cysE Gene Research Articles

The Structure of the LysR-type Transcriptional Regulator, CysB, Bound to the Inducer, N-acetylserine

In Escherichia coli and Salmonella typhimurium, cysteine biosynthesis requires the products of 20 or more cys genes co-ordinately regulated by CysB. Under conditions of sulphur limitation and in the presence of the inducer, N-acetylserine, CysB binds to cys promoters and activates the transcription of the downstream coding sequences. CysB is a homotetramer, comprising an N-terminal DNA binding domain (DBD) and a C-terminal effector binding domain (EBD). The crystal structure of a dimeric EBD fragment of CysB from Klebsiella aerogenes revealed a protein fold similar to that seen in Lac repressor but with a different symmetry in the dimer so that the mode of DNA binding was not apparent. To elucidate the subunit arrangement in the tetramer, we determined the crystal structure of intact CysB in complex with N-acetylserine. The tetramer has two subunit types that differ in the juxtaposition of their winged helix-turn-helix DNA binding domains with respect to the effector binding domain. In the assembly, the four EBDs form a core with the DNA binding domains arranged in pairs on the surface. N-acetylserine makes extensive polar interactions in an enclosed binding site, and its binding is accompanied by substantial conformational rearrangements of surrounding residues that are propagated to the protein surface where they appear to alter the arrangement of the DNA binding domains. The results are (i) discussed in relation to the extensive mutational data available for CysB and (ii) used to propose a structural mechanism of N-acetylserine induced CysB activation.

Alkyl gallates inhibit serine O-acetyltransferase in bacteria and enhance susceptibility of drug-resistant Gram-negative bacteria to antibiotics.

A principal concept in developing antibacterial agents with selective toxicity is blocking metabolic pathways that are critical for bacterial growth but that mammalian cells lack. Serine O-acetyltransferase (CysE) is an enzyme in many bacteria that catalyzes the first step in l-cysteine biosynthesis by transferring an acetyl group from acetyl coenzyme A (acetyl-CoA) to l-serine to form O-acetylserine. Because mammalian cells lack this l-cysteine biosynthesis pathway, developing an inhibitor of CysE has been thought to be a way to establish a new class of antibacterial agents. Here, we demonstrated that alkyl gallates such as octyl gallate (OGA) could act as potent CysE inhibitors in vitro and in bacteria. Mass spectrometry analyses indicated that OGA treatment markedly reduced intrabacterial levels of l-cysteine and its metabolites including glutathione and glutathione persulfide in Escherichia coli to a level similar to that found in E. coli lacking the cysE gene. Consistent with the reduction of those antioxidant molecules in bacteria, E. coli became vulnerable to hydrogen peroxide-mediated bacterial killing in the presence of OGA. More important, OGA treatment intensified susceptibilities of metallo-β-lactamase-expressing Gram-negative bacteria (E. coli and Klebsiella pneumoniae) to carbapenem. Structural analyses showed that alkyl gallate bound to the binding site for acetyl-CoA that limits access of acetyl-CoA to the active site. Our data thus suggest that CysE inhibitors may be used to treat infectious diseases caused by drug-resistant Gram-negative bacteria not only via direct antibacterial activity but also by enhancing therapeutic potentials of existing antibiotics.

Extracellular Production of Glutathione by Recombinant Escherichia coli K-12

The goal of this study was to produce a sufficient amount of glutathione in the fermentation medium without the addition of cysteine. This would simplify and reduce the cost of its purification. In addition to reducing the cost of cysteine, it also avoids the inhibition of bacterial growth by cysteine. The gshA, gshB, and cysE genes of Escherichia coli were cloned under the control of the strong T5 promoter of the pQE-80L plasmid and introduced into an E. coli strain knocked out for the genes encoding γ-glutamyltranspeptidase and the GsiABCD glutathione transporter, which are responsible for the recycling of excreted glutathione. The overexpression of the gshA and gshB genes, genes for γ-glutamylcysteine synthetase and glutathione synthetase, and the cysEV95R D96P gene, a gene for serine acetyltransferase with the V95R D96P mutation that makes it insensitive to cysteine, were effective on glutathione production. Na2S2O3 was a good sulfur source for glutathione production, while the addition of Na2SO4 did not affect the glutathione production. With the addition of 50 mM glutamic acid and 75 mM glycine, but without the addition of cysteine, to the simplified SM1 medium, 4.6 mM and 0.56 mM of the reduced and oxidized glutathione, respectively, were accumulated in the extracellular space after 36 h of batch culture. This can eliminate the need to extract glutathione from the bacterial cells for purification.

GhCYS2 governs the tolerance against cadmium stress by regulating cell viability and photosynthesis in cotton

Cysteine, an early sulfur-containing compound in plants, is of significant importance in sulfur metabolism. CYS encodes cysteine synthetase that further catalyzes cysteine synthesis. In this investigation, CYS genes, identified from genome-wide analysis of Gossypium hirsutum bioinformatically, led to the discovery of GhCYS2 as the pivotal gene responsible for Cd2+ response. The silencing of GhCYS2 through virus-induced gene silencing (VIGS) rendered plants highly susceptible to Cd2+ stress. Silencing GhCYS2 in plants resulted in diminished levels of cysteine and glutathione while leading to the accumulation of MDA and ROS within cells, thereby impeding the regular process of photosynthesis. Consequently, the stomatal aperture of leaves decreased, epidermal cells underwent distortion and deformation, intercellular connections are dramatically disrupted, and fissures manifested between cells. Ultimately, these detrimental effected culminating in plant wilting and a substantial reduction in biomass. The association established between Cd2+ and cysteine in this investigation offered a valuable reference point for further inquiry into the functional and regulatory mechanisms of cysteine synthesis genes.

Applications of Genomics, Metabolomics, Fourier Transform Infrared in the Evaluation of Spoilage Targets of Shewanella putrefaciens from Spoiled Bigeye Tuna.

Shewanella putrefaciens is a typical spoiler that is commonly found in seafood and has high spoilage potential. However, the spoilage mechanism against Shewanella putrefaciens at the gene and metabolism levels has not been well elucidated. This work determined the spoilage targets on Shewanella putrefaciens XY07 from spoiled bigeye tuna by genome sequencing, metabolomics, and Fourier transform infrared (FTIR) analysis. Shewanella putrefaciens XY07 contained some genes on spoilage regulating of cys genes, his genes, spe genes and rpoS gene involved in sulfur metabolism, histidine metabolism, arginine and proline degradation, and biofilm formation at the genome level, respectively. Some spoilage genes like speC, cysM, trxB genes were identified. In addition, ABC transporters, arginine and proline metabolism; beta-alanine metabolism; glycine, serine, and threonine metabolism; histidine metabolism; sulfur metabolism; and lipid metabolism were identified as important pathways related to aquatic food during spoilage, which indicated the functions of amino acid degradation in S. putrefaciens XY 07 by metabolomics analysis. The metabolites of l-ornithine, 5-aminopentanoate, and 4-aminobutyraldehyde could be further metabolized to spermidine and spermine, producing a spoilage odor, and were involved in arginine and proline metabolism serving as key spoilage regulating metabolisms. Therefore, Shewanella putrefaciens XY07 was applied to genomics, metabolomics analysis, and FTIR to provide comprehensive insight into the investigation of spoilage targets.

Deciphering the biodesulfurization pathway employing marine mangrove Bacillus aryabhattai strain NM1-A2 according to whole genome sequencing and transcriptome analyses

In the biogeochemical cycle, sulfur oxidation plays a vital role and is typically referred to as the elemental sulfur or reductive sulfide oxidation process. This study aimed to characterize a subtropical mangrove-isolated bacterial strain using biochemical, whole-genome, and transcriptome sequencing analyses to enhance our understanding of sulfur metabolism and biodegradation from a molecular genetic perspective. Strain NM1-A2 was characterized as Gram-positive and found to have a close molecular phylogenetic relationship with Bacillus aryabhattai. NM1-A2 efficiently converted dibenzothiophene (DBT) into 2-hydroxybiphenyl (2-HBP) via a 4S pathway with 95% efficiency, using enzymes encoded by the dsz operon (dszA, dszB, and dszC), which determine monooxygenases (DszA & DszC) and desulfinase (DszB). The whole-genome sequence of NM1-A2 had a length of approximately 5,257,678 bp and included 16 sulfur metabolism-related genes, featuring the ABC transport system, small subunit (ssu) and cysteine (cys) gene families, and adenosine 5′-phosphosulfate (APS) and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) biosynthesis-related genes. Transcriptomic analysis of NM1-A2 using three sulfur groups—magnesium sulfate (MS), sulfur powder (SP), and sodium thiosulfate (ST) resulted in a significant number of differentially expressed genes (1200, 2304, and 2001, respectively). This analysis revealed that intracellular cysteine concentration directly regulated the expression of cys and ssu genes. Sulfate did not directly affect cys gene expression but repressed ssu gene expression. The cys gene expression levels decreased during the conversion of sulfate to sulfide and cysteine. The transcriptomic data was validated by analyzing the expression patterns of NM1-A2 using real-time quantitative PCR validation analysis. The expression levels of cysl, mccB, and nrnA were significantly upregulated, while cysH, metB, and sat were downregulated in the SP, ST, and MS groups, respectively. This research contributes to our understanding of marine mangrove microorganisms' bacterial efficiency through characterization, whole-genome, and transcriptome sequencing-based molecular degradation of organic compounds in the mangrove ecosystem, which may enhance nutrient availability.

Bioinformatics Analysis of Cysteine Transmembrane Gene CYS in Transgenic Tobacco

Overexpression of MYB transcription factor LrAN2  in tobacco can activate up-regulation of anthocyanin biosynthetic pathways, but the expression of cysteine-rich transmembrane domain protein A (CYS) is significantly reduced. AS an important biological amino acid in plants, the biological research of CYS is rarely reported. In this study, the cloned tobacco CYS gene is 282 bp long and encoding 94 amino acids. The predicted molecular weight and isoelectric point of the encoded protein are 24 019.84 Da and 5.23 respectively, and there are no conserved regions in the sequence. The experimental results show that the protein is hydrophilic, mainly composed of α-helix (19.35%) and irregular coil (65.59%), CYS protein does not pass through the transmembrane region and acts outside the membrane. Phylogenetic tree shows that the CYS gene in tobacco was closely related to the CYS gene in pepper, followed by HTC gene and WIH2 gene in tomato. The results of this study cloned and analyzed the sequence of the coding region of CYS gene to provide a reference for the functional study of CYS gene.

Carbon-Nitrogen-Sulfur-Related Microbial Taxa and Genes Maintained the Stability of Microbial Communities in Coals.

Coal microbes are the predominant form of life in the subsurface ecosystem, which play a vital role in biogeochemical cycles. However, the systematic information about carbon–nitrogen–sulfur (C–N–S)-related microbial communities in coal seams is limited. In this study, 16S rRNA gene data from a total of 93 microbial communities in coals were collected for meta-analysis. The results showed that 718 functional genera were related to the C–N–S cycle, wherein N2 fixation, denitrification, and C degradation groups dominated in relative abundance, Chao1 richness, Shannon diversity, and niche width. Genus Pseudomonas having the most C–N–S-related functions showed the highest relative abundance, and genus Herbaspirillum with a higher abundance participated in C degradation, CH4 oxidation, N2 fixation, ammoxidation, and denitrification. Such Herbaspirillum was a core genus in the co-occurrence network of microbial prokaryotes and showed higher levels in weight degree, betweenness centrality, and eigenvector centrality. In addition, most of the methanogens could fix N2 and dominated in the N2 fixation groups. Among them, genera Methanoculleus and Methanosaeta showed higher levels in the betweenness centrality index. In addition, the genus Clostridium was linked to the methanogenesis co-occurrence network module. In parallel, the S reduction gene was present in the highest total relative abundance of genes, followed by the C degradation and the denitrification genes, and S genes (especially cys genes) were the main genes linked to the co-occurrence network of the C–N–S-related genes. In summary, this study strengthened our knowledge regarding the C–N–S-related coal microbial communities, which is of great significance in understanding the microbial ecology and geochemical cycle of coals.

Cbl upregulates cysH for hydrogen sulfide production in Aeromonas veronii.

Endogenous hydrogen sulfide (H2S) is generated in many metabolism pathways, and has been recognized as a second messenger against antibiotics and reactive oxygen species (ROS). In Aeromonas veronii, Small Protein B (SmpB) plays an important role in resisting stress. The absence of smpB could trigger sulfate assimilation pathway to adapt the nutrient deficiency, of which was mediated by up-regulation of cbl and cys genes and followed with enhancing H2S production. To figure out the mutual regulations of cbl and cys genes, a series of experiments were performed. Compared with the wild type, cysH was down-regulated significantly in cbl deletion by qRT-PCR. The fluorescence analysis further manifested that Cbl had a positive regulatory effect on the promoter of cysJIH. Bacterial one-hybrid analysis and electrophoretic mobility shift assay (EMSA) verified that Cbl bound with the promoter of cysJIH. Collectively, the tolerance to adversity could be maintained by the production of H2S when SmpB was malfunctioned, of which the activity of cysJIH promoter was positively regulated by upstream Cbl protein. The outcomes also suggested the enormous potentials of Aeromonas veronii in environmental adaptability.

Highly Cr(vi)-tolerant Staphylococcus simulans assisting chromate evacuation from tannery effluent

Abstract Chromium(vi) contaminated sites have been targeted for studying highly chromate-resistant bacteria. From a total of 23 Cr(vi)-resistant bacteria isolated on Luria-Bertani agar medium supplemented with K2CrO4 (1,500 μg/mL), only one strain UT8 (Staphylococcus simulans) was able to tolerate high concentration of chromate, i.e., up to 200 mg/mL in agar medium from tannery effluent. In acetate minimal medium (AMM), it showed maximum tolerance of up to 2,500 μg/mL. Cr(vi) removal potential was 94.6% after 24 h (K2CrO4 1,500 μg/mL). Parametric conditions were optimized in AMM to attain maximum chromate removal. Exopolysaccharides extracted from bacterial cells exposed to chromate exhibited major absorption shifts from 2,500 to 500 cm−1 revealed by Fourier transform infrared spectroscopy. Energy-disperse X-ray spectroscopy further confirmed the adsorption of oxyanions to the bacterial cells. Surface topography of the Cr(vi) treated cells showed transformation into concave shape by scanning electron microscopy. The presence of resistance genes, i.e., chromate reductase (chrR) and class I integrase (intI1), further confirmed tolerance toward chromate. Microarray data analysis of transcriptional gene expression suggested upregulation of cys gene cluster under chromate exposure. Concisely, the present investigations revealed the potential of S. simulans to be an effective candidate for chromate reclamation of wastewater.

Analysis of tRNA Cys processing under salt stress in Bacillus subtilis spore outgrowth using RNA sequencing data

Background: In spore-forming bacteria, the molecular mechanisms of accumulation of transfer RNA (tRNA) during sporulation must be a priority as tRNAs play an essential role in protein synthesis during spore germination and outgrowth. However, tRNA processing has not been extensively studied in these conditions, and knowledge of these mechanisms is important to understand long-term stress survival. Methods:To gain further insight into tRNA processing during spore germination and outgrowth, the expression of the single copy tRNA Cys gene was analyzed in the presence and absence of 1.2 M NaCl in Bacillus subtilis using RNA-Seq data obtained from the Gene Expression Omnibus (GEO) database. The CLC Genomics work bench 12.0.2 (CLC Bio, Aarhus, Denmark, https://www.qiagenbioinformatics.com/) was used to analyze reads from the tRNA Cys gene. Results:The results show that spores store different populations of tRNA Cys-related molecules. One such population, representing 60% of total tRNA Cys, was composed of tRNA Cys fragments. Half of these fragments (3´-tRF) possessed CC, CCA or incorrect additions at the 3´end. tRNA Cys with correct CCA addition at the 3´end represented 23% of total tRNA Cys, while with CC addition represented 9% of the total and with incorrect addition represented 7%. While an accumulation of tRNA Cys precursors was induced by upregulation of the rrnD operon under the control of σ A -dependent promoters under both conditions investigated, salt stress produced only a modest effect on tRNA Cys expression and the accumulation of tRNA Cys related species. Conclusions:The results demonstrate that tRNA Cys molecules resident in spores undergo dynamic processing to produce functional molecules that may play an essential role during protein synthesis.

Improving the freeze-drying survival rate of Lactobacillus plantarum LIP-1 by increasing biofilm formation based on adjusting the composition of buffer salts in medium

Lactic acid bacteria can improve their resistance to adverse environments through the formation of biofilm. This study found that adding different buffer salts in culture medium had a great impact on the freeze-drying survival rate of the Lactobacillus plantarum LIP-1, which could be linked to biofilm formation. Transcriptome data showed that potassium ions in buffer salt increased the expression of the luxS gene in the LuxS/autoinducer-2 (AI-2) quorum sensing system and increase synthesis of the quorum sensing signal AI-2. The AI-2 signal molecules up-regulated the cysE gene, which helps to promote biofilm formation. By adding a biofilm inhibitor, d-galactose, and performing a real-time quantitative polymerase chain reaction experiment, we found that d-galactose could down-regulated the luxS and cysE genes, reduced biofilm formation, and decreased the freeze-drying survival rate. The results of this study showed that promoting biofilm formation using appropriate buffer salts may lead to better freeze-drying survival rates.

Analysis of tRNA Cys processing under salt stress in Bacillus subtilis spore outgrowth using RNA sequencing data.

Background: In spore-forming bacteria, the molecular mechanisms of accumulation of transfer RNA (tRNA) during sporulation must be a priority as tRNAs play an essential role in protein synthesis during spore germination and outgrowth. However, tRNA processing has not been extensively studied in these conditions, and knowledge of these mechanisms is important to understand long-term stress survival. Methods:To gain further insight into tRNA processing during spore germination and outgrowth, the expression of the single copy tRNA Cys gene was analyzed in the presence and absence of 1.2 M NaCl in Bacillus subtilis using RNA-Seq data obtained from the Gene Expression Omnibus (GEO) database. The CLC Genomics work bench 12.0.2 (CLC Bio, Aarhus, Denmark, https://www.qiagenbioinformatics.com/) was used to analyze reads from the tRNA Cys gene. Results:The results show that spores store different populations of tRNA Cys-related molecules. One such population, representing 60% of total tRNA Cys, was composed of tRNA Cys fragments. Half of these fragments (3´-tRF) possessed CC, CCA or incorrect additions at the 3´end. tRNA Cys with correct CCA addition at the 3´end represented 23% of total tRNA Cys, while with CC addition represented 9% of the total and with incorrect addition represented 7%. While an accumulation of tRNA Cys precursors was induced by upregulation of the rrnD operon under the control of σ A -dependent promoters under both conditions investigated, salt stress produced only a modest effect on tRNA Cys expression and the accumulation of tRNA Cys related species. Conclusions:The results demonstrate that tRNA Cys molecules resident in spores undergo dynamic processing to produce functional molecules that may play an essential role during protein synthesis.

Identification and inoculation of fungal strains from Cedrus deodara rhizosphere involve in growth and alleviation of high nitrogen stress.

Cedrus deodara is economically and ethnobotanically an important forest tree and is shown to be at decline in Northern areas of Pakistan in recent years mainly due to high concentration of Nitrogen in forests. Ectomycorrhizal (ECM) association forming fungi enables the forest trees to develop optimally by absorbing water from the rhizosphere through their absorptive hyphae and by making available the nutrients by mobilization of N and P from the organic substrates. This study was conducted to identify the ECM strains from C. deodara rhizosphere and to analyse the impact of high N load on the C. deodara seedlings to establish N critical load value for coniferous forests of Pakistan. Six new fungal strains were identified from the rhizosphere of C. deodara and were registered at GenBank (NCBI) as Emmia latemarginata strain ACE1, Aspergillus terreus strain ACE2, Purpureocillium lilacinum strain ACE3, Talaromyces pinophilus strain ACE4, A. fumigatus strain ACE5 and T. pinophilus strain ACE6 with accession numbers MH145426, MH145427, MH145428, MH145429, MH145430 and MH547115. Four out of six isolated strains were inoculated with seedlings of C. deodara singly and in consortium (CN) in combination with nitrogen load of 0 (C), 25 (T1), 50 (T2), 100 kg N ha−1 yr−1 (T3). Agronomic, physiological and gene expression studies for ExpansinA4 (EXPA4) and Cystatins (Cys) were made to analyse the impact of fungal strains in relation to high N stress. This study suggests a positive impact of T1 (25 kg N ha−1 yr−1) Nitrogen load and a negative impact of T3 (100 kg N ha−1 yr−1) on growth parameters and expression patterns of EXPA4 and Cys genes. Peroxidase (POX) activity decreased in the order ACE5 > ACE2 > C > ACE3 > ACE1 > CN. However, the results of Superoxide dismutase (SOD) showed decreasing trend in the order ACE5 > C > CN > ACE1 > ACE2 > ACE3. Strain ACE3 was shown to have a positive impact on the seedlings in terms of growth, physiology and expression of genes. Present study suggests that newly identified fungal strains showing positive impact on the growth and physiology of C. deodara could be used for the propagation of this economically important plant in Pakistan after pathogenicity test.

L-Cysteine production by metabolically engineered Corynebacterium glutamicum.

L-Cysteine is a commercially important amino acid. Here, we report the construction of L-cysteine-producing Corynebacterium glutamicum using a metabolic engineering approach. L-Serine O-acetyltransferase (SAT), encoded by cysE gene, is a key enzyme of L-cysteine biosynthesis, because of its feedback inhibition by L-cysteine. Therefore, we introduced a mutation into the C. glutamicum cysE gene, which appeared to desensitize SAT against feedback inhibition by L-cysteine. We successfully produced L-cysteine by overexpressing this mutant cysE gene in C. glutamicum, while the wild-type strain scarcely produced L-cysteine. To enhance the biosynthesis of L-serine (a substrate for SAT), a mutant serA gene, encoding D-3-phosphoglycerate dehydrogenase to desensitize it against feedback inhibition by L-serine, was additionally overexpressed in the mutant cysE-overexpressing strain and its L-cysteine production was indeed improved. Moreover, we disrupted the ldh gene encoding L-lactate dehydrogenase and the aecD gene encoding cysteine desulfhydrase to prevent the formation of lactic acid as a by-product and degradation of L-cysteine produced at the stationary phase, respectively, which resulted in enhanced L-cysteine production. However, since the concentration of L-cysteine produced still decreased at the stationary phase despite the aecD disruption, NCgl2463 encoding a possible cystine importer protein was further disrupted to prevent cystine import, because the produced L-cysteine is immediately oxidized to cystine. As a result, the time before the start of the decrease in L-cysteine concentration was successfully prolonged. Approximately 200mg/L of L-cysteine production was achieved by overexpression of mutant cysE and serA genes and disruption of aecD and NCgl2463 genes in C. glutamicum.

In Salmonella enterica, OatA (Formerly YjgM) Uses O-Acetyl-Serine and Acetyl-CoA to Synthesize N,O-Diacetylserine, Which Upregulates Cysteine Biosynthesis.

L-Cysteine biosynthesis has been extensively analyzed in Salmonella enterica. The cysteine regulon contains the genes whose protein products are necessary to convert sulfate to sulfide, which is eventually reacted with O-acetyl-serine (OAS) to generate cysteine. The LysR type regulator, CysB, is required for activation of the cysteine regulon, and its interaction with various cys genes has been thoroughly characterized. Results from previous studies by others, suggested that OAS undergoes a spontaneous O- to N- migration to produce N-acetyl-serine (NAS), and that NAS is the true signal sensed by CysB. It was unclear, however, whether such migration occurred spontaneously in vivo or if NAS was generated enzymatically. Work reported herein characterizes a S. enterica N-acetyltransferase, OatA (formerly YjgM), which acetylates the Nα-amino group of OAS, producing N,O-diacetyl-serine (DAS) at the expense of acetyl-CoA. We isolated OatA to homogeneity and performed its initial biochemical characterization. The product of the OatA reaction was isolated by HPLC and confirmed by mass spectrometry to be DAS; OatA did not acetylate NAS, consistent with the conclusion that OatA is an N-acetyltransferase, not an O-acetyltransferase. Binding of OAS to OatA appears to be positively cooperative with the apparent K0.5 for OAS determined to be 0.74 mM, the kcat was 1.05 s-1, and the catalytic efficiency of the enzyme (kcat/K0.5) was 1.4 × 103 M-1 s-1. Size exclusion chromatography indicated that OatA was a monomer in solution. In S. enterica, overexpression of oatA led to shorter lag times on sulfate-limiting medium and that these delayed lag times were due to increased expression of the cysteine regulon, as indicated by RT-qPCR results. OatA is the first Gcn5-related N-acetyltransferase (aka GNAT) involved in the regulation of amino acid biosynthetic genes in Salmonella. On the basis of results of transcriptomics studies performed by other investigators, we hypothesize that DAS may play a role in biofilm formation in S. enterica and other bacteria.

The New Role of СysB Transcription Factor in Cysteine Degradation and Production of Hydrogen Sulfide in E. coli

The paradoxical effect of deletion of the Escherichia coli genes cysK and cysM encoding cysteine synthase enzymes has been studied: such cysteine auxotrophs actively degrade the excess of cysteine transported from the medium to form H2S. We have shown that deletions of any of the known genes controlling the degradation of exogenous cysteine, including the genes aspC, mstA, cysK, cysM, tnaA, metC, and malY, as well as the newly discovered genes yciW, cyuA, cyuP, and cyuR, do not deprive the cysteine auxotrophs ΔcysK ΔcysM of the ability to degrade cysteine. Cysteine degradation in the ΔcysK ΔcysM mutant is positively regulated by the products of the cysB and cysE genes. It is significant that the ΔcysK ΔcysM mutant shows an increased transcription of the genes opposing the oxidative stress (sodA, catG, arcA, and cydD). We assume that oxidative stress in cells of the ΔcysK ΔcysM mutant is provoked by restriction of cysteine resynthesis, while cysB-dependent degradation of exogenous cysteine and generated H2S provide protection against oxidative stress.

KRAS mutations and M2PK upregulation in stool samples from individuals with positive fecal occult blood tests screened for colorectal cancer.

Screening for colorectal cancer (CRC) requires non-invasive methods of high diagnostic sensitivity and specificity. We evaluated the measurement of genetic and protein biomarkers of CRC in stool samples with the aim of testing their clinical utility in a CRC screening program. Individuals aged 53-75 years who were at risk of CRC and immunochemical fecal occult blood test (iFOBT) positive were invited to submit stool samples for molecular testing prior to colonoscopy. KRAS codon 12 Gly→Asp, Gly, Val, and codon 13 Gly→Cys gene mutations were tested using an in-house real-time ARMS PCR method. M2PK levels in stool samples were measured utilizing a commercial ELISA kit. At colonoscopy, 7.6% of patients were found to have CRC, 50% had adenomas, 10.6% had hyperplastic polyps, 20.2% had diverticulosis and hemorrhoids, and 11.6% had normal mucosa. The best sensitivity for CRC (50%) was found in those cases where M2PK and KRAS abnormalities coexisted. M2PK showed a detection rate of 40.3% for adenomas but the combination of M2PK and KRAS abnormalities was found in only 5.7% of adenomas (P <0.01). iFOBT was false positive in 31.8% of cases in which colonoscopy excluded neoplastic lesions, while the coexistence of molecular and enzymatic abnormalities was more specific with false positive rates between 8.3% and 9.0% (P <0.05). Our molecular screening approach demonstrates that detection of cancer-associated biomarkers measured in iFOBT-positive stool samples could help separate true from false positives in a FOBT-based screening process. M2PK showed particular promise for the detection of CRC and adenomas.

Cysteine Biosynthesis Controls Serratia marcescens Phospholipase Activity.

Serratia marcescens causes health care-associated opportunistic infections that can be difficult to treat due to a high incidence of antibiotic resistance. One of the many secreted proteins of S. marcescens is the PhlA phospholipase enzyme. Genes involved in the production and secretion of PhlA were identified by screening a transposon insertion library for phospholipase-deficient mutants on phosphatidylcholine-containing medium. Mutations were identified in four genes (cyaA, crp, fliJ, and fliP) that are involved in the flagellum-dependent PhlA secretion pathway. An additional phospholipase-deficient isolate harbored a transposon insertion in the cysE gene encoding a predicted serine O-acetyltransferase required for cysteine biosynthesis. The cysE requirement for extracellular phospholipase activity was confirmed using a fluorogenic phospholipase substrate. Phospholipase activity was restored to the cysE mutant by the addition of exogenous l-cysteine or O-acetylserine to the culture medium and by genetic complementation. Additionally, phlA transcript levels were decreased 6-fold in bacteria lacking cysE and were restored with added cysteine, indicating a role for cysteine-dependent transcriptional regulation of S. marcescens phospholipase activity. S. marcescenscysE mutants also exhibited a defect in swarming motility that was correlated with reduced levels of flhD and fliA flagellar regulator gene transcription. Together, these findings suggest a model in which cysteine is required for the regulation of both extracellular phospholipase activity and surface motility in S. marcescensIMPORTANCESerratia marcescens is known to secrete multiple extracellular enzymes, but PhlA is unusual in that this protein is thought to be exported by the flagellar transport apparatus. In this study, we demonstrate that both extracellular phospholipase activity and flagellar function are dependent on the cysteine biosynthesis pathway. Furthermore, a disruption of cysteine biosynthesis results in decreased phlA and flagellar gene transcription, which can be restored by supplying bacteria with exogenous cysteine. These results identify a previously unrecognized role for CysE and cysteine in the secretion of S. marcescens phospholipase and in bacterial motility.

L-Cysteine Metabolism and Fermentation in Microorganisms.

L-Cysteine is an important amino acid both biologically and commercially. Although most amino acids are industrially produced by microbial fermentation, L-cysteine has been mainly produced by protein hydrolysis. Due to environmental and safety problems, synthetic or biotechnological products have been preferred in the market. Here, we reviewed L-cysteine metabolism, including biosynthesis, degradation, and transport, and biotechnological production (including both enzymatic and fermentation processes) of L-cysteine. The metabolic regulation of L-cysteine including novel sulfur metabolic pathways found in microorganisms is also discussed. Recent advancement in biochemical studies, genome sequencing, structural biology, and metabolome analysis has enabled us to use various approaches to achieve direct fermentation of L-cysteine from glucose. For example, worldwide companies began to supply L-cysteine and its derivatives produced by bacterial fermentation. These companies successfully optimized the original metabolism of their private strains. Basically, a combination of three factors should be required for improving L-cysteine fermentation: that is, (1) enhancing biosynthesis: overexpression of the altered cysE gene encoding feedback inhibition-insensitive L-serine O-acetyltransferase (SAT), (2) weakening degradation: knockout of the genes encoding L-cysteine desulfhydrases, and (3) exploiting export system: overexpression of the gene involved in L-cysteine transport. Moreover, we found that "thiosulfate" is much more effective sulfur source than commonly used "sulfate" for L-cysteine production in Escherichia coli, because thiosulfate is advantageous for saving consumption of NADPH and relating energy molecules.