Cysteine Biosynthesis Research Articles

Combatting antimicrobial resistance via the cysteine biosynthesis pathway in bacterial pathogens.

Antibiotics are the cornerstone of modern medicine and agriculture, and rising antibiotic resistance is one the biggest threats to global health and food security. Identifying new and different druggable targets for the development of new antibiotics is absolutely crucial to overcome resistance. Adjuvant strategies that either enhance the activity of existing antibiotics or improve clearance by the host immune system provide another mechanism to combat antibiotic resistance. Targeting a combination of essential and non-essential enzymes that play key roles in bacterial metabolism is a promising strategy to develop new antimicrobials and adjuvants, respectively. The enzymatic synthesis of L-cysteine is one such strategy. Cysteine plays a key role in proteins and is crucial for the synthesis of many biomolecules important for defense against the host immune system. Cysteine synthesis is a two-step process, catalyzed by two enzymes. Serine acetyltransferase (CysE) catalyzes the first step to synthesize the pathway intermediate O-acetylserine, and O-acetylserine sulfhydrylase (CysK/CysM) catalyzes the second step using sulfide or thiosulfate to produce cysteine. Disruption of the cysteine biosynthesis pathway results in dysregulated sulfur metabolism, altering the redox state of the cell leading to decreased fitness, enhanced susceptibility to oxidative stress and increased sensitivity to antibiotics. In this review, we summarize the structure and mechanism of characterized CysE and CysK/CysM enzymes from a variety of bacterial pathogens, and the evidence that support targeting these enzymes for the development of new antimicrobials or antibiotic adjuvants. In addition, we explore and compare compounds identified thus far that target these enzymes.

Investigation of the bactericidal mechanism of Penicilazaphilone C on Escherichia coli based on 4D label-free quantitative proteomic analysis

There is an urgent need to find new antibiotics to fight against the increasing drug resistance of microorganisms. A novel natural compound, Penicilazaphilone C (PAC), was isolated from a marine-derived fungus. It has displayed broad bactericidal activities against Gram-negative and Gram-positive bacteria. However, its bactericidal mechanism is still unknown. Herein, time-kill assays verified that PAC is a fast and efficient bactericidal agent. Furthermore, data from 4D label-free quantitative proteome assays revealed that PAC significantly influences over 898 proteins in Escherichia coli. Combining the results of biofilm formation, β-galactosidase measurement, TEM observation, soft agar plate swimming, reactive oxygen species measurement, qRT-PCR, and west-blotting, the mode of PAC action against E. coli was to block respiration, inhibit assimilatory nitrate reduction and dissimilar sulfur reduction, facilitate assimilatory sulfate reduction, suppress cysteine and methionine biosynthesis, down-regulate antioxidant protein expression and induced intracellular ROS accumulation, weaken bacterial chemotaxis, destroy flagellar assembly, etc., and finally cause the bacteria's death. Our findings suggest that PAC could have a multi-target regulatory effect on E. coli and could be used as a new antibiotic in medicine.

The coral Acropora loripes genome reveals an alternative pathway for cysteine biosynthesis in animals.

The metabolic capabilities of animals have been derived from well-studied model organisms and are generally considered to be well understood. In animals, cysteine is an important amino acid thought to be exclusively synthesized through the transsulfuration pathway. Corals of the genus Acropora have lost cystathionine β-synthase, a key enzyme of the transsulfuration pathway, and it was proposed that Acropora relies on the symbiosis with dinoflagellates of the family Symbiodiniaceae for the acquisition of cysteine. Here, we identify the existence of an alternative pathway for cysteine biosynthesis in animals through the analysis of the genome of the coral Acropora loripes. We demonstrate that these coral proteins are functional and synthesize cysteine in vivo, exhibiting previously unrecognized metabolic capabilities of animals. This pathway is also present in most animals but absent in mammals, arthropods, and nematodes, precisely the groups where most of the animal model organisms belong to, highlighting the risks of generalizing findings from model organisms.

Functional characterization of the Serine acetyltransferase family genes uncovers the diversification and conservation of cysteine biosynthesis in tomato.

Sulfur-containing compounds are essential for plant development and environmental adaptation, and closely related to the flavor and nutrition of the agricultural products. Cysteine, the first organic sulfur-containing molecule generated in plants, is the precursor for most of these active substances. Serine acetyltransferase (SERAT) catalyzes the rate-limiting step of its formation. However, despite their importance, systematic analyses of these enzymes in individual species, especially in economically important crops, are still limited. Here, The SERAT members (SlSERATs, four in total) were identified and characterized in tomato. Phylogenetically, the four SlSERAT proteins were classified into three subgroups with distinct genomic structures and subcellular localizations. On the function, it was interesting to find that SlSERAT3;1, possessed a high ability to catalyze the formation of OAS, even though it contained a long C-terminus. However, it retained the essential C-terminal Ile, which seems to be a characteristic feature of SERAT3 subfamily members in Solanaceae. Besides, SlSERAT1;1 and SlSERAT2;2 also had high activity levels and their catalyzing abilities were significantly improved by the addition of an OAS-(thiol)-lyase protein. At the transcriptional level, the four SlSERAT genes had distinct expression patterns during tomato plant development. Under abiotic stress conditions, the chloroplast-localized SlSERATs were the main responders, and the SlSERATs adopted different strategies to cope with osmotic, ion toxicity and other stresses. Finally, analyses in the loss-of-function and overexpression lines of SlSERAT1;1 suggested that function redundancy existed in the tomato SERAT members, and the tomato SERAT member was ideal target for S-assimilation manipulating in molecular breeding.

Loss of the Rhodobacter capsulatus Serine Acetyl Transferase Gene, cysE1, Impairs Gene Transfer by Gene Transfer Agents and Biofilm Phenotypes.

ABSTRACTBiofilms are widespread in the environment, where they allow bacterial species to survive adverse conditions. Cells in biofilms are densely packed, and this proximity is likely to increase the frequency of horizontal gene transfer. Gene transfer agents (GTAs) are domesticated viruses with the potential to spread any gene between bacteria. GTA production is normally restricted to a small subpopulation of bacteria, and regulation of GTA loci is highly coordinated, but the environmental conditions that favor GTA production are poorly understood. Here, we identified a serine acetyltransferase gene, cysE1, in Rhodobacter capsulatus that is required for optimal receipt of GTA DNA, accumulation of extracellular polysaccharide, and biofilm formation. The cysE1 gene is directly downstream of the core Rhodobacter-like GTA (RcGTA) structural gene cluster and upregulated in an RcGTA overproducer strain, although it is expressed on a separate transcript. The data we present suggest that GTA production and biofilm are coregulated, which could have important implications for the study of rapid bacterial evolution and understanding the full impact of GTAs in the environment.IMPORTANCE Direct exchange of genes between bacteria leads to rapid evolution and is the major factor underlying the spread of antibiotic resistance. Gene transfer agents (GTAs) are an unusual but understudied mechanism for genetic exchange that are capable of transferring any gene from one bacterium to another, and therefore, GTAs are likely to be important factors in genome plasticity in the environment. Despite the potential impact of GTAs, our knowledge of their regulation is incomplete. In this paper, we present evidence that elements of the cysteine biosynthesis pathway are involved in coregulation of various phenotypes required for optimal biofilm formation by Rhodobacter capsulatus and successful infection by the archetypal RcGTA. Establishing the regulatory mechanisms controlling GTA-mediated gene transfer is a key stepping stone to allow a full understanding of their role in the environment and wider impact.

Scaling-up and proteomic analysis reveals photosynthetic and metabolic insights toward prolonged H2 photoproduction in Chlamydomonas hpm91 mutant lacking proton gradient regulation 5 (PGR5)

Clean and sustainable H2 production is crucial to a carbon–neutral world. H2 generation by Chlamydomonas reinhardtii is an attractive approach for solar-H2 from H2O. However, it is currently not large-scalable because of lacking desirable strains with both optimal H2 productivity and sufficient knowledge of underlying molecular mechanism. We hereby carried out extensive and in-depth investigations of H2 photoproduction of hpm91 mutant lacking PGR5 (Proton Gradient Regulation 5) toward its up-scaling and fundamental mechanism issues. We show that hpm91 is at least 100-fold scalable (up to 10 L) with continuous H2 collection of 7287 ml H2/10L-HPBR in averagely 26 days under sulfur deprivation. Also, we show that hpm91 is robust and active during sustained H2 photoproduction, most likely due to decreased intracellular ROS relative to wild type. Moreover, we obtained quantitative proteomic profiles of wild type and hpm91 at four representing time points of H2 evolution, leading to 2229 and 1350 differentially expressed proteins, respectively. Compared to wild type, major proteome alterations of hpm91 include not only core subunits of photosystems and those related to anti-oxidative responses but also essential proteins in photosynthetic antenna, C/N metabolic balance, and sulfur assimilation toward both cysteine biosynthesis and sulfation of metabolites during sulfur-deprived H2 production. These results reveal not only new insights of cellular and molecular basis of enhanced H2 production in hpm91 but also provide additional candidate gene targets and modules for further genetic modifications and/or in artificial photosynthesis mimics toward basic and applied research aiming at advancing solar-H2 technology.

M6A regulator-based methylation modification patterns and characterization of tumor microenvironment in acute myeloid leukemia.

RNA N6-methyladenosine (m6A) is the most common and intensively studied RNA modification that critically regulates RNA metabolism, cell signaling, cell survival, and differentiation. However, the overall role of multiple m6A regulators in the tumor microenvironment (TME) has not yet been fully elucidated in acute myeloid leukemia (AML). In our study, we explored the genetic and transcriptional alterations of 23 m6A regulators in AML patients. Three distinct molecular subtypes were identified and associated with prognosis, patient clinicopathological features, as well as TME characteristics. The TME characterization revealed that m6A patterns were highly connected with metabolic pathways such as biosynthesis of unsaturated fatty acids, cysteine and methionine metabolism, and citrate cycle TCA cycle. Then, based on the differentially expressed genes (DEGs) related to m6A molecular subtypes, our study categorized the entire cohort into three m6A gene clusters. Furthermore, we constructed the m6Ascore for quantification of the m6A modification pattern of individual AML patients. It was found that the tumor-infiltrating lymphocyte cells (TILs) closely correlated with the three m6A clusters, three m6A gene clusters, and m6Ascore. And many biological processes were involved, including glycogen degradation, drug metabolism by cytochrome P450, pyruvate metabolism, and so on. Our comprehensive analysis of m6A regulators in AML demonstrated their potential roles in the clinicopathological features, prognosis, tumor microenvironment, and particularly metabolic pathways. These findings may improve our understanding of m6A regulators in AML and offer new perspectives on the assessment of prognosis and the development of anticancer strategy.

The ΔCysK2 mutant of Mycobacterium tuberculosis is sensitive to vancomycin associated with changes in cell wall phospholipid profile

Cysteine plays a versatile role in cellular physiology and has previously been shown to be instrumental to Mycobacterium tuberculosis (M.tb) pathophysiology. In this study, we have generated mutants deficient in CysK2 and CysH, the key Cysteine, biosynthetic enzymes. In contrast to the ΔcysH mutant, the ΔcysK2 mutant is not an auxotroph and as such not essential for cysteine biosynthesis. Interestingly, the ΔcysK2 mutant shows increased sensitivity to cumene hydroperoxide, vitamin C, diamide, rifampicin and Vancomycin and shows alterations in phospholipid profile of Mtb cell wall. Our findings suggest that alteration in phospholipids content of M.tb cell wall by CysK2 may form a mode of defence against selected antibiotics and oxidative stress.

H2S biogenesis by cystathionine beta-synthase: mechanism of inhibition by aminooxyacetic acid and unexpected role of serine

Cystathionine beta-synthase (CBS) is a pivotal enzyme of the transsulfuration pathway responsible for diverting homocysteine to the biosynthesis of cysteine and production of hydrogen sulfide (H2S). Aberrant upregulation of CBS and overproduction of H2S contribute to pathophysiology of several diseases including cancer and Down syndrome. Therefore, pharmacological CBS inhibition has emerged as a prospective therapeutic approach. Here, we characterized binding and inhibitory mechanism of aminooxyacetic acid (AOAA), the most commonly used CBS inhibitor. We found that AOAA binds CBS tighter than its respective substrates and forms a dead-end PLP-bound intermediate featuring an oxime bond. Surprisingly, serine, but not cysteine, replaced AOAA from CBS and formed an aminoacrylate reaction intermediate, which allowed for the continuation of the catalytic cycle. Indeed, serine rescued and essentially normalized the enzymatic activity of AOAA-inhibited CBS. Cellular studies confirmed that AOAA decreased H2S production and bioenergetics, while additional serine rescued CBS activity, H2S production and mitochondrial function. The crystal structure of AOAA-bound human CBS showed a lack of hydrogen bonding with residues G305 and Y308, found in the serine-bound model. Thus, AOAA-inhibited CBS could be reactivated by serine. This difference may be important in a cellular environment in multiple pathophysiological conditions and may modulate the CBS-inhibitory activity of AOAA. In addition, our results demonstrate additional complexities of using AOAA as a CBS-specific inhibitor of H2S biogenesis and point to the urgent need to develop a potent, selective and specific pharmacological CBS inhibitor.

Combined Analysis of BSA-Seq Based Mapping, RNA-Seq, and Metabolomic Unraveled Candidate Genes Associated with Panicle Grain Number in Rice (Oryza sativa L.).

Rice grain yield is a complex and highly variable quantitative trait consisting of several key components, including the grain weight, the effective panicles per unit area, and the grain number per panicle (GNPP). The GNPP is a significant contributor to grain yield controlled by multiple genes (QTL) and is crucial for improvement. Attempts have been made to find genes for this trait, which has always been a challenging and arduous task through conventional methods. We combined a BSA analysis, RNA profiling, and a metabolome analysis in the present study to identify new candidate genes involved in the GNPP. The F2 population from crossing R4233 (high GNPP) and Ce679 (low GNPP) revealed a frequency distribution fitting two segregated genes. Three pools, including low, middle, and high GNPP, were constructed and a BSA analysis revealed six candidate regions spanning 5.38 Mb, containing 739 annotated genes. Further, a conjunctive analysis of BSA-Seq and RNA-Seq showed 31 differentially expressed genes (DEGs) in the candidate intervals. Subsequently, a metabolome analysis showed 1024 metabolites, with 71 significantly enriched, including 44 up and 27 downregulated in Ce679 vs. R4233. A KEGG enrichment analysis of these 31 DEGs and 71 differentially enriched metabolites (DEMs) showed two genes, Os12g0102100 and Os01g0580500, significantly enriched in the metabolic pathways’ biosynthesis of secondary metabolites, cysteine and methionine metabolism, and fatty acid biosynthesis. Os12g0102100, which encodes for the alcohol dehydrogenase superfamily and a zinc-containing protein, is a novel gene whose contribution to the GNPP is not yet elucidated. This gene coding for mitochondrial trans-2-enoyl-CoA reductase is involved in the biosynthesis of myristic acid, also known as tetradecanoic acid. The Os01g0580500 coding for the enzyme 1-aminoclopropane-1-carboxylate oxidase (OsACO7) is responsible for the final step of the ethylene biosynthesis pathway through the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) into ethylene. Unlike Os12g0102100, this gene was significantly upregulated in R4233, downregulated in Ce679, and significantly enriched in two of the three metabolite pathways. This result pointed out that these two genes are responsible for the difference in the GNPP in the two cultivars, which has never been identified. Further validation studies may disclose the physiological mechanisms through which they regulate the GNPP in rice.

Transcriptome analysis reveals the genes involved in S-alk(en)ylcysteine sulfoxide biosynthesis and its biosynthetic location in postharvest chive (Allium schoenoprasum L.)

The biosynthetic mechanism of S-alk(en)ylcysteine sulfoxides (CSOs), a flavor precursor and nonvolatile medicinal compound in chive is still poorly understood. In the present study, transcriptomic analysis was used to investigate the biosynthetic mechanism of S-alk(en)ylcysteine sulfoxides (CSOs) in green leaves of postharvest chive stored under normal temperature (20 °C) for 5 d and low-temperature (3 °C) for 12 d. The de novo assembly of the transcriptome enabled the identification of unigenes involved in the sulfur assimilation and CSOs biosynthesis. The RNA-seq data showed that the unigenes related to sulfur assimilation were down-regulated during storage under 20 °C and 3 °C. The low temperature did not affect cysteine biosynthesis and the expression of γ-glutamyl transpeptidase (GGT) and flavin-containing monooxygenase (FMO) involved in CSOs biosynthesis; nonetheless, it prolonged CSOs synthesis by sustaining the chive quality during the storage period. The qPCR data revealed that the expressions of genes related to sulfur assimilation were mainly in the white stalk. In contrast, CSOs biosynthetic genes had higher expression levels in green leaf. The results indicate the CSOs were mainly synthesized in green leaf while cysteine, the primary substrate for CSOs synthesis, was from de novo synthesis and proteolysis. The study presents discrete evidence that CSOs biosynthesis in postharvest chives occurs in green leaves and is translocated to the white stalk for storage.

Impact of intracellular response regulator QseB in quorum sensing regulatory network in a clinical isolate SC1401 of Glaesserella parasuis

Two component systems (TCS) mediate specific responses to different conditions and/or pressures. In the quorum sensing Glaesserella parasuis (QSE) BC TCS, qseB, as a response regulator, is closely related to the transcriptional regulation of multiple downstream genes. In this study, the effects of qseB gene deletion, which encodes the response regulator of population density sensing in G. parasuis, were studied through biological characteristics and metabolomic analysis. Based on previous research, we further explored the virulence of ΔqseB mutant strains through cell morphology, adhesion and invasion. The ΔqseB mutant and parent strains were sequenced by metabolome and combined with the previous transcriptome sequencing results for joint analysis. This study aims to clarify the regulatory effect of QseB on the virulence of G. parasuis and lay the foundation for revealing the pathogenic mechanism of G. parasuis. We detected 476 different metabolites, of which 30 metabolites (6.3%) had a significant difference in abundance between SC1401 and ΔqseB (p < 0.05). We conducted a comparative analysis of pathway enrichment on the transcriptome and metabolome, and found that the two omics participate in seven metabolic pathways together. The top 10 KEGG pathways with the largest number of genes and metabolites identified in this experiment are ABC transporters, Biosynthesis of secondary metabolites, Cysteine and methionine metabolism, Purine metabolism, Pyrimidine metabolism, Metabolic pathways, and Nicotinate and nicotinamide metabolism. Analysis of metabolome sequencing results showed that differential metabolites were also enriched in metabolic pathways, such as Purine metabolism, cGMP-PKG signaling pathway and cAMP signaling pathway, which were not found in transcriptome sequencing data. The internal coloration of the mutant strain ΔqseB was uneven, and the adhesion and invasion ability of PAM cell lines were significantly reduced. We speculate that QseB may affect the adhesion and invasion ability of Glaesserella parasuis by influencing substance transport and signal transduction.

In silico screening, molecular dynamic simulations, and in vitro activity of selected natural compounds as an inhibitor of Leishmania donovani 3-mercaptopyruvate sulfurtransferase.

In Leishmania sp., the enzymes of de novo cysteine biosynthesis pathway require sulfide. Other organisms utilize sulfide through the sulfide reduction pathway, but Leishmania lacks the gene that encodes these enzymes. Hence, the major source of sulfide for Leishmania is believed to be from the action of 3-mercaptopyruvate sulfurtransferase (3MST) on 3-mercapto-pyruvate (3MP). There has been no effort reported in the past to screen inhibitors against L. donovani 3MST (Ld3MST). As a result, this study examines natural compounds that are potent against Ld3MST and validates it by in vitro activity and cytotoxicity tests. Initially, a library of ~ 5000 natural compounds was subjected to molecular docking approach for screening, and the best hit was validated using a long-term molecular dynamic simulation (MD). Among the docking results, quercetine-3-rutinoside (Rutin) was deemed the best hit. The results of the MD indicated that Rutin was highly capable of interacting with the varied active site segments, possibly blocking substrate access. Additionally, promastigotes and amastigotes were tested for Rutin activity and the IC50 was found to be 40.95 and 90.09 μM, respectively. Similarly, the cytotoxicity assay revealed that Rutin was not toxic even at a concentration of 819.00 μM to THP-1 cell lines. Additionally, the Ld3MST was cloned, purified, and evaluated for enzyme activity in the presence of Rutin. Reduction in the enzyme activity (~ 85%) was observed in the presence of ~ 40 μM Rutin. Thus, this study suggests that Rutin may act as a potent inhibitor of Ld3MST. With further in vivo investigations, Rutin could be a small molecule of choice for combating leishmaniasis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00436-022-07532-5.

Surviving in the Brine: A Multi-Omics Approach for Understanding the Physiology of the Halophile Fungus Aspergillus sydowii at Saturated NaCl Concentration.

Although various studies have investigated osmoadaptations of halophilic fungi to saline conditions, only few analyzed the fungal mechanisms occurring at saturated NaCl concentrations. Halophilic Aspergillus sydowii is a model organism for the study of molecular adaptations of filamentous fungi to hyperosmolarity. For the first time a multi-omics approach (i.e., transcriptomics and metabolomics) was used to compare A. sydowii at saturated concentration (5.13 M NaCl) to optimal salinity (1 M NaCl). Analysis revealed 1,842 genes differentially expressed of which 704 were overexpressed. Most differentially expressed genes were involved in metabolism and signal transduction. A gene ontology multi-scale network showed that ATP binding constituted the main network node with direct interactions to phosphorelay signal transduction, polysaccharide metabolism, and transferase activity. Free amino acids significantly decreased and amino acid metabolism was reprogrammed at 5.13 M NaCl. mRNA transcriptional analysis revealed upregulation of genes involved in methionine and cysteine biosynthesis at extreme water deprivation by NaCl. No modifications of membrane fatty acid composition occurred. Upregulated genes were involved in high-osmolarity glycerol signal transduction pathways, biosynthesis of β-1,3-glucans, and cross-membrane ion transporters. Downregulated genes were related to the synthesis of chitin, mannose, cell wall proteins, starvation, pheromone synthesis, and cell cycle. Non-coding RNAs represented the 20% of the total transcripts with 7% classified as long non-coding RNAs (lncRNAs). The 42% and 69% of the total lncRNAs and RNAs encoding transcription factors, respectively, were differentially expressed. A network analysis showed that differentially expressed lncRNAs and RNAs coding transcriptional factors were mainly related to the regulation of metabolic processes, protein phosphorylation, protein kinase activity, and plasma membrane composition. Metabolomic analyses revealed more complex and unknown metabolites at saturated NaCl concentration than at optimal salinity. This study is the first attempt to unravel the molecular ecology of an ascomycetous fungus at extreme water deprivation by NaCl (5.13 M). This work also represents a pioneer study to investigate the importance of lncRNAs and transcriptional factors in the transcriptomic response to high NaCl stress in halophilic fungi.

Transcriptome analysis reveals decreased accumulation and toxicity of Cd in upland rice inoculated with arbuscular mycorrhizal fungi

There have been a few reports on the impacts of arbuscular mycorrhizal fungi (AMF) on the expression of genes associated with the uptake, transport and detoxification of Cd in upland rice, but there is no study on the impacts of AMF on the transcriptome of upland rice grown in Cd-contaminated soil. In the present work, a pot experiment was conducted to study the influences of the AMF-Glomus versiforme (Gv) on the Cd accumulation, photosynthesis, antioxidant enzymes and transcriptome in upland rice grown in soils supplemented with 10 μg Cd g−1 soil. Gv symbiosis evidently promoted the plant growth, P acquisition and photosynthetic characteristics in upland rice. In addition, 229 differentially expressed genes (DEGs) were identified in upland rice root inoculated with Gv, of which 149 DEGs were upregulated and 80 DEGs were downregulated. More importantly, one Cd transporter gene Nramp5 was significantly downregulated, thus reducing Cd absorption, transfer and accumulation in mycorrhizal upland rice. Moreover, four genes in the Gene Ontology (GO) term of cysteine biosynthesis and two metallothionein genes were upregulated, which could have decreased Cd phytotoxicity in upland rice inoculated with Gv. Meanwhile, one peroxidase gene, two genes in the GO term of peroxisome and one gene in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of carotenoid biosynthesis were significantly upregulated, and enhanced the activities of catalase (CAT) and peroxidase (POD) in Gv-inoculated upland rice. Our results of transcriptome analysis initially elucidated the molecular mechanism by which AMF decreased the accumulation and toxicity of Cd in upland rice.

Microbiology and Epidemiology of Escherichia albertii-An Emerging Elusive Foodborne Pathogen.

Escherichia albertii, a close relative of E. coli, is an emerging zoonotic foodborne pathogen associated with watery diarrhea mainly in children and immunocompromised individuals. E. albertii was initially classified as eae-positive Hafnia alvei, however, as more genetic and biochemical information became available it was reassigned to its current novel taxonomy. Its infections are common under conditions of poor hygiene with confirmed transmission via contaminated water and food, mainly poultry-based products. This pathogen has been isolated from various domestic and wild animals, with most isolates being derived from birds, implying that birds among other wild animals might act as its reservoir. Due to the absence of standardized isolation and identification protocols, E. albertii can be misidentified as other Enterobacteriaceae. Exploiting phenotypes such as its inability to ferment rhamnose and xylose and PCR assays targeting E. albertii-specific genes such as the cytolethal distending toxin and the DNA-binding transcriptional activator of cysteine biosynthesis encoding genes can be used to accurately identify this pathogen. Several gaps exist in our knowledge of E. albertii and need to be bridged. A deeper understanding of E. albertii epidemiology and physiology is required to allow the development of effective measures to control its transmission and infections. Overall, current data suggest that E. albertii might play a more significant role in global infectious diarrhea cases than previously assumed and is often overlooked or misidentified. Therefore, simple, and efficient diagnostic tools that cover E. albertii biodiversity are required for effective isolation and identification of this elusive agent of diarrhea.

Transcriptomic Analysis of E. coli after Exposure to a Sublethal Concentration of Hydrogen Peroxide Revealed a Coordinated Up-Regulation of the Cysteine Biosynthesis Pathway.

Hydrogen peroxide (H2O2) is a key defense component of host-microbe interaction. However, H2O2 concentrations generated by immune cells or epithelia are usually insufficient for bacterial killing and rather modulate bacterial responses. Here, we investigated the impact of sublethal H2O2 concentration on gene expression of E. coli BW25113 after 10 and 60 min of exposure. RNA-seq analysis revealed that approximately 12% of bacterial genes were strongly dysregulated 10 min following exposure to 2.5 mM H2O2. H2O2 exposure led to the activation of a specific antioxidant response and a general stress response. The latter was characterized by a transient down-regulation of genes involved in general metabolism, such as nucleic acid biosynthesis and translation, with a striking and coordinated down-regulation of genes involved in ribosome formation, and a sustained up-regulation of the SOS response. We confirmed the rapid transient and specific response mediated by the transcription factor OxyR leading to up-regulation of antioxidant systems, including the catalase-encoding gene (katG), that rapidly degrade extracellular H2O2 and promote bacterial survival. We documented a strong and transient up-regulation of genes involved in sulfur metabolism and cysteine biosynthesis, which are under the control of the transcription factor CysB. This strong specific transcriptional response to H2O2 exposure had no apparent impact on bacterial survival, but possibly replenishes the stores of oxidized cysteine and glutathione. In summary, our results demonstrate that different stress response mechanisms are activated by H2O2 exposure and highlight the cysteine synthesis as an antioxidant response in E. coli.

Cysteine dependence of Lactobacillus iners is a potential therapeutic target for vaginal microbiota modulation.

Vaginal microbiota composition affects many facets of reproductive health. Lactobacillus iners-dominated microbial communities are associated with poorer outcomes, including higher risk of bacterial vaginosis (BV), compared with vaginal microbiota rich in L. crispatus. Unfortunately, standard-of-care metronidazole therapy for BV typically results in dominance of L. iners, probably contributing to post-treatment relapse. Here we generate an L. iners isolate collection comprising 34 previously unreported isolates from 14 South African women with and without BV and 4 previously unreported isolates from 3 US women. We also report an associated genome catalogue comprising 1,218 vaginal Lactobacillus isolate genomes and metagenome-assembled genomes from >300 women across 4 continents. We show that, unlike L. crispatus, L. iners growth is dependent on L-cysteine in vitro and we trace this phenotype to the absence of canonical cysteine biosynthesis pathways and a restricted repertoire of cysteine-related transport mechanisms. We further show that cysteine concentrations in cervicovaginal lavage samples correlate with Lactobacillus abundance in vivo and that cystine uptake inhibitors selectively inhibit L. iners growth in vitro. Combining an inhibitor with metronidazole promotes L. crispatus dominance of defined BV-like communities in vitro by suppressing L. iners growth. Our findings enable a better understanding of L. iners biology and suggest candidate treatments to modulate the vaginal microbiota to improve reproductive health for women globally.

Construction and optimization of ergothioneine-producing Escherichia coli

Ergothioneine (ERG) is a natural antioxidant that has been widely used in the fields of food, medicine and cosmetics. Compared with traditional plant extraction and chemical synthesis approaches, microbial synthesis of ergothioneine has many advantages, such as the short production cycle and low cost, and thus has attracted intensive attention. In order to engineer an ergothioneine high-yielding Escherichia coli strain, the ergothioneine synthesis gene cluster egtABCDE from Mycobacterium smegmatis and egt1 from Schizosaccharomyces pombe were introduced into E. coli BL21(DE3) to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway. As a result, (95.58±3.2) mg/L ergothioneine was produced in flask cultures. To further increase ergothioneine yield, the relevant enzymes for biosynthesis of histidine, methionine, and cysteine, the three precursor amino acids of ergothioneine, were overexpressed. Individual overexpression of serAT410STOP and thrA resulted in an ergothioneine titer of (134.83±4.22) mg/L and (130.26±3.34) mg/L, respectively, while co-overexpression of serAT410STOP and thrA increased the production of ergothioneine to (144.97±5.40) mg/L. Eventually, by adopting a fed-batch fermentation strategy in 3 L fermenter, the optimized strain E1-A1-thrA-serA* produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium, respectively.

Cysteine supplementation enhanced inhibitor tolerance of Zymomonas mobilis for economic lignocellulosic bioethanol production

Inhibitors in lignocellulosic hydrolysates are toxic to Zymomonas mobilis and reduce its bioethanol production. This study revealed cysteine supplementation enhanced furfural tolerance in Z. mobilis with a 2-fold biomass increase. Transcriptomic study illustrated that cysteine biosynthesis pathway was down-regulated while cysteine catabolism was up-regulated with cysteine supplementation. Mutants for genes involved in cysteine metabolism were constructed, and metabolites in cysteine metabolic pathway including methionine, glutathione, NaHS, glutamate, and pyruvate were supplemented into media. Cysteine supplementation boosted glutathione synthesis or H2S release effectively in Z. mobilis leading to the reduced accumulation of reactive oxygen species (ROS) induced by furfural, while pyruvate and glutamate produced in the H2S generation pathway promoted cell growth by serving as the carbon or nitrogen source. Finally, cysteine supplementation was confirmed to enhance Z. mobilis tolerance against ethanol, acetate, and corncob hydrolysate with an enhanced ethanol productivity from 0.38 to 0.55 g−1∙L−1∙h−1.