Biosynthesis of Sulfur-Containing Small Biomolecules in Plants.

Sulfur is an essential element required for plant growth. It can be found as a thiol group of proteins or non-protein molecules, and as various sulfur-containing small biomolecules, including iron-sulfur (Fe/S) clusters, molybdenum cofactor (Moco), and sulfur-modified nucleotides. Thiol-mediated redox regulation has been well investigated, whereas biosynthesis pathways of the sulfur-containing small biomolecules have not yet been clearly described. In order to understand overall sulfur transfer processes in plant cells, it is important to elucidate the relationships among various sulfur delivery pathways as well as to investigate their interactions. In this review, we summarize the information from recent studies on the biosynthesis pathways of several sulfur-containing small biomolecules and the proteins participating in these processes. In addition, we show characteristic features of gene expression in Arabidopsis at the early stage of sulfate depletion from the medium, and we provide insights into sulfur transfer processes in plant cells.

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

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

Methioninase Cell-Cycle Trap Cancer Chemotherapy.

Cancer cells are methionine (MET) dependent compared to normal cells as they have an elevated requirement for MET in order to proliferate. MET restriction selectively traps cancer cells in the S/G2 phase of the cell cycle. The cell cycle phase can be visualized by color coding with the fluorescence ubiquitination-based cell cycle indicator (FUCCI). Recombinant methioninase (rMETase) is an enzyme that effectively degrades MET. rMETase induces S/G2-phase blockage of cancer cells which is identified by the cancer cells' green fluorescence with FUCCI imaging. Cancer cells in G1/G0 are the majority of the cells in solid tumors and are resistant to the chemotherapy. Treatment of cancer cells with standard chemotherapy drugs only led to the majority of the cancer cell population being arrested in G0/G1 phase, identified by the cancer cells' red fluorescence in the FUCCI system. The G0/G1-phase cancer cells are chemo-resistant. Tumor targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) was used to decoy quiescent G0/G1 stomach cancer cells growing in nude mice to cycle, with subsequent rMETase treatment to selectively trap the decoyed cancer cells in S/G2 phase, which made them highly sensitive to chemotherapy. Subsequent cisplatinum (CDDP) or paclitaxel (PTX) chemotherapy was then administered to kill the decoyed and trapped cancer cells, which completely prevented or regressed tumor growth. In a subsequent experiment, a patient-derived orthotopic xenograft (PDOX) model of recurrent CDDP-resistant metastatic osteosarcoma was eradicated by the combination of Salmonella typhimurium A1-R decoy, rMETase S/G2-phase cell cycle trap, and CDDP cell kill. Salmonella typhimurium A1-R and rMETase pre-treatment thereby overcame CDDP resistance. These results demonstrate the effectiveness of the new chemotherapy paradigm of "decoy, trap, and kill" chemotherapy.

Afterword: Oral Methioninase-Answer to Cancer and Fountain of Youth?

The elevated methionine (MET) requirement of cancer cells is termed MET dependence and is possibly the only known general metabolic defect in cancer. Targeting MET by recombinant methioninase (rMETase) can arrest the growth of cancer cells in vitro and in vivo due to their elevated requirement for MET. rMETase can also potentiate chemotherapy drugs active in S phase due to the selective arrest of cancer cells in S/G2 phase during MET restriction (MR). We previously reported that rMETase, administrated by intraperitoneal injection (ip-rMETase), could inhibit tumor growth in mouse models of cancer including patient-derived orthotopic xenograft (PDOX) mouse models. We subsequently compared ip-rMETase and oral rMETase (o-rMETase) on a melanoma PDOX mouse model. o-rMETase was significantly more effective than ip-rMETase to inhibit tumor growth without overt toxicity. The combination of o-rMETase+ip-rMETase was significantly more effective than either monotherapy and completely arrested tumor growth. Thus, o-rMETase is effective as an anticancer agent with the potential of clinical development for chronic cancer therapy as well as for cancer prevention. o-rMETase may also have potential as an antiaging agent for healthy people, since MR has been shown to extend the life span of a variety of different organisms.

Potential of exogenously sourced kinetin in protecting Solanum lycopersicum from NaCl-induced oxidative stress through up-regulation of the antioxidant system, ascorbate-glutathione cycle and glyoxalase system.

The protective role of exogenously applied kinetin (10 µM KN, a cytokinin) against the adverse effects caused by NaCl-induced (150 mM) stress in Solanum lycopersicum was investigated. Application of KN significantly enhanced growth and biomass production of normally grown plants (non-stressed) and also mitigated the adverse effect of NaCl on stressed plants to a considerable extent. Among the examined parameters, chlorophyll and carotenoid contents, photosynthetic parameters, components of the antioxidant system (both enzymatic and non-enzymatic), osmotica accumulation, and mineral uptake exhibited a significant increase following the application of KN. Furthermore, KN application reduced the generation of reactive free radical hydrogen peroxide, coupled with a significant reduction in lipid peroxidation and an increase in membrane stability. The activities of antioxidant enzymes, and glyoxylase system were found to be promoted in plants exposed to NaCl, and the activities were further promoted by KN application, thereby protecting S. lycopersicum plants against NaCl-induced oxidative damage. Further strengthening of the antioxidant system in KN supplied plants was ascribed to regulation of ascorbate-glutathione cycle, phenols and flavonoids in them. The levels of proline and glycine betaine increased considerably in KN-treated plants, thereby maintaining relative water content. Moreover, exogenous KN application reduced the inhibitory effects of NaCl on K+ and Ca2+ uptake, which resulted in a considerable reduction in tissue Na+/K+ ratio.

Hyaluronic acid-modified polyamidoamine dendrimer G5-entrapped gold nanoparticles delivering METase gene inhibits gastric tumor growth via targeting CD44+ gastric cancer cells.

BACKGROUND: Gastric cancer (GC) is the second most common leading cause of cancer-related death. Cancer stem cell (CSC) with the mark of CD44 played an important role in GC. rMETase was wildly exploited as chemotherapeutic option for GC. Polymers synthetic nanoparticle drug delivery systems have been commonly used for cancer therapy. With the decorating of Hyaluronic acid (HA), a receptor of CD44, nanoparticles exhibit with good biocompatibility and aqueous solubility. METHODS: The characteristic of nanoparticles (NPs) was analyzed by TEM and DLS. The viability and proliferation of GC cells were examined by MTT assays. The levels of CD44, Cyt C, and c-caspase 3 were examined by Western blot. The level of ROS was measured by DCFH-DA assays. The morphology of tissues was detected using hematoxylin-eosin (H&E) stain. Nude mice xenograft models were used to evaluate the effect of HA-PAMAM-Au-METase on GC. RESULTS: The transfection of rMETase carried by HA-G5 PAMAM-Au visibly inhibited the proliferation and tumorsphere formation of GC cells through obviously enhancing METase activity. Elevation of METase activity suppressed the proliferation of CD44(+) GC cells through down-regulating MET in cellular supernatant that resulted in the increase of Cyc C and ROS levels. The number of CD44(+) GC cells in nude mice injected with G5 PAMAM-Au-METase decorated by HA was markly declined resulting in the inhibition of tumor growth. CONCLUSION: HA-G5 PAMAM-Au-METase significantly suppressed tumor growth of GC by targeted damaging the mitochondrial function of CD44(+) gastric CSCs.

Targeting altered cancer methionine metabolism with recombinant methioninase (rMETase) overcomes partial gemcitabine-resistance and regresses a patient-derived orthotopic xenograft (PDOX) nude mouse model of pancreatic cancer.

Pancreatic cancer is a recalcitrant disease. Gemcitabine (GEM) is the most widely-used first-line therapy for pancreatic cancer, but most patients eventually fail. Transformative therapy is necessary to significantly improve the outcome of pancreatic cancer patients. Tumors have an elevated requirement for methionine and are susceptible to methionine restriction. The present study used a patient-derived orthotopic xenograft (PDOX) nude mouse model of pancreatic cancer to determine the efficacy of recombinant methioninase (rMETase) to effect methionine restriction and thereby overcome GEM-resistance. A pancreatic cancer obtained from a patient was grown orthotopically in the pancreatic tail of nude mice to establish the PDOX model. Five weeks after implantation, 40 pancreatic cancer PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n = 10); GEM (100 mg/kg, i.p., once a week for 5 weeks, n = 10); rMETase (100 units, i.p., 14 consecutive days, n = 10); GEM+rMETase (GEM: 100 mg/kg, i.p., once a week for 5 weeks, rMETase: 100 units, i.p., 14 consecutive days, n = 10). Although GEM partially inhibited PDOX tumor growth, combination therapy (GEM+rMETase) was significantly more effective than mono therapy (GEM: p = 0.0025, rMETase: p = 0.0010). The present study is the first demonstrating the efficacy of rMETase combination therapy in a pancreatic cancer PDOX model to overcome first-line therapy resistance in this recalcitrant disease.

Exogenous nitric oxide pretreatment protects Brassica napus L. seedlings from paraquat toxicity through the modulation of antioxidant defense and glyoxalase systems.

To investigate the physiological and biochemical mechanisms of nitric oxide (NO)-induced paraquat (PQ) tolerance in plants, we pretreated a set of 10-day-old Brassica napus seedlings with 500 µM sodium nitroprusside (SNP - a NO donor) for 24 h. Then, three doses of PQ (62.5, 125 and 250 µM) were applied separately, as well as to SNP-pretreated seedlings, and the seedlings were allowed to grow for an additional 48 h. The seedlings treated with PQ showed clear, dose-dependent signs of oxidative stress, with elevated levels of lipid peroxidation (MDA, malondialdehyde), H2O2 and O2- generation, and lipoxygenase (LOX) activity. Paraquat treatment disrupted pools of water-soluble antioxidants (ascorbate-AsA and reduced glutathione-GSH). Paraquat had different effects on the activities of antioxidant enzymes. The activities of glutathione reductase (GR) and catalase (CAT) decreased after PQ treatment in a dose-dependent manner, while the activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glyoxalase (Gly I and Gly II) decreased only with high doses of PQ (125 and 250 µM). By contrast, the activities of monodehydroascorbate reductase (MDHAR), glutathione S-transferase (GST) and glutathione peroxidase (GPX) increased after PQ treatment. A higher dose of PQ reduced chlorophyll and leaf water content but increased the methylglyoxal (MG) and proline (Pro) content. Compared to PQ alone, PQ supplemented with exogenous NO reduced LOX activity, the AsA-GSH pool, and the activities of APX, DHAR, GR, GPX, Gly I and Gly II. These effects helped to reduce oxidative stress and MG toxicity and were accompanied by reduced chlorosis and increased relative water content. Given these results, exogenous NO was found to be a key player in the mitigation of PQ toxicity in plants.

Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways.

Fe-S cofactors are composed of iron and inorganic sulfur in various stoichiometries. A complex assembly pathway conducts their initial synthesis and subsequent binding to recipient proteins. In this minireview, we discuss how discovery of the role of the mammalian cytosolic aconitase, known as iron regulatory protein 1 (IRP1), led to the characterization of the function of its Fe-S cluster in sensing and regulating cellular iron homeostasis. Moreover, we present an overview of recent studies that have provided insights into the mechanism of Fe-S cluster transfer to recipient Fe-S proteins.

The Influences of Bacillus subtilis on the Virulence of Aeromonas hydrophila and Expression of luxS Gene of Both Bacteria Under Co-cultivation.

The aim of this study was to explore the influence of Bacillus subtilis CH9 on Aeromonas hydrophila SC2005. The transcription level of virulence genes of A. hydrophila SC2005 and its hemolysin activity as well as its cytotoxicity were analyzed when B. subtilis CH9 and A. hydrophila SC2005 were co-cultured. The results indicated that the transcription levels of four virulence genes of A. hydrophila, including aer, ahyB, hcp, and emp, decreased when A. hydrophila was cultured with B. subtilis CH9. Furthermore, the extracellular products of A. hydrophila showed attenuated hemolysin activity as well as cytotoxicity when A. hydrophila was cultured with B. subtilis CH9. Finally, the transcriptional levels of luxS genes of B. subtilis CH9 and A. hydrophila SC2005 were determined when these two species were co-cultured. RT-qPCR results suggested that the transcription level of A. hydrophila was down-regulated significantly. On the contrary, the transcription level of B. subtilis CH9 was up-regulated significantly. These results suggested that the probiotic role of B. subtilis CH9 is related to the inhibition of growth and virulence of A. hydrophila SC2005, and quorum sensing may be involved.

Emodin affects biofilm formation and expression of virulence factors in Streptococcus suis ATCC700794.

Streptococcus suis (S. suis) is a swine pathogen and also a zoonotic agent. In this study, the effects of subinhibitory concentrations (sub-MICs) of emodin on biofilm formation by S. suis ATCC700794 were evaluated. As quantified by crystal violet staining, biofilm formation by S. suis ATCC700794 was dose-dependently decreased after growth with 1/2 MIC, 1/4 MIC, or 1/8 MIC of emodin. By scanning electron microscopy, the structural architecture of the S. suis ATCC700794 biofilms was examined following growth in culture medium supplemented with 1/2 MIC, 1/4 MIC, 1/8 MIC, or 1/16 MIC of emodin. Scanning electron microscopy analysis revealed the potential effect of emodin on biofilm formation by S. suis ATCC700794. The expression of luxS gene and virulence genes in S. suis ATCC700794 was investigated by quantitative RT-PCR. It was found that sub-MICs of emodin significantly decreased the expression of gapdh, sly, fbps, ef, and luxS. However, it was found that sub-MICs of emodin significantly increased the expression of cps2J, mrp, and gdh. These findings showed that sub-MICs of emodin could cause the difference in the expression level of the virulence genes.

The dependence of quorum sensing in Serratia marcescens JG on the transcription of luxS gene.

Bacteria communicate with one another using chemical signal molecules. This phenomenon termed quorum sensing enables the bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to cell density. Serratia marcescens JG, a quorum sensing bacterium, can secrete a furanosyl borate diester autoinducer (AI-2) in the exponential phase of growth. In this study, to further investigate the regulation of AI-2 production in S. marcescens JG, the pfs and luxS promoter fusions to an operon luxCDABE reporter were constructed in a low-copy-number vector pBR322K, which allows an examination of transcription of the genes in the pathway for signal synthesis. The results show that the luxS expression is constitutive, and the transcription of luxS is tightly correlated with AI-2 production in S. marcescens JG because the peaks of AI-2 production and transcriptional level of luxS appear at the same time point. The close relation of the profiles of luxS transcription and AI-2 production was also confirmed with real-time PCR technology. These results support the hypothesis that the quorum sensing in S. marcescens JG is luxS dependent.

Reactive oxygen species regulates expression of iron-sulfur cluster assembly protein IscS of Leishmania donovani.

The cysteine desulfurase, IscS, is a highly conserved and essential component of the mitochondrial iron-sulfur cluster (ISC) system that serves as a sulfur donor for Fe-S clusters biogenesis. Fe-S clusters are versatile and labile cofactors of proteins that orchestrate a wide array of essential metabolic processes, such as energy generation and ribosome biogenesis. However, no information regarding the role of IscS or its regulation is available in Leishmania, an evolving pathogen model with rapidly developing drug resistance. In this study, we characterized LdIscS to investigate the ISC system in AmpB-sensitive vs resistant isolates of L. donovani and to understand its regulation. We observed an upregulated Fe-S protein activity in AmpB-resistant isolates but, in contrast to our expectations, LdIscS expression was upregulated in the sensitive strain. However, further investigations showed that LdIscS expression is positively correlated with ROS level and negatively correlated with Fe-S protein activity, independent of strain sensitivity. Thus, our results suggested that LdIscS expression is regulated by ROS level with Fe-S clusters/proteins acting as ROS sensors. Moreover, the direct evidence of a mechanism, in support of our results, is provided by dose-dependent induction of LdIscS-GFP as well as endogenous LdIscS in L. donovani promastigotes by three different ROS inducers: H2O2, menadione, and Amphotericin B. We postulate that LdIscS is upregulated for de novo synthesis or repair of ROS damaged Fe-S clusters. Our results reveal a novel mechanism for regulation of IscS expression that may help parasite survival under oxidative stress conditions encountered during infection of macrophages and suggest a cross talk between two seemingly unrelated metabolic pathways, the ISC system and redox metabolism in L. donovani.

In silico and in vitro studies of cinnamaldehyde and their derivatives against LuxS in Streptococcus pyogenes: effects on biofilm and virulence genes.

The LuxS-based signalling pathway has an important role in physiological and pathogenic functions that are capable of causing different infections. In the present study, cinnamaldehyde (CN) and their derivatives were evaluated for their inhibitory efficiency against LuxS by molecular modelling, docking, dynamics and free-energy calculations. Sequence and structure-similarity analysis of LuxS protein, five different amino acids were found to be highly conserved, of which GLY128 was identified as the key residue involved in the effective binding of the ligands. Quantum-polarized ligand docking protocol showed that 2nitro and 4nitro CN has a higher binding efficiency than CN, which very well corroborates with the in vitro studies. COMSTAT analysis for the microscopic images of the S. pyogenes biofilm showed that the ligands have antibiofilm potential. In addition, the results of quantitative polymerase chain reaction (qPCR) analysis revealed that the transcripts treated with the compounds showed decrease in luxS expression, which directly reflects with the reduction in expression of speB. No substantial effect was observed on the virulence regulator (srv) transcript. These results confirm that speB is controlled by the regulation of luxS. The decreased rate of S. pyogenes survival in the presence of these ligands envisaged the fact that the compounds could readily enhance opsonophagocytosis with the reduction of virulence factor secretion. Thus, the overall data supports the use of CN derivatives against quorum sensing-mediated infections caused by S. pyogenes.

Production of methionine γ- lyase in recombinant Citrobacter freundii bearing the hemoglobin gene.

The production of antileukemic enzyme methionine γ-lyase (MGL) in distinctly related bacteria, Citrobacter freundii and in their recombinants expressing the Vitresocilla hemoglobin (VHb) has been studied. This study concerns the potential of Citrobacter freundii expressing the Vitreoscilla hemoglobin gene (vgb) for the methionine γ- liyase production. Methionine γ- liyase production by Citrobacter freundii and its vgb(-) and vgb(+) bearing recombinant strain was studied in shake-flasks under 200 rpm agitation, culture medium and 30 °C in a time-course manner. The vgb(+) and especially the carbon type had a dramatic effect on methionine γ- liyase production. The vgb(+) strain of C. freundii had about 2-fold and 3.1-fold higher levels of MGL than the host and vgb(-) strain, respectively.

Regulation of the Edwardsiella tarda hemolysin gene and luxS by EthR.

Edwardsiella tarda is a pathogen with a broad host range that includes human and animals. The E. tarda hemolysin (Eth) system, which comprises EthA and EthB, is a noted virulence element that is widely distributed in pathogenic isolates of E. tarda. Previous study has shown that the expression of ethB is regulated by iron, which suggests the possibility that the ferric uptake regulator (Fur) is involved in the regulation of ethB. The work presented in this report supports the previous findings and demonstrates that ethB expression was decreased under conditions when the E. tarda Fur (Fur(Et)) was overproduced, and enhanced when FurEt was inactivated. We also identified a second ethB regulator, EthR, which is a transcription regulator of the GntR family. EthR represses ethB expression by direct interaction with the ethB promoter region. In addition to ethB, EthR also modulates, but positively, luxS expression and AI-2 production by binding to the luxS promoter region. The expression of ethR itself is subject to negative autoregulation; interference with this regulation by overexpressing ethR during the process of infection caused (i) drastic changes in ethB and luxS expressions, (ii) vitiation in the tissue dissemination and survival ability of the bacterium, and (iii) significant attenuation of the overall bacterial virulence. These results not only provide new insights into the regulation mechanisms of the Eth hemolysin and LuxS/AI-2 quorum sensing systems but also highlight the importance of these systems in bacterial virulence.

L-methioninase production by Aspergillus flavipes under solid-state fermentation.

Solid-state fermentation was carried out for the production of extra-cellular L-methioninase by Aspergillus flavipes (Bain and Sart.) using nine agro-industrial residues, namely wheat bran, rice bran, wheat flour, coconut seeds, cotton seeds, ground nut cake, lentil hulls, soya beans and chicken feathers. Chicken feathers were selected as solid substrate for L-methioninase production by A. flavipes. The maximum L-methioninase productivity (71.0 U/mg protein) and growth (11 mg protein/ml) of A. flavipes was obtained using alkali pretreated chicken feathers of 50% initial moisture content as substrate supplemented with D-glucose (1.0% w/v) and L-methionine (0.2% w/v). External supplementation of the fermentation medium with various vitamin sources has no overinductive effect on L-methioninase biosynthesis. The partially purified A. flavipes L-methioninase preparation showed highest activity (181 U/ml) at pH 8.0 with stability over a pH range (pH 6-8) for 2 h. L-methioninase activity was increased by preincubation of the enzyme for 2 h with Co(2+), Mn(2+), Cu(2+) and Mg(2+) and strongly inhibited by the presence of EDTA, NaN(3), Li(2+), Cd(2+), DMSO and 2-mercaptoethanol. The enzyme preparation has a broad substrate spectrum showing a higher affinity to deaminate L-glycine, N -acetylglucosamine and glutamic acid, in addition to their proteolytic activity against bovine serum albumin, casein, gelatin and keratin. The partially purified enzyme was found to be glyco-metalloproteinic in nature as concluded from the analytical and spectroscopic profiles of the enzyme preparation. The demethiolating activity of the enzyme was also visualized chromogenially.

Heterologous production of methionine-gamma-lyase from Brevibacterium linens in Lactococcus lactis and formation of volatile sulfur compounds.

The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor improvement. A synthetic mgl gene encoding methionine-gamma-lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade L-methionine as well as other sulfur-containing compounds such as L-cysteine, L-cystathionine, and L-cystine. Overproduction of MGL in recombinant L. lactis also resulted in an increase in the degradation of these compounds compared to the wild-type strain. Importantly, gas chromatography-mass spectrometry analysis identified considerably higher formation of methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) in reactions containing either purified protein, whole cells, or cell extracts from the heterologous L. lactis strain. This is the first report of production of MGL from B. linens in L. lactis. Given their significance in cheese flavor development, the use of lactic acid bacteria with enhanced VSC-producing abilities could be an efficient way to enhance cheese flavor development.

Iron-sulfur cluster biosynthesis: characterization of IscU-IscS complex formation and a structural model for sulfide delivery to the [2Fe-2S] assembly site.

Recent work on the bacterial iron-sulfur cluster (isc) family of gene products, and eukaryotic homologs, has advanced the molecular understanding of cellular mechanisms of iron-sulfur cluster biosynthesis. Members of the IscS family are pyridoxyl-5'-phosophate dependent proteins that deliver inorganic sulfide during assembly of the [2Fe-2S] cluster on the IscU scaffold protein. Herein it is demonstrated through calorimetry, fluorescence, and protein stability measurements that Thermotoga maritima IscS forms a 1:1 complex with IscU in a concentration-dependent manner (K(D) varying from 6 to 34 microM, over an IscS concentration range of approximately 2-50 microM). Docking simulations of representative IscU and IscS proteins reveal critical contact surfaces at the N-terminal helix of IscU and a C-terminal loop comprising a chaperone binding domain. Consistent with the isothermal titration calorimetry results described here, an overall dominant contribution of charged surfaces with a change in the molar heat capacity of binding, DeltaC(p) approximately 199.8 kcal K(-1) mol(-1), is observed that accounts for approximately 10% of the total accessible surface area at the binding interface. Both apo and holo IscUs and homologs were found to bind to IscS in an enthalpically driven reaction with comparable K(D) values. Both helix and loop regions are highly conserved among phylogenetically diverse organisms from a pool of archael, bacterial, fungal, and mammalian representatives.

Cysteine metabolism-related genes and bacterial resistance to potassium tellurite.

Tellurite exerts a deleterious effect on a number of small molecules containing sulfur moieties that have a recognized role in cellular oxidative stress. Because cysteine is involved in the biosynthesis of glutathione and other sulfur-containing compounds, we investigated the expression of Geobacillus stearothermophilus V cysteine-related genes cobA, cysK, and iscS and Escherichia coli cysteine regulon genes under conditions that included the addition of K2TeO3 to the culture medium. Results showed that cell tolerance to tellurite correlates with the expression level of the cysteine metabolic genes and that these genes are up-regulated when tellurite is present in the growth medium.