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Homoserine dehydrogenase (HSD) is a key enzyme in the biosynthesis of aspartate-family amino acids such as L-homoserine and L-threonine. But HSD exhibited low activity and is feedback inhibited by L-threonine, which severely restricts the biosynthesis level of L-homoserine and L-threonine. In this study, eight HSDs from different species were mined through database search. Among of them, BdHSD derived from Brachypodium distachyon had the highest catalytic activity with 7. 6 U/ mg, and was not feedback-inhibited by L-threonine. The optimal catalytic pH of BdHSD was 10.5, and the optimal catalytic temperature was 38 °C . To improve the catalytic activity of BdHSD, this study further performed the directed evolution of BdHSD, and three BdHSD mutants T186A, N283K, and A137T/ I188V with higher catalytic activity were obtained through multiple rounds of screening. The enzyme activity of the mutant T186A reached 10.3 U/ mg, which was 35. 6% higher than that of the wild type. The L-homoserine fermentation analysis suggested that the BdHSD mutant could effectively enhance the synthesis level of L-homoserine. In summary, this study had mined and evolved the BdHSD with high efficient catalytic, which provided a powerful catalytic element for the efficient biosynthesis of L-homoserine, L-threonine, L-methionine, and other aspartate-family amino acids. [ABSTRACT FROM AUTHOR]

Site-directed mutagenesis was used to improve the catalytic activity of homoserine dehydrogenase (HSD) to reduce its feedback inhibition and repression by metabolites in the pathway. HSD was docked with the substrate homoserine, and its spatial structure was analyzed. Two key sites, Gly25 and Asp61, were selected for site-directed saturation mutation. The activity screening showed that the mutants A61L and G25G had significantly increased enzyme activity when compared to the wild type (WT). The kinetics and enzymatic properties of these two mutants were studied. It was found that compared to the WT enzyme, the Km values of G25G and A61L decreased, the substrate affinity increased, and the enzyme activity increased by 1.21 and 1.35 times, respectively; the n value decreased, and the positive synergy increased. The optimum temperature for A61L and G25G was 40 ℃, the same as that for WT; the optimum pH for A61L and WT was 8.0, which was lower than that (8.5) for G25G. The half-lives of A61L and G25G were 1 h longer and 0.5 h shorter than that of WT, respectively. Low concentrations of K+, Mg2+, and Ca2+ could activate the mutants and WT, while different concentrations of methanol, ethanol, acetonitrile and dimethyl sulfoxide had significantly inhibitory effects on the mutants and WT. At inhibitor concentrations of 1–25 mmol/L, the inhibitory effect was significantly weaker on the mutants than on WT. The mutants G25G and A61L showed improved enzyme activity and weakened allosteric inhibition. This study provides a reference for optimizing the biosynthetic pathway of HSD and constructing strains capable of producing high yield of methionine, threonine and isoleucine.

Allosteric regulation by pathway products plays a vital role in amino acid metabolism. Homoserine dehydrogenase (HSD), the key enzyme for the biosynthesis of various aspartate family amino acids, is subject to feedback inhibition by l-threonine and l-isoleucine. The desensitized mutants with the potential for amino acid production remain limited. Herein, a semi-rational approach was proposed to relieve the feedback inhibition. HSD from Corynebacterium glutamicum (CgHSD) was first characterized as a homotetramer, and nine conservative sites at the tetramer interface were selected for saturation mutagenesis by structural simulations and sequence analysis. Then, we established a high-throughput screening (HTS) method based on resistance to l-threonine analog and successfully acquired two dominant mutants (I397V and A384D). Compared with the best-ever reported desensitized mutant G378E, both new mutants qualified the engineered strains with higher production of CgHSD-dependent amino acids. The mutant and wild-type enzymes were purified and assessed in the presence or absence of inhibitors. Both purified mutants maintained >90% activity with 10 mM l-threonine or 25 mM l-isoleucine. Moreover, they showed >50% higher specific activities than G378E without inhibitors. This work provides two competitive alternatives for constructing cell factories of CgHSD-related amino acids and derivatives. Moreover, the proposed approach can be applied to engineering other allosteric enzymes in the amino acid synthesis pathway.

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The enzyme Homoserine dehydrogenase from Paracoccidioides brasiliensis (Pb HSD), an interesting enzyme in the search for new antifungal drugs against paracoccidioidomycosis, was expressed by E. coli. Thirty milligrams of Pb HSD with 94% of purity were obtained per liter of culture medium. The analysis by CD spectroscopy indicates a composition of 45.5 ± 7.3% of α-helices and 10.5 ± 7.0% β-strands. Gel filtration chromatography indicates a homodimer as biological unity. Fluorescence emission spectroscopy has shown stability of Pb HSD in the presence of urea until C m of 4.13 ± 0.21 M, and a broad pH range in which there is no conformational change. The protein analysis by differential scanning calorimetry indicates high stability at room temperature, but low stability at high temperatures, suffering irreversible denaturation, with T m = 58.65 ± 0.87 °C. Kinetic studies of Pb HSD by molecular absorption spectroscopy in UV/Vis have shown an optimum pH between 9.35 and 9.50, with Michaelian behavior, presenting K M of 224 ± 15 μM and specific activity at optimum pH of 2.10 ± 0.07 μmol/min/mg for homoserine. Therefore, protein expression and purification were efficient, and the structural characterization has shown that Pb HSD presents native conformation with enzymatic activity in kinetic assays. • Homoserine dehydrogenase from P. brasiliensis was functionally expressed in E. coli. • The specific activity was 2.10 μmol/min/mg and K M was 224 μM for homoserine. • Pb HSD is a homodimer with 45.5% of α-helices, 10.5% β-strands, and T m of 57.65 °C. • The enzyme was not sensitive to l -Threonine nor dl -Aspartate. • Pb HSD is a target for antifungal drugs which may help to identify new inhibitors. [ABSTRACT FROM AUTHOR]

Paracoccidioidomycosis is a systemic mycosis endemic in Latin America, and one of the etiological agents of the disease is Paracoccidioides brasiliensis. Currently, available treatments present adversities, such as duration, side effects, and drug interactions. In search of new therapy possibilities, this study evaluates drugs approved for use against the homoserine dehydrogenase enzyme, by an in silico approach, which performs an important biosynthesis phase for the fungus and is not present in the human body. The three-dimensional structure of the homoserine dehydrogenase enzyme from Paracoccidioides brasiliensis was obtained by homology modeling. The model was validated, and simulations were performed for virtual screening of molecules of drugs approved from the Drugs-libs database by the MTiOpenScreen web server. Molecular dynamics in three replicas were used for four drugs with better results, and in two more molecules as a control, the HS9 with inhibition against enzyme and HON which shows inhibition against mold structure. Based on the results of molecular dynamics and the comparison of binding free energy, the drug that obtained the best result was Bemcentinib. In comparison with the controls, it presented a highly relevant affinity with − 44.63 kcal/mol, in addition to good structural stability and occupation of the active site. Therefore, Bemcentinib is a promising molecule for the inhibition of PbHSD protein (homoserine dehydrogenase of Paracoccidioides brasiliensis) and a therapeutic option to be investigated. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel homoserine dehydrogenase (An HSD) from A. nicotinovorans was identified. • An HSD was not homologous to HSDs enzymes which are part of the aspartate pathway. • An HSD was insensitive to the feedback inhibition of l -threonine and l -methionine. • This study provided possibility for the biosynthesis of l -aspartate-β-semialdehyde. Homoserine dehydrogenase (HSD) is a key enzyme in the synthesis pathway of the aspartate family of amino acids. HSD can catalyze the reversible reaction of l -aspartate-β-semialdehyde (l -Asa) to l -homoserine (l -Hse). In this study, one putative homoserine dehydrogenase from Arthrobacter nicotinovorans , which was named An HSD, was different from those HSDs that from the aspartic acid metabolic pathway, and might be responsible for the specific oxidation of l -Hse. Surprisingly, the analysis showed that the purified An HSD exhibited high oxidation activity of l -Hse. At pH 10.0 and 40 °C, the K m and k cat of An HSD was 5.97 ± 1.10 mM and 3.61 s−1, respectively. In terms of cofactor reliance, An HSD preferred NAD+ than NADP+ as a cofactor. The physiological role of An HSD under natural pH is also discussed in A. nicotinovorans. Collectively, these findings provide a novel insight for a better understanding of a novel HSD, and a possible biotransformation method for preparing l -Asa. [ABSTRACT FROM AUTHOR]

Lysine, a nutritionally important amino acid, is involved in adaptation and tolerance to environmental stresses in various organisms. Previous studies reported that lysine accumulation occurs in response to stress and that lysine supplementation enhances stress tolerance; however, the effect of lysine biosynthesis enhancement on stress tolerance has yet to be elucidated. In this study, we confirmed that lysine supplementation to the culture medium increased intracellular lysine content and improved cell growth of Escherichia coli at high temperature (42.5 °C). Lysine-overproducing strains were then isolated from the lysine analogue S-adenosylmethionine-resistant mutants by conventional mutagenesis and exhibited higher tolerance to high-temperature stress than the wild-type strain. We identified novel amino acid substitutions Gly474Asp and Cys554Tyr on ThrA, a bifunctional aspartate kinase/homoserine dehydrogenase (AK/HSDH), in the lysine-overproducing mutants. Interestingly, the Gly474Asp and Cys554Tyr variants of ThrA induced lysine accumulation and conferred high-temperature stress tolerance to E. coli cells. Enzymatic analysis revealed that the Gly474Asp substitution in ThrA reduced HSDH activity, suggesting that the intracellular level of aspartate semialdehyde, which is a substrate for HSDH and an intermediate for lysine biosynthesis, is elevated by the loss of HSDH activity and converted to lysine in E. coli. The present study demonstrated that both lysine supplementation and lysine biosynthesis enhancement improved the high-temperature stress tolerance of E. coli cells. Our findings suggest that lysine-overproducing strains have the potential as stress-tolerant microorganisms and can be applied to robust host cells for microbial production of useful compounds. Key points: • Lysine supplementation improved the growth of E. coli cells at high temperature. • The G474D and C554Y variant ThrA increased lysine productivity in E. coli cells. • The G474D substitution in ThrA reduced homoserine dehydrogenase activity. • E. coli cells that overproduce lysine exhibited high-temperature stress tolerance. [ABSTRACT FROM AUTHOR]

Homoserine dehydrogenase (HSD) catalyzes the reduction of l‐aspatate‐4‐semaldehyde (l‐ASA) to l‐homoserine (l‐HSE) or oxidation reversely, which is a part of the aspartate pathway synthesizing threonine, isoleucine, and methionine in vivo. HSD has gained much interest in medical application since HSD is a well‐established target for pesticides and antibiotics. In addition, HSD is also valuable in industrial application for l‐lysine production. In this study, HSD from Pseudomonas aeruginosa (PaHSD) was overexpressed in Escherichia coli, and purified to homogeneity for molecular and biochemical characterization. PaHSD exhibits the comparable activity of l‐HSE oxidation for both types of cofactors, NAD+ and NADP+, but at the same time shows interesting differences. The kcat/Km value of NADP+ was ~1.8 times larger than that of NAD+, while the kcat/Km value of l‐HSE was ~1.4 times larger with NAD+ than with NADP+. Notably, the Km value is about three times smaller for NADP+, while Vmax is about 1.7 times larger for NAD+, which implies that while NADP+ binds more strongly to the active site, NAD+ has a faster turnover. Size‐exclusion chromatography analysis showed PaHSD forms tetramers, as opposed to HSDs from other species forming dimers. In the circular dichroism analysis, PaHSD showed a typical spectrum of α/β structure, and its melting temperature was determined to be 50.5 °C from the thermal denaturation test at 222 nm. The molecular and biochemical features of PaHSD revealed in this study provide the basis for the future study of amino acid metabolism, and its application to industrial and medical purposes. [ABSTRACT FROM AUTHOR]

Allosteric regulation by pathway products plays a vital role in amino acid metabolism. Homoserine dehydrogenase (HSD), the key enzyme for the biosynthesis of various aspartate family amino acids, is subject to feedback inhibition by l-threonine and l-isoleucine. The desensitized mutants with the potential for amino acid production remain limited. Herein, a semi-rational approach was proposed to relieve the feedback inhibition. HSD from Corynebacterium glutamicum ( Cg HSD) was first characterized as a homotetramer, and nine conservative sites at the tetramer interface were selected for saturation mutagenesis by structural simulations and sequence analysis. Then, we established a high-throughput screening (HTS) method based on resistance to l-threonine analog and successfully acquired two dominant mutants (I397V and A384D). Compared with the best-ever reported desensitized mutant G378E, both new mutants qualified the engineered strains with higher production of Cg HSD-dependent amino acids. The mutant and wild-type enzymes were purified and assessed in the presence or absence of inhibitors. Both purified mutants maintained >90% activity with 10 mM l-threonine or 25 mM l-isoleucine. Moreover, they showed >50% higher specific activities than G378E without inhibitors. This work provides two competitive alternatives for constructing cell factories of Cg HSD-related amino acids and derivatives. Moreover, the proposed approach can be applied to engineering other allosteric enzymes in the amino acid synthesis pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Liu, Liu, Qiao, Ni, Yang, Sun, Li, Zhou, Guo, Chen, Jia, Zheng, Zheng and Sun.)

Aim: To search for potential inhibitors to homoserine dehydrogenase (HSD) in Paracoccidioides brasiliensis the causative agent of paracoccidioidomycosis, an infection with a high mortality rate in Brazil. Materials & methods: The enzyme was modeled and used in the virtual screening of the compounds. The library was first screened by the Autodock, in which 66 molecules were better ranked than substrate, and then, also evaluated by the Molegro and Gold programs. Results: The HS23 and HS87 molecules were selected in common by the three programs, and ADME/Tox evaluation indicates they are not toxic. The molecular dynamics of Pb HSD bonded to ligands showed stable complexes until 50 ns. To validate the results, compounds were purchased for assays of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), synergic profile with Amphotericin B (AmB) and cytotoxicity. The two molecules presented MIC of 32 μg/ml and MFC of 64 μg/ml against the P. brasiliensis (strain Pb18). They also showed synergistic activity with AmB and a lack of toxicity against Hela and Vero cell lines. Conclusion: These results suggest that the HS23 and HS87 are promising candidates as Pb HSD inhibitors and may be used as hits for the development of new drugs against paracoccidioidomycosis.

Homoserine dehydrogenase from Thermus thermophilus (Tt HSD) is a key enzyme in the aspartate pathway that catalyses the reversible conversion of l -aspartate-β-semialdehyde to l -homoserine (l -Hse) with NAD(P)H. We determined the crystal structures of unliganded Tt HSD, Tt HSD complexed with l -Hse and NADPH, and Lys99Ala and Lys195Ala mutant Tt HSDs, which have no enzymatic activity, complexed with l -Hse and NADP+ at 1.83, 2.00, 1.87 and 1.93 Å resolutions, respectively. Binding of l -Hse and NADPH induced the conformational changes of Tt HSD from an open to a closed form: the mobile loop containing Glu180 approached to fix l -Hse and NADPH, and both Lys99 and Lys195 could make hydrogen bonds with the hydroxy group of l -Hse. The ternary complex of Tt HSDs in the closed form mimicked a Michaelis complex better than the previously reported open form structures from other species. In the crystal structure of Lys99Ala Tt HSD, the productive geometry of the ternary complex was almost preserved with one new water molecule taking over the hydrogen bonds associated with Lys99, while the positions of Lys195 and l -Hse were significantly retained with those of the wild-type enzyme. These results propose new possibilities that Lys99 is the acid–base catalytic residue of HSDs. [ABSTRACT FROM AUTHOR]

The orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. L-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K0.5 = 37 μM) than that needed to inhibit the A. thaliana enzyme (K0.5 = 500 μM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by L-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to L-threonine. Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK-HseDH: bifunctional aspartate kinase-homoserine dehydrogenase; AsaDH: aspartate-β-semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T). [ABSTRACT FROM AUTHOR]

Dihydrodipicolinate synthase (DHDPS) catalyzes the first step in the pathway for the biosynthesis of L-lysine in most bacteria and plants. The substrates for the enzyme are pyruvate and L-aspartate-β-semialdehyde (ASA). The product of the reaction was originally proposed to be 2,3-dihydrodipicolinate (DHDP), but has now generally been assumed to be (4 S )-4-hydroxy-2,3,4,5-tetrahydro-(2 S )-dipicolinate (HTPA). ASA is unstable at high pH and it is proposed that ASA reacts with itself. At high pH ASA also reacts with Tris buffer and both reactions are largely reversible at low pH. It is proposed that the basic un-protonated form of the amine of Tris or the α-amine of ASA reacts with the aldehyde functional group of ASA to generate an imine product. Proton NMR spectra of ASA done at different pH values shows new NMR peaks at high pH, but not at low pH, confirming the presence of reaction products for ASA at high pH. The enzymatic product of the DHDPS reaction was examined at low pH by proton NMR starting with either 3 h-pyruvate or 3 d-pyruvate and identical NMR spectra were obtained with four new NMR peaks observed at 1.5, 2.3, 3.9 and 4.1 ppm in both cases. The NMR results were most consistent with DHDP as the reaction product. The UV -spectral studies of the DHDPS reaction shows the formation of an initial product with a broad spectral peak at 254 nM. The DHDPS reaction product was further examined by reduction of the enzymatic reaction components with borohydride followed by GC-MS analysis of the mixture. Three peaks were found at 88, 119 and 169  m/z , consistent with pyruvate, homoserine (reduction product of ASA), and the reduction product of DHDP (1,2,3,6-tetrahydropyridine-2,6-dicarboxylate). There was no indication for a peak associated with the reduced form of HTPA. [ABSTRACT FROM AUTHOR]

Salmonella belongs to the Enterobacteriaceae family which is widely distributed in the environment due to its adaptive capacity to stress conditions. In addition, Salmonella is able to perform a type of cell-to-cell communication called quorum sensing, which leads to differential gene expression. The quorum sensing system mediated by AI-1, acyl homoserine lactones (AHLs), is incomplete in Salmonella because the luxI homolog gene, which encodes for AI-1 synthase, is missing in the genome. However, a homologue of LuxR, known as SdiA, is present and allows the detection of signaling molecules produced by other species of bacteria, leading to regulation of gene expression, mainly related to virulence and biofilm formation. Thus, in view of the importance of quorum sensing on the physiology regulation of microorganisms, the aim of the present study was to perform a virtual screening of plant compounds and nonsteroidal anti-inflammatory drugs (NASIDs) for inhibition of quorum sensing by molecular docking and biofilm formation in Salmonella. In general, most plant compounds and all NSAIDs bound in, at least, one of the three modeled structures of SdiA proteins of Salmonella Enteritidis PT4 578. In addition, many tested compounds had higher binding affinities than the AHLs and the furanones which are inducers and inhibitors of quorum sensing, respectively. The Z-phytol and lonazolac molecules were good candidates for the in vitro inhibition tests of quorum sensing mediated by AI-1 and biofilm formation in Salmonella . Thus, this study directs future prospecting of plant extracts for inhibition of quorum sensing mechanism depending on AHL and biofilm formation. In addition, the use of inhibitors of quorum sensing and biofilm formation can be combined with antibiotics for better treatment efficacy, as well as the use of these compounds to design new drugs. [ABSTRACT FROM AUTHOR]

Two lab-scale nitrifying sequencing batch biofilm reactors, with (SBBR_CN) or without the addition of organics (SBBR_N), were operated to investigate potential roles of acyl homoserine lactone (AHL) based quorum sensing. AHLs of N-[(RS)-3-Hydroxybutyryl]-L-homoserine lactone, N-hexanoyl-L-homoserine lactone (C 6 -HSL) and N-octanoyl-L-homoserine lactone (C 8 -HSL) were detected in both reactors. C 6 -HSL and C 8 -HSL were also detected in batch experiments, especially with stimulated nitrite oxidizing bacteria activities. Quorum sensing affected biofilm formation mainly through the regulation of extracellular protein production. By the metagenomics analysis, many identified genera and species could participate in quorum sensing, quorum quenching and extracellular polymeric substances (EPS) production. A high quorum quenching activity was obtained in SBBR_CN, whereas a high quorum sensing activity in SBBR_N. Nitrosomonas -like ammonia oxidizing bacteria, Nitrospira -like nitrite oxidizing bacteria and Comammox harbored genes for AHL synthesis and EPS production. Possible relationships among AHLs synthesis, biofilm formation and nitrifiers activity were proposed. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Novel fouling control with quorum quenching bacteria (QQ) entrapped PAC-beads. • QQ affected microbial communities in both bulk sludge and biocake. • QQ largely reduced AHL levels in bulk sludge and fully depleted AHLs in biocake. • QQ-PAC-beads greatly increased floc size, hence enhancing sludge dewaterability. • PAC facilitated high removal efficiencies of trace amounts of pharmaceuticals. Abstract This study aimed to integrate the quorum quenching (QQ) strategy with powdered activated carbon (PAC) adsorption for fouling control and simultaneous removal of trace amounts of selected pharmaceutically active compounds (PhACs) in laboratory membrane bioreactors (MBRs). With the addition of QQ strains immobilized in PAC-alginate beads, a 4.6-folds delay in fouling was achieved. The QQ strains not only altered the sludge properties, but also influenced the microbial communities in the MBRs, thus leading to distinct quorum sensing signal molecule (acyl-homoserine lactones, AHLs) profiles. Despite the difference in AHL profiles, the total AHL concentrations were greatly reduced in bulk sludge, and completely quenched in biocake with QQ supplementation. PAC addition enabled high removals of all PhACs, and also resulted in a prominent increase in sludge floc size, which further enhanced sludge filterability. These QQ-entrapped PAC-alginate beads provide a novel MBR setting with less biofouling and high removal efficiency of PhACs. [ABSTRACT FROM AUTHOR]

The antifungal activity of 5‐hydroxy‐4‐oxo‐ l‐norvaline (HONV), exhibited under conditions mimicking human serum, may be improved upon incorporation of this amino acid into a dipeptide structure. Several HONV‐containing dipeptides inhibited growth of human pathogenic yeasts of the Candida genus in the RPMI‐1640 medium, with minimal inhibitory concentration values in the 32 to 64 μg mL−1 range. This activity was not affected by multidrug resistance that is caused by overexpression of genes encoding drug efflux proteins. The mechanism of antifungal action of HONV dipeptides involved uptake by the oligopeptide transport system, subsequent intracellular cleavage by cytosolic peptidases, and inhibition of homoserine dehydrogenase by the released HONV. The relative transport rates determined the anticandidal activity of HONV dipeptides. [ABSTRACT FROM AUTHOR]

In their 1 H NMR spectra in CDCl 3 3-oxo- N -acyl homoserine lactones (OHLs) show significant downfield chemical shifts of the amide N H proton when compared to the parent N -acyl homoserine lactones (AHLs). NMR spectroscopic and DFT calculation studies have shown that this is most likely due to the presence of a stabilising intramolecular H-bond from the N H to the 3-oxo group. The 1 H NMR spectra also show evidence for the enol tautomers and that the amount of enol present for a range of OHLs is 4.1–4.5% in CDCl 3 and 6.5–7.2% in CD 3 CN. In contrast, DFT calculations show that the lowest energy enol tautomer and the keto tautomer are of equal energy in the gas phase, but that the keto tautomer is more stable in chloroform, acetonitrile and water solution. The calculations also show that there is no evidence for any n → π ∗ or C5H-bonding interactions being present in either the lowest energy keto or enol tautomer of the OHLs in solution or the gas phase, which is in contrast to the reported solid-state structure. [ABSTRACT FROM AUTHOR]

Background: Agrobacterium tumefaciens strain P4 is atypical, as the strain is not pathogenic and produces a for this species unusual quorum sensing signal, identified as N-(3-hydroxy-octanoyl)-homoserine lactone (3OH,C8-HSL). Results: By sequence analysis and cloning, a functional luxI-like gene, named cinI, has been identified on the At plasmid of A. tumefaciens strain P4. Insertion mutagenesis in the cinI gene and transcriptome analyses permitted the identification of 32 cinI-regulated genes in this strain, most of them encoding proteins responsible for the conjugative transfer of pAtP4. Among these genes were the avhB genes that encode a type 4 secretion system (T4SS) involved in the formation of the conjugation apparatus, the tra genes that encode the DNA transfer and replication (Dtr) machinery and cinI and two luxR orthologs. These last two genes, cinR and cinX, exhibit an unusual organization, with the cinI gene surrounded by the two luxR orthologs. Conjugation experiments confirmed that the conjugative transfer of pAtP4 is regulated by 3OH,C8-HSL. Root colonization experiments indicated that the quorum sensing regulation of the conjugation of the pAtP4 does not confer a gain or a loss of fitness to the bacterial host in the tomato plant rhizosphere. Conclusion: This work is the first identification of the occurrence of a quorum sensing regulation of the pAt conjugation phenomenon in Agrobacterium. [ABSTRACT FROM AUTHOR]

Traditional therapeutics to treat bacterial infections have given rise to multi-drug resistant pathogens, which pose a major threat to human and animal health. In several pathogens, quorum sensing (QS)-a cell-cell communication system in bacteria-controls the expression of genes responsible for pathogenesis, thus representing a novel target in the fight against bacterial infections. Based on the structure of the autoinducers responsible for QS activity and other QS inhibitors, we hypothesize that β-keto esters with aryl functionality could possess anti-QS activity. A panel of nineteen β-keto ester analogs was tested for the inhibition of bioluminescence (a QS-controlled phenotype) in the marine pathogen Vibrio harveyi. Initial screening demonstrated the need of a phenyl ring at the C-3 position for antagonistic activity. Further additions to the phenyl ring with 4-substituted halo groups or a 3- or 4-substituted methoxy group resulted in the most active compounds with IC50 values ranging from 23 -M to 53 -M. The compounds additionally inhibit green fluorescent protein production by E. coli JB525. Evidence is presented that aryl β-keto esters may act as antagonists of bacterial quorum sensing by competing with N-acyl homoserine lactones for receptor binding. Expansion of the β-keto ester panel will enable us to obtain more insight into the structure-activity relationships needed to allow for the development of novel anti-virulence agents. [ABSTRACT FROM AUTHOR]

Background: Traditional medicinal plants have been used as an alternative medicine in many parts of the world, including Ethiopia. There are many documented scientific reports on antimicrobial activities of the same. To our knowledge, however, there is no report on the anti-Quorum Sensing (Quorum Quenching, QQ) potential of traditional Ethiopian medicinal plants. As many of the opportunistic pathogenic bacteria depend on Quorum Sensing (QS) systems to coordinate their virulence expression, interference with QS could be a novel approach to control bacterial infections. Thus, the aim of this study was to evaluate selected medicinal plants from Ethiopia for their antimicrobial activities against bacterial and fungal pathogens; and to assess the interference of these plant extracts with QS of bacteria. Methods: Antimicrobial activities of plant extracts (oil, resins and crude extracts) were evaluated following standard agar diffusion technique. The minimum inhibitory concentrations (MIC) of potent extracts were determined using 96 well micro-titer plates and optical densities were measured using an ELISA Microplate reader. Interference with Quorum Sensing activities of extracts was determined using the recently established E. coli based reporter strain AI1-QQ.1 and signaling molecule N-(ß-ketocaproyl)-L-homoserine lactone (3-oxo-C6-HSL). Results: Petroleum ether extract of seed of Nigella sativa exhibited the highest activity against both the laboratory isolated Bacillus cereus [inhibition zone (IZ), 44 ± 0.31 mm] and B. cereus ATCC 10987 (IZ, 40 ± 2.33 mm). Similarly, oil extract from mature ripe fruit husk of Aframomum corrorima and mature unripe fruit of A. corrorima revealed promising activities against Candida albicans ATCC 90028 (IZ, 35 ± 1.52 mm) and Staphylococcus aureus DSM 346 (IZ, 25 ± 1.32 mm), respectively. Antimicrobial activities of oil extract from husk of A. corrorima and petroleum ether extract of seed of N. sativa were significantly higher than that of the control antibiotic [Gentamycin sulfate, (IZ, 25–30 mm)]. The lowest MIC value (12.5 mg/mL) was recorded for oil from husk of A. corrorima against Pseudomonas aeruginosa. Of the total eighteen extracts evaluated, two of the extracts [Methanol extract of root of Albiza schimperiana (ASRM) and petroleum ether extract of seed of Justica schimperiana (JSSP)] interfered with cell-cell communication most likely by interacting with the signaling molecules. Conclusion: Traditional medicinal plants from Ethiopia are potential source of alternative medicine for the local community and scientific research in search for alternative drugs to halt challenges associated with the emerging antimicrobial resistance. Furthermore, the Quorum Quenching activities observed in two of the plant extracts calls for more comprehensive evaluation of medicinal plants for the control of many bacterial processes and phenotypic behaviors such as pathogenicity, swarming, and biofilm formation. Being the first assessment of its kind on the potential application of Ethiopian traditional medicinal plants for interference in microbial cell-cell communication (anti-Quorum Sensing activities), the detailed chemistry of the active compounds and possible mechanism(s) of actions of the bio-molecules responsible for the observed interference were not addressed in the current study. Thus, further evaluation for the nature of those active compounds (bio-molecules) and detailed mechanism(s) of their interaction with microbial processes are recommended. [ABSTRACT FROM AUTHOR]

Ectoine is a protective agent and stabilizer whose synthesis pathway exclusively exists in select moderate halophiles. A novel established process called “ bacterial milking ” efficiently synthesized ectoine in moderate halophiles, however, this method places high demands on equipment and is cost prohibitive. In this study, we constructed an ectoine producing strain by introducing the ectoine synthesis pathway into Escherichia coli and improved its production capacity. Firstly, the ectABC gene cluster from Halomonas elongata was introduced into E. coli W3110 and the resultant strain synthesized 4.9 g/L ectoine without high osmolarity. Subsequently, thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was deleted to weaken the competitive l -threonine branch, resulting in an increase of ectoine titer by 109%. Furthermore, a feedback resistant lysC from Corynebacterium glutamicum encoding the aspartate kinase was introduced to complement the enzymatic activity deficiency caused by thrA deletion and a 9% increase of ectoine titer was obtained. Finally, the promoter of ppc that encodes phosphoenolpyruvate carboxylase was replaced by a trc promoter, and iclR , a glyoxylate shunt transcriptional repressor gene, was deleted. The oxaloacetate pool, was thus reinforced and ectoine titer increased by 21%. The final engineered strain ECT05 (pTrcECT, pSTVLysC-CG) produced 25.1 g/L ectoine by fed-batch fermentation in low salt concentration with glucose as a carbon source. The specific ectoine production and productivity was 0.8 g/g DCW and 0.84 g L − 1 h − 1 respectively. The overall ectoine yield was 0.11 g/g of glucose. [ABSTRACT FROM AUTHOR]

Previously we have characterized a threonine dehydratase mutant TD (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best l-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK (encoded by lysC1) and a homoserine dehydrogenase mutant HD (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK is completely resistant to feed-back inhibition by l-threonine and l-lysine, and that HD is partially resistant to l-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive l-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from l-lysine (decreased by 50.1 %) to l-threonine (4.85 g/L) with minor l-isoleucine and no l-homoserine accumulation, further co-expressing ilvA1 completely depleted l-threonine and strongly shifted carbon flux from l-lysine (decreased by 83.0 %) to l-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD might be the main driving force for l-isoleucine over-synthesis in this case, and the partially feed-back resistant HD might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering. [ABSTRACT FROM AUTHOR]

Quorum sensing (QS) is a widespread intercellular form of communication to coordinate physiological processes and cooperative activities of bacteria at the population level, and it depends on the production, secretion, and detection of small diffusible autoinducers, such as acyl-homoserine lactones (AHLs), auto-inducing oligo-peptides (AIPs) and autoinducer 2. In this review, the function of QS autoinducers of gram-negative bacteria in different aspects of wastewater treatment systems is examined. Based on research primarily performed over the past 10 years, QS involvement in the formation of biofilm and aerobic granules and changes of the microbial community and degradation/transformation pathways is discussed. In particular, the QS pathway in the role of bacterial infections and disease prevention in aquaculture is addressed. Interference of QS autoinducer-regulated pathways is considered potential treatment for a variety of environmentally related problems. This review is expected to serve as a stepping stone for further study and development strategies based on the mediation of QS-regulated pathways to enhance applications in both wastewater treatment systems and aquaculture. [ABSTRACT FROM AUTHOR]