the flux-control genes in the L-homoserine degradation pathway were omitted to redistribute carbon flux. To drive more carbon flux into L-homoserine production, the phosphoenolpyruvate-pyruvate-oxaloacetate loop was redrawn. Subsequently, the cell division was engineered by using the self-regulated promoters to coordinate cell growth and L-homoserine production. The ultimate strain HOM23 produced 101.31 g/L L-homoserine with a productivity of 1.91 g/L/h, which presented the highest L-homoserine titer and productivity to date from plasmid-free strains. The strategies used in this study could be applied to constructing cell factories for producing other L-aspartate derivatives.
Asunto(s)
Escherichia coli , Homoserina , Escherichia coli/genética , Escherichia coli/metabolismo , Homoserina/genética , Homoserina/metabolismo , Ingeniería Metabólica , Fermentación , División CelularRESUMEN
L-Homoserine is a valuable amino acid as a platform chemical in the synthesis of various important compounds. Development of microbial strains for high-level L-homoserine production is an attractive research direction in recent years. Herein, we converted a wild-type Escherichia coli to a non-auxotrophic and plasmid-free hyperproducer of L-homoserine using systematically metabolic engineer strategies. First, an initial strain was obtained through regulating L-homoserine degradation pathway and enhancing synthetic flow. To facilitate L-homoserine production, flux-control genes were tuned by optimizing the copy numbers in chromosome, and transport system was modified to promote L-homoserine efflux. Subsequently, a strategy of cofactors synergistic utilization was proposed and successfully applied to achieve L-homoserine hyperproduction. The final engineered strain could efficiently produce 85.29 g/L L-homoserine, which was the highest production level ever reported from a plasmid-free, antibiotic-free, inducer-free and nonauxotrophic strain. These strategies used here can be considered for developing microbial cell factory of other L-aspartate derivatives.
Asunto(s)
Proteínas de Escherichia coli , Ingeniería Metabólica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Homoserina/genética , Homoserina/metabolismo , Plásmidos/genéticaRESUMEN
Prevalence of toxigenic Vibrio cholerae O1 in aquatic reservoirs in Bangladesh apparently increases coinciding with the occurrence of seasonal cholera epidemics. In between epidemics, these bacteria persist in water mostly as dormant cells, known as viable but non-culturable cells (VBNC), or conditionally viable environmental cells (CVEC), that fail to grow in routine culture. CVEC resuscitate to active cells when enriched in culture medium supplemented with quorum sensing autoinducers CAI-1 or AI-2 which are signal molecules that regulate gene expression dependent on cell density. V. cholerae O1 mutant strains with inactivated cqsS gene encoding the CAI-1 receptor has been shown to overproduce AI-2 that enhance CVEC resuscitation in water samples. Since V. cholerae non-O1 non-O139 (non-cholera-vibrios) are abundant in aquatic ecosystems, we identified and characterized naturally occurring variant strains of V. cholerae non-O1 non-O139 which overproduce AI-2, and monitored their co-occurrence with V. cholerae O1 in water samples. The nucleotide sequence and predicted protein products of the cqsS gene carried by AI-2 overproducing variant strains showed divergence from that of typical V. cholerae O1 or non-O1 strains, and their culture supernatants enhanced resuscitation of CVEC in water samples. Furthermore, prevalence of V. cholerae O1 in the aquatic environment was found to coincide with an increase in AI-2 overproducing non-O1 non-O139 strains. These results suggest a possible role of non-cholera vibrios in the environmental biology of the cholera pathogen, in which non-O1 non-O139 variant strains overproducing AI-2 presumably contribute in resuscitation of the latent pathogen, leading to seasonal cholera epidemics. Importance. Toxigenic Vibrio cholerae which causes seasonal epidemics of cholera persists in aquatic reservoirs in endemic areas. The bacteria mostly exist in a dormant state during inter-epidemic periods, but periodically resuscitate to the active form. The resuscitation is enhanced by signal molecules called autoinducers (AIs). Toxigenic V. cholerae can be recovered from water samples that normally test negative for the organism in conventional culture, by supplementing the culture medium with exogenous AIs. V. cholerae belonging to the non-O1 non-O139 serogroups which do not cause cholera are also abundant in natural waters, and they are capable of producing AIs. In this study we characterized V. cholerae non-O1 non-O139 variant strains which overproduce an autoinducer called AI-2, and found that the abundance of the cholera pathogen in aquatic reservoirs correlates with an increase in the AI-2 overproducing strains. Our results suggest a probable role of these variant strains in the environmental biology and epidemiology of toxigenic V. cholerae, and may lead to novel means for surveillance, prevention and control of cholera.
Asunto(s)
Microbiología Ambiental , Variación Genética , Homoserina/análogos & derivados , Vibrio cholerae O1/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Bangladesh , Genoma Bacteriano , Homoserina/genética , Lactonas , Luminiscencia , Mutación/genética , Prevalencia , Microbiología del AguaRESUMEN
Escherichia coli can survive improper disinfection processes, which is a potential source of contamination of food products. Benzalkonium chloride (BC) is a common disinfectant widely used in food industry. Bacterial quorum sensing (QS) plays a major role in food spoilage, biofilm formation and food-related pathogenesis. Understanding QS can help to control the growth of undesirable food-related bacteria. The LuxS/AI-2 QS system of E. coli has been confirmed to regulate many important phenotypes including biofilm formation and motility. In the current study, we aimed to investigate the effect of sublethal concentrations of BC on the LuxS/AI-2 system of E. coli isolates from retail meat samples, as well as bacterial biofilm formation and motility. Our results showed that sublethal concentrations of BC promoted AI-2 production in four test E. coli isolates. The results from microplate assay and confocal laser scanning microscopy (CLSM) analysis indicated that sublethal concentrations of BC enhanced biofilm formation of E. coli. When treated with sublethal concentrations of BC, exopolysaccharides (EPS) production during biofilm development increased significantly and swimming motility of tested isolates was also promoted. The expression levels of luxS, biofilm-associated genes and flagellar motility genes were increased by BC at sublethal concentrations. Our findings underline the importance of proper use of the disinfectant BC in food processing environments to control food contamination by E. coli.
Asunto(s)
Escherichia coli , Percepción de Quorum , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Compuestos de Benzalconio/farmacología , Biopelículas/efectos de los fármacos , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/efectos de la radiación , Homoserina/análogos & derivados , Homoserina/genética , Homoserina/metabolismo , Lactonas/metabolismo , Percepción de Quorum/efectos de los fármacosRESUMEN
OBJECTIVE: The LuxS/AI-2 quorum sensing (QS) system has critical roles in Streptococcus mutans cariogenicity. Whereas the molecular and cellular mechanisms of the LuxS/AI-2 QS system are not thoroughly understood. Given that LuxS has roles in QS and methyl cycle, its mutation can cause QS deficiency and methyl cycle disruption. The aim of this study was to investigate effects of the LuxS/AI-2 QS system on gene expression in Streptococcus mutans when methyl cycle was recovered with exogenous sahH gene. METHODS: Our previous study introduced the exogenous sahH gene from Pseudomonas aeruginosa into an S. mutans luxS-null strain to restore the disrupted methyl cycle, and this produced the solely the LuxS/AI-2 QS system deficient strain. Here, we analyzed the transcriptomics of this strain to get insights into the molecular mechanisms of the LuxS/AI-2 QS system applying RNA-seq. RESULTS: With recovery of methyl cycle, 84 genes didn't change in expression trends in S. mutans luxS-null strain. These genes mainly encode the ABC transporters, sugar transporter EII and enzymes of carbohydrate metabolism, and are rich in the Phosphotransferase system, Fructose and mannose metabolism, Amino sugar and nucleotide sugar metabolism, Galactose metabolism, Glycolysis/Gluconeogenesis, RNA degradation, Lysine biosynthesis, and Glycine, serine and threonine metabolism. CONCLUSIONS: The LuxS/AI-2 QS system may mainly affect ABC transporters and carbohydrate transport, transformation and metabolism via EII subunits and enzymes to influence virulence-associated traits without effects of methyl cycle inStreptococcus mutans.
Asunto(s)
Percepción de Quorum , Streptococcus mutans , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Liasas de Carbono-Azufre/genética , Regulación Bacteriana de la Expresión Génica , Homoserina/genética , Homoserina/metabolismo , Lactonas , Percepción de Quorum/genética , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , TranscriptomaRESUMEN
Quorum sensing is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. Quorum sensing depends on the production, release, and detection of extracellular signal molecules called autoinducers (AIs) that accumulate with increasing cell density. While most AIs are species specific, the AI called AI-2 is produced and detected by diverse bacterial species, and it mediates interspecies communication. We recently reported that mammalian cells produce an AI-2 mimic that can be detected by bacteria through the AI-2 receptor LuxP, potentially expanding the role of the AI-2 system to interdomain communication. Here, we describe a second molecule capable of interdomain signaling through LuxP, 4-hydroxy-5-methylfuran-3(2H)-one (MHF), that is produced by the yeast Saccharomyces cerevisiae Screening the S. cerevisiae deletion collection revealed Cff1p, a protein with no known role, to be required for MHF production. Cff1p is proposed to be an enzyme, with structural similarity to sugar isomerases and epimerases, and substitution at the putative catalytic residue eliminated MHF production in S. cerevisiae Sequence analysis uncovered Cff1p homologs in many species, primarily bacterial and fungal, but also viral, archaeal, and higher eukaryotic. Cff1p homologs from organisms from all domains can complement a cff1ΔS. cerevisiae mutant and restore MHF production. In all cases tested, the identified catalytic residue is conserved and required for MHF to be produced. These findings increase the scope of possibilities for interdomain interactions via AI-2 and AI-2 mimics, highlighting the breadth of molecules and organisms that could participate in quorum sensing.IMPORTANCE Quorum sensing is a cell-to-cell communication process that bacteria use to monitor local population density. Quorum sensing relies on extracellular signal molecules called autoinducers (AIs). One AI called AI-2 is broadly made by bacteria and used for interspecies communication. Here, we describe a eukaryotic AI-2 mimic, 4-hydroxy-5-methylfuran-3(2H)-one, (MHF), that is made by the yeast Saccharomyces cerevisiae, and we identify the Cff1p protein as essential for MHF production. Hundreds of viral, archaeal, bacterial, and eukaryotic organisms possess Cff1p homologs. This finding, combined with our results showing that homologs from all domains can replace S. cerevisiae Cff1p, suggests that like AI-2, MHF is widely produced. Our results expand the breadth of organisms that may participate in quorum-sensing-mediated interactions.
Asunto(s)
Bacterias/metabolismo , Furanos/metabolismo , Homoserina/análogos & derivados , Lactonas/metabolismo , Percepción de Quorum , Saccharomyces cerevisiae/metabolismo , Proteínas Bacterianas/metabolismo , Furanos/análisis , Homoserina/genética , Homoserina/metabolismo , Saccharomyces cerevisiae/genética , Transducción de SeñalRESUMEN
OBJECTIVE: O-acetylhomoserine (OAH) is an important platform chemical to produce high-valuable chemicals. To improve the production of O-acetylhomoserine from glycerol, the glycerol-oxidative pathway was investigated and the optimization of fermentation with crude glycerol was carried out. RESULTS: The glycerol-uptake system and glycerol-oxidative pathway were modified and O-acetyltransferase from Corynebacterium glutamicum was introduced into the engineered strain to produce O-acetylhomoserine. It was found that overexpression of glycerol 3-phosphate dehydrogenase improved the OAH production to 6.79 and 4.21 g/L from pure and crude glycerol, respectively. And the higher OAH production depending on higher level of transcription of glpD. Two-step statistical approach was employed to optimize the fermentation conditions. The significant effects of glycerol, ammonium chloride and yeast extract were screened applying Plackett-Burman design and were optimized further by employing the Response Surface Methodology. Under optimized conditions, the OAH production was up to 9.42 and 7.01 g/L when pure and crude glycerol were used in shake flask cultivations, respectively. CONCLUSIONS: The enzymatic step catalyzing the oxidation of glycerol through GlpD was the key step for OAH production, which served the foundation for realization of a consistent OAH production from crude glycerol in the future.
Asunto(s)
Escherichia coli , Glicerol/metabolismo , Homoserina , Ingeniería Metabólica/métodos , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/enzimología , Corynebacterium glutamicum/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación/genética , Homoserina/análogos & derivados , Homoserina/análisis , Homoserina/genética , Homoserina/metabolismo , Redes y Vías Metabólicas/genética , Oxidación-ReducciónRESUMEN
Autoinducer-2 (AI-2) is a quorum sensing signal that mediates communication within and between many bacterial species. However, its known receptors (LuxP and LsrB families) are not found in all the bacteria capable of responding to this signaling molecule. Here, we identify a third type of AI-2 receptor, consisting of a dCACHE domain. AI-2 binds to the dCACHE domain of chemoreceptors PctA and TlpQ of Pseudomonas aeruginosa, thus inducing chemotaxis and biofilm formation. Boron-free AI-2 is the preferred ligand for PctA and TlpQ. AI-2 also binds to the dCACHE domains of histidine kinase KinD from Bacillus subtilis and diguanylate cyclase rpHK1S-Z16 from Rhodopseudomonas palustris, enhancing their enzymatic activities. dCACHE domains (especially those belonging to a subfamily that includes the AI-2 receptors identified in the present work) are present in a large number of bacterial and archaeal proteins. Our results support the idea that AI-2 serves as a widely used signaling molecule in the coordination of cell behavior among prokaryotic species.
Asunto(s)
Quimiotaxis/fisiología , Homoserina/análogos & derivados , Homoserina/metabolismo , Lactonas/metabolismo , Células Procariotas/metabolismo , Proteínas Arqueales , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Proteínas Portadoras/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Homoserina/química , Homoserina/genética , Lactonas/química , Ligandos , Liasas de Fósforo-Oxígeno , Pseudomonas aeruginosa/metabolismo , Percepción de Quorum , Rhodopseudomonas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Root colonization of beneficial rhizobacteria is critical for their beneficial effects. Quorum sensing (QS) has been reported to affect the colonization of many plant pathogens. However, how QS signals regulate root colonization of beneficial rhizobacteria is unclear. In this study, the QS signal AI-2 synthetase-encoding gene luxS was completely deleted from the genome of the plant beneficial rhizobacterium Bacillus velezensis SQR9, and bioluminescence experiments showed that AI-2 production was blocked. Deletion of luxS reduced biofilm formation, motility, and root colonization of B. velezensis SQR9, while addition of exogenous AI-2 to the mutant restored this phenomenon. These results indicated that AI-2 positively affects the root colonization of B. velezensis SQR9. This study provided new insights for enhancing the colonization of beneficial rhizobacteria. KEY POINTS: ⢠LuxS participated in the synthesis of the quorum sensing signal AI-2 in B. velezensis. ⢠AI-2 enhanced motility, biofilm formation, and root colonization of B. velezensis. ⢠AI-2 stimulated the production of γ-polyglutamic acid by B. velezensis.
Asunto(s)
Bacillus/genética , Bacillus/fisiología , Biopelículas/crecimiento & desarrollo , Homoserina/análogos & derivados , Raíces de Plantas/microbiología , Percepción de Quorum , Proteínas Bacterianas/genética , Liasas de Carbono-Azufre/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Homoserina/genética , Lactonas , Mediciones Luminiscentes , MovimientoRESUMEN
The engineering of advanced multicellular behaviors, such as the programmed growth of biofilms or tissues, requires cells to communicate multiple aspects of physiological information. Unfortunately, few cell-cell communication systems have been developed for synthetic biology. Here, we engineer a genetically encoded channel selector device that enables a single communication system to transmit two separate intercellular conversations. Our design comprises multiplexer and demultiplexer sub-circuits constructed from a total of 12 CRISPRi-based transcriptional logic gates, an acyl homoserine lactone-based communication module, and three inducible promoters that enable small molecule control over the conversations. Experimentally parameterized mathematical models of the sub-components predict the steady state and dynamical performance of the full system. Multiplexed cell-cell communication has applications in synthetic development, metabolic engineering, and other areas requiring the coordination of multiple pathways among a community of cells.
Asunto(s)
Sistemas CRISPR-Cas , Comunicación Celular/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Ingeniería Metabólica/métodos , Percepción de Quorum/genética , Biología Sintética/métodos , Escherichia coli/metabolismo , Homoserina/genética , Homoserina/metabolismo , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida , Proteínas Recombinantes , Bibliotecas de Moléculas PequeñasRESUMEN
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.
Asunto(s)
Escherichia coli/metabolismo , Homoserina/biosíntesis , Redes y Vías Metabólicas/genética , Técnicas de Cultivo Celular por Lotes , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentación , Homoserina/genética , Ingeniería MetabólicaRESUMEN
luxS-mediated autoinducer 2 (AI-2)-dependent quorum sensing (QS) has been demonstrated to affect many bacterial phenotypes, including virulence. Streptococcus agalactiae harbors a functional luxS gene required for the biosynthesis of AI-2. In this study, we investigated the regulation effect and mechanism of the luxS/AI-2 QS system in the pathogenicity of the piscine S. agalactiae strain GD201008-001. We found that inactivation of luxS caused a marked decrease in biofilm formation, hemolytic activity, antiphagocytosis and intracellular survival of S. agalactiae. Except for hemolytic activity, the altered phenotypes due to the luxS deletion were AI-2-independent. Further investigation indicated that high levels of the proinflammatory cytokines IL-1ß and IL-6 could be induced in macrophages co-incubated with the luxS deletion mutant and synthetic AI-2, single or combined. Also, the results of tilapia infection showed that inactivation of luxS significantly decreased the virulence of S. agalactiae but upregulated the expression of cytokines in spleens and brains. Increased proinflammatory effects of the luxS mutant were restored in the luxS complemented strain but could not be restored by AI-2 addition. All the findings suggest that luxS is involved in virulence-associated phenotypes and immunological evasion of S. agalactiae, and furthermore, this involvement is mostly AI-2-independent. This study will provide valuable insights into our understanding of the role of the LuxS/AI-2 QS system in the pathogenesis of S. agalactiae.
Asunto(s)
Proteínas Bacterianas/metabolismo , Liasas de Carbono-Azufre/metabolismo , Homoserina/análogos & derivados , Lactonas/metabolismo , Streptococcus agalactiae/metabolismo , Streptococcus agalactiae/patogenicidad , Animales , Proteínas Bacterianas/genética , Biopelículas , Liasas de Carbono-Azufre/genética , Supervivencia Celular , Cíclidos , Citocinas/genética , Citocinas/metabolismo , Enfermedades de los Peces/microbiología , Eliminación de Gen , Regulación de la Expresión Génica , Homoserina/genética , Homoserina/metabolismo , Ratones , Mutación , Células RAW 264.7 , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/veterinaria , VirulenciaRESUMEN
MvaT and MvaU are global transcriptional regulators belonging to the H-NS family, and pyocyanin is an important virulence factor produced by Pseudomonas aeruginosa. mvaT mvaU double knockout mutant of P. aeruginosa PAO1 demonstrated pyocyanin abolishment in the previous study. Here, we further explored the mechanism. Two main directions were studied: pyocyanin biosynthesis pathway and QS system. The effect on the expression of the pyocyanin biosynthesis genes was evaluated by promoter strength determination and Real-Time PCR assay, and significant changes leading to low pyocyanin production were found. The effect on the QS system was studied by signal molecule quantification using LC-MS/MS and related gene expression measurements using Real-Time PCR. In mvaT mvaU double knockout, the production of 3-oxo-C12-HSL obviously increased, while those of C4-HSL and PQS obviously decreased, and the changes can be recovered by mvaT or mvaU complementation. The expressions of transcriptional activator genes binding with QS system signal molecules were all decreased, resulting in decreased formation of signal-transcriptional activator complexes. And the decreased expression of rhlR and pqsE also led to the lower expression of phzA1 and phzA2. Further exploration found that QS system downregulation may be related to QsrO, a QS system repressor, which was highly upregulated with mvaT mvaU double knockout. Hence, the synthesis of pyocyanin was suffocated and the biofilm formation ability was decreased. These results were also confirmed by transcriptome analysis, which demonstrated similar gene expression changes of the aforementioned genes together with decreased expression of other virulence factor genes regulated by QS system.
Asunto(s)
Proteínas Bacterianas/genética , Pseudomonas aeruginosa/genética , Piocianina/biosíntesis , Transactivadores/genética , Factores de Virulencia/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/genética , 4-Butirolactona/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Cromatografía Liquida , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Técnicas de Silenciamiento del Gen , Homoserina/análogos & derivados , Homoserina/genética , Homoserina/metabolismo , Ratones , Piocianina/genética , Percepción de Quorum/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en TándemRESUMEN
The function of Autoinducer-2 (AI-2) which acts as the signal molecule of LuxS-mediated quorum sensing, is regulated through the lsr operon (which includes eight genes: lsrK, lsrR, lsrA, lsrC, lsrD, lsrB, lsrF, and lsrG). However, the functions of the lsr operon remain unclear in avian pathogenic Escherichia coli (APEC), which causes severe respiratory and systemic diseases in poultry. In this study, the presence of the lsr operon in 60 APEC clinical strains (serotypes O1, O2, and O78) was investigated and found to be correlated with serotype and has the highest detection rate in O78. The AI-2 binding capacity of recombinant protein LsrB of APEC (APEC-LsrB) was verified and was found to bind to AI-2 in vitro. In addition, the lsr operon was mutated in an APEC strain (APEC94Δlsr(Cm)) and the mutant was found to be defective in motility and AI-2 uptake. Furthermore, deletion of the lsr operon attenuated the virulence of APEC, with the LD50 of APEC94Δlsr(Cm) decreasing 294-fold compared with wild-type strain APEC94. The bacterial load in the blood, liver, spleen, and kidneys of ducks infected with APEC94Δlsr(Cm) decreased significantly (p < 0.0001). The results of transcriptional analysis showed that 62 genes were up-regulated and 415 genes were down-regulated in APEC94Δlsr(Cm) compared with the wild-type strain and some of the down-regulated genes were associated with the virulence of APEC. In conclusion, our study suggests that lsr operon plays a role in the pathogenesis of APEC.
Asunto(s)
Proteínas Portadoras/metabolismo , Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Homoserina/análogos & derivados , Lactonas/metabolismo , Enfermedades de las Aves de Corral/microbiología , Percepción de Quorum , Animales , Biopelículas , Proteínas Portadoras/genética , China/epidemiología , Escherichia coli/genética , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Homoserina/genética , Homoserina/metabolismo , Aves de Corral , Enfermedades de las Aves de Corral/epidemiología , SerogrupoRESUMEN
BACKGROUND: Toxigenic Vibrio cholerae resides in aquatic reservoirs of cholera-endemic areas mostly in a dormant form known as conditionally viable environmental cells (CVEC) in which the bacteria remain embedded in an exopolysaccharide matrix, and fail to grow in routine bacteriological culture. The CVEC can be resuscitated by supplementing culture media with either of two autoinducers CAI-1 and AI-2, which are signal molecules controlling quorum sensing, a regulatory network of bacterial gene expression dependent on cell density. This study investigated possible existence of variant strains that overproduce AIs, sufficient to resuscitate CVEC in environmental waters. METHODS: Environmental V. cholerae isolates and Tn insertion mutants of a V. cholerae strain C6706 were screened for production of AIs using bioluminescent reporter strains. Relevant mutations in environmental strains which overproduced AI-2 were characterized by nucleotide sequencing and genetic complementation studies. Effect of AIs produced in culture supernatants of relevant strains on reactivation of CVEC in water was determined by resuscitation assays. RESULTS: Two of 54 environmental V. cholerae isolates were found to overproduce AI-2. Screening of a Tn-insertion library of V. cholerae strain C6706, identified a mutant which overproduced AI-2, and carried Tn insertion in the cqsS gene. Nucleotide sequencing also revealed mutations inactivating the cqsS gene in environmental isolates which overproduced AI-2, and this property was reversed when complemented with a wild type cqsS gene. Culture of river water samples supplemented with spent medium of these mutants resuscitated dormant V. cholerae cells in water. SIGNIFICANCE: V. cholerae strains with inactivated cqsS gene may offer a convenient source of AI-2 in enhanced assays for monitoring bacteriological quality of water. The results also suggest a potential role of naturally occurring cqsS mutants in the environmental biology of V. cholerae. Furthermore, similar phenomenon may have relevance in the ecology of other waterborne bacterial pathogens beyond V. cholerae.
Asunto(s)
Cólera/genética , Homoserina/análogos & derivados , Cetonas , Vibrio cholerae/genética , Biopelículas , Cólera/epidemiología , Cólera/microbiología , Microbiología Ambiental , Regulación Bacteriana de la Expresión Génica/genética , Homoserina/genética , Humanos , Lactonas , Mutación/genética , Percepción de Quorum/genética , Vibrio cholerae/patogenicidadRESUMEN
Increasing resistance to fluoroquinolones (FQs), such as norfloxacin and enrofloxacin, supports the need for the discovery of novel molecules and alternative approaches in antimicrobial therapy. Quorum sensing (QS) is a promising target for next-generation anti-infective agents designed to address the evolving drug resistance in bacterial pathogens. Given that the LuxS/autoinducer-2 (AI-2) quorum-sensing system regulates microbial group behaviors, we hypothesized that this system influences the FQ susceptibility in Streptococcus suis. It was found that a luxS mutant (ΔluxS) of S. suis possesses an increased susceptibility to FQs compared to the wild type strain. When grown in the presence of sub-MIC of antibiotics, the ΔluxS strain showed a significant decrease in growth rate and biofilm formation. These results suggest that the FQ resistance in S. suis could involve a signaling mechanism associated with the LuxS/AI-2 quorum-sensing system. HPLC (High Performance Liquid Chromatography) analyses showed a signiï¬cant increase in the intracellular accumulation of enrofloxacin in the ΔluxS strain compared to the wild type strain. This increase was less pronounced in the presence of exogenous AI-2. Moreover, the expression of satA and satB genes was decreased in the ΔluxS strain. Exogenous AI-2 reversed the down-regulated gene expression observed in the ΔluxS strain. Our study brought strong evidence that the LuxS/AI-2 system in S. suis is involved in FQ susceptibility by regulating the efflux pump SatAB. LuxS is highly conserved among Gram-positive bacteria and may therefore represent a novel antimicrobial target for an alternative approach in antimicrobial therapy.
Asunto(s)
Proteínas Bacterianas/genética , Liasas de Carbono-Azufre/genética , Fluoroquinolonas/farmacología , Homoserina/análogos & derivados , Proteínas de Transporte de Membrana/genética , Streptococcus suis/efectos de los fármacos , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica , Homoserina/genética , Lactonas , Streptococcus suis/genéticaRESUMEN
The link between quorum sensing in Vibrio campbellii and its virulence towards tiger grouper (Epinephelus fuscoguttatus) was investigated using V. campbellii wild type and quorum-sensing mutants with inactive quorum sensing or constitutively maximal quorum-sensing activity, and signal molecule synthase mutants. The results showed that wild-type V. campbellii is pathogenic to grouper larvae, causing more than 50% mortality after 4 days of challenge. Furthermore, the mortality of larvae challenged with the mutant with maximally active quorum sensing was significantly higher than that of larvae challenged with the wild type, whereas a higher survival was observed in the larvae challenged to the mutant with a completely inactive quorum-sensing system. Grouper larvae challenged with either the signal molecule synthase triple mutant, the harveyi autoinducer-1 (HAI-1) synthase mutant and the autoinducer-2 (AI-2) synthase mutant showed higher survival than larvae challenged with the wild type. In contrast, larvae challenged with the cholerae autoinducer-1 (CAI-1) synthase mutant showed high mortality. This indicates that HAI-1 and AI-2, but not CAI-1, are required for full virulence of V. campbellii towards grouper larvae. Our data suggest that quorum-sensing inhibition could be an effective strategy to control V. campbellii infections in tiger grouper.
Asunto(s)
Lubina/microbiología , Enfermedades de los Peces/microbiología , Percepción de Quorum , Vibrio/metabolismo , Vibrio/patogenicidad , 4-Butirolactona/análogos & derivados , 4-Butirolactona/genética , Animales , Homoserina/análogos & derivados , Homoserina/genética , Lactonas , Larva/microbiología , Mutación , Vibrio/genética , VirulenciaRESUMEN
Bacteria use quorum sensing to monitor cell density and coordinate group behaviours. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, quorum sensing is connected to virulence gene expression via the two autoinducer molecules, AI-2 and CAI-1. Both autoinducers share one signal transduction pathway to control the production of AphA, a key transcriptional activator of biofilm formation and virulence genes. In this study, we demonstrate that the recently identified autoinducer, DPO, also controls AphA production in V. cholerae. DPO, functioning through the transcription factor VqmA and the VqmR small RNA, reduces AphA levels at the post-transcriptional level and consequently inhibits virulence gene expression. VqmR-mediated repression of AphA provides an important link between the AI-2/CAI-1 and DPO-dependent quorum sensing pathways in V. cholerae. Transcriptome analyses comparing the effect of single autoinducers versus autoinducer combinations show that quorum sensing controls the expression of â¼400 genes in V. cholerae and that all three autoinducers are required for a full quorum sensing response. Together, our data provide a global view on autoinducer interplay in V. cholerae and highlight the importance of RNA-based gene control for collective functions in this major human pathogen.