The Identified Hub Gene GlcN in Osteoarthritis Progression and Treatment.

BACKGROUND: As a chronic disease, osteoarthritis has caused great trouble to the health of middle-aged and elderly people. Studies have shown that glucosamine (GlcN) can be used to abate the progression and improve this disease. Based on this point of view, we try to verify the connection between GlcN and osteoarthritis and find more effective biomarkers. METHODS: We downloaded the GSE72575 data set from the GEO database, and used the R language to perform DEG analysis on the gene expression profile of the samples. Next, the GO function and the KEGG signaling pathways were analyzed through the DAVID database, and then, the KEGG pathways enriched in the gene set were analyzed based on GSEA. Then, the PPI network of DEGs was constructed based on the STRING online database, and finally, the hub genes were selected by Cytoscape. RESULTS: Three GlcN-treated MH7A cell treatment groups and 3 control groups in the GSE72575 data set were studied. Through analysis, there were 52 DEGs in these samples. Then, through GO, KEGG, and GSEA, regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway, FoxO signaling pathway, JAK-STAT signaling pathway, PI3K-Akt signaling pathway, TGF-beta signaling pathway, and ECM receptor interaction were involved in the regulatory mechanisms of the osteoarthritis pathogenesis. After that, the hub genes IL6 and DDIT3 were identified through PPI network construction and analysis. And it was found that IL6 was lowly expressed in the group with GlcN-treated MH7A cells, while DDIT3 was highly expressed. CONCLUSION: The above results provide a basis for GlcN to participate in the treatment of osteoarthritis and a possibility for finding effective therapeutic targets.

A unique cell wall synthetic response evoked by glucosamine determines pathogenicity-associated fungal cellular differentiation.

The yeast-to-hypha transition is tightly associated with pathogenicity in many human pathogenic fungi, such as the model fungal pathogen Cryptococcus neoformans, which is responsible for approximately 180,000 deaths annually. In this pathogen, the yeast-to-hypha transition can be initiated by distinct stimuli: mating stimulation or glucosamine (GlcN), the monomer of cell wall chitosan. However, it remains poorly understood how the signal specificity for Cryptococcus morphological transition by disparate stimuli is ensured. Here, by integrating temporal expression signature analysis and phenome-based clustering evaluation, we demonstrate that GlcN specifically triggers a unique cellular response, which acts as a critical determinant underlying the activation of GlcN-induced filamentation (GIF). This cellular response is defined by an unusually hyperactive cell wall synthesis that is highly ATP-consuming. A novel cell surface protein Gis1 was identified as the indicator molecule for the GlcN-induced cell wall response. The Mpk1-directed cell wall pathway critically bridges global cell wall gene induction and intracellular ATP supply, ensuring the Gis1-dependent cell wall response and the stimulus specificity of GIF. We further reveal that the ability of Mpk1 to coordinate the cell wall response and GIF activation is conserved in different Cryptococcus pathogens. Phosphoproteomics-based profiling together with genetic and phenotypic analysis revealed that the Mpk1 kinase mediates the regulatory specificity of GIF through a coordinated downstream regulatory network centered on Skn7 and Crz1. Overall, our findings discover an unprecedented and conserved cell wall biosynthesis-dependent fungal differentiation commitment mechanism, which enables the signal specificity of pathogenicity-related dimorphism induced by GlcN in Cryptococcus pathogens.

Redundant targeting of Isr1 by two CDKs in mitotic cells.

Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, integrating a variety of environmental signals to drive cellular growth. Isr1 is a negative regulator of the hexosamine biosynthesis pathway (HBP), which produces UDP-GlcNAc, an essential carbohydrate that is the building block of N-glycosylation, GPI anchors and chitin. Isr1 was recently shown to be regulated by phosphorylation by the nutrient-responsive CDK kinase Pho85, allowing it to be targeted for degradation by the SCFCDC4. Here, we show that while deletion of PHO85 stabilizes Isr1 in asynchronous cells, Isr1 is still unstable in mitotically arrested cells in a pho85∆ strain. We provide evidence to suggest that this is through phosphorylation by CDK1. Redundant targeting of Isr1 by two distinct kinases may allow for tight regulation of the HBP in response to different cellular signals.

Genome analysis of 1-deoxynojirimycin (1-DNJ)-producing Bacillus velezensis K26 and distribution of Bacillus sp. harboring a 1-DNJ biosynthetic gene cluster.

1-Deoxynojirumycin (1-DNJ) is a representative iminosugar with α-glucosidase inhibition (AGI) activity. In this study, the full genome sequencing of 1-DNJ-producing Bacillus velezensis K26 was performed. The genome consists of a circular chromosome (4,047,350 bps) with two types of putative virulence factors, five antibiotic resistance genes, and seven secondary metabolite biosynthetic gene clusters. Genomic analysis of a wide range of Bacillus species revealed that a 1-DNJ biosynthetic gene cluster was commonly present in four Bacillus species (B. velezensis, B. pseudomycoides, B. amyloliquefaciens, and B. atrophaeus). In vitro experiments revealed that the increased mRNA expression levels of the three 1-DNJ biosynthetic genes were closely related to increased AGI activity. Genomic comparison and alignment of multiple gene sequences indicated the conservation of the 1-DNJ biosynthetic gene cluster in each Bacillus species. This genomic analysis of Bacillus species having a 1-DNJ biosynthetic gene cluster could provide a basis for further research on 1-DNJ-producing bacteria.

Glucosamine regulates hepatic lipid accumulation by sensing glucose levels or feeding states of normal and excess.

Dose-dependent lipid accumulation was induced by glucose in HepG2 cells. GlcN also exerted a promotory effect on lipid accumulation in HepG2 cells under normal glucose conditions (NG, 5 mM) and liver of normal fed zebrafish larvae. High glucose (HG, 25 mM)-induced lipid accumulation was suppressed by l-glutamine-d-fructose 6-phosphate amidotransferase inhibitors. ER stress inhibitors did not suppress HG or GlcN-mediated lipid accumulation. HG and GlcN stimulated protein expression, DNA binding and O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP). Furthermore, both HG and GlcN increased nuclear sterol regulatory element-binding protein-1 (SREBP-1) levels in HepG2 cells. In contrast to its stimulatory effect under NG, GlcN suppressed lipid accumulation in HepG2 cells under HG conditions. Similarly, GlcN suppressed lipid accumulation in livers of overfed zebrafish. In addition, GlcN activity on DNA binding and O-GlcNAcylation of ChREBP was stimulatory under NG and inhibitory under HG conditions. Moreover, GlcN enhanced ChREBP, SREBP-1c, ACC, FAS, L-PK and SCD-1 mRNA expression under NG but inhibited HG-induced upregulation in HepG2 cells. The O-GlcNAc transferase inhibitor, alloxan, reduced lipid accumulation by HG or GlcN while the O-GlcNAcase inhibitor, PUGNAc, enhanced lipid accumulation in HepG2 cells and liver of zebrafish larvae. GlcN-induced lipid accumulation was inhibited by the AMPK activator, AICAR. Phosphorylation of AMPK (p-AMPK) was suppressed by GlcN under NG while increased by GlcN under HG. PUGNAc downregulated p-AMPK while alloxan restored GlcN- or HG-induced p-AMPK inhibition. Our results collectively suggest that GlcN regulates lipogenesis by sensing the glucose or energy states of normal and excess fuel through AMPK modulation.

Enhancement of Production of D-Glucosamine in Escherichia coli by Blocking Three Pathways Involved in the Consumption of GlcN and GlcNAc.

D-Glucosamine is a commonly used dietary supplement that promotes cartilage health in humans. Metabolic flux analysis showed that D-glucosamine production could be increased by blocking three pathways involved in the consumption of glucosamine-6-phosphate and acetylglucosamine-6-phosphate. By homologous single-exchange, two key genes (nanE and murQ) of Escherichia coli BL21 were knocked out, respectively. The D-glucosamine yields of the engineered strains E. coli BL21ΔmurQ and E. coli BL21ΔnanE represented increases by factors of 2.14 and 1.79, respectively. Meanwhile, for bifunctional gene glmU, we only knocked out its glucosamine-1-phosphate acetyltransferase domain by 3D structural analysis to keep the engineered strain E. coli BL21glmU-Δgpa survival, which resulted in an increase in the production of D-glucosamine by a factor of 2.16. Moreover, for further increasing D-glucosamine production, two genes encoding rate-limiting enzymes, named glmS and gna1, were coexpressed by an RBS sequence in those engineered strains. The total concentrations of D-glucosamine in E. coli BL21 glmU-Δgpa', E. coli BL21ΔmurQ', and E. coli BL21ΔnanE' were 2.65 g/L, 1.73 g/L, and 1.38 g/L, which represented increases by factors of 8.83, 5.76, and 3.3, respectively.

Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis.

Dynamic regulation is an effective strategy for fine-tuning metabolic pathways in order to maximize target product synthesis. However, achieving dynamic and autonomous up- and down-regulation of the metabolic modules of interest simultaneously, still remains a great challenge. In this work, we created an autonomous dual-control (ADC) system, by combining CRISPRi-based NOT gates with novel biosensors of a key metabolite in the pathway of interest. By sensing the levels of the intermediate glucosamine-6-phosphate (GlcN6P) and self-adjusting the expression levels of the target genes accordingly with the GlcN6P biosensor and ADC system enabled feedback circuits, the metabolic flux towards the production of the high value nutraceutical N-acetylglucosamine (GlcNAc) could be balanced and optimized in Bacillus subtilis. As a result, the GlcNAc titer in a 15-l fed-batch bioreactor increased from 59.9 g/l to 97.1 g/l with acetoin production and 81.7 g/l to 131.6 g/l without acetoin production, indicating the robustness and stability of the synthetic circuits in a large bioreactor system. Remarkably, this self-regulatory methodology does not require any external level of control such as the use of inducer molecules or switching fermentation/environmental conditions. Moreover, the proposed programmable genetic circuits may be expanded to engineer other microbial cells and metabolic pathways.

Skp1 isoforms are differentially modified by a dual function prolyl 4-hydroxylase/N-acety lglucosaminyltransferase in a plant pathogen.

Skp1 is hydroxylated by an O2-dependent prolyl hydroxylase (PhyA) that contributes to O2-sensing in the social amoeba Dictyostelium and the mammalian pathogen Toxoplasma gondii. HO-Skp1 is subject to glycosylation and the resulting pentasaccharide affects Skp1 conformation in a way that influences association of Skp1 with F-box proteins, and potentially the assembly of E3(SCF) ubiquitin ligase complexes that mediate the polyubiquitination of target proteins that are degraded in the 26S-proteasome. To investigate the conservation and specificity of these modifications, we analyzed proteins from the oomycete Pythium ultimum, an important crop plant pathogen. Putative coding sequences for Pythium's predicted PhyA and first glycosyltransferase in the predicted five-enzyme pathway, a GlcNAc-transferase (Gnt1), predict a bifunctional enzyme (Phgt) that, when expressed in Dictyostelium, rescued a knockout of phyA but not gnt1. Though recombinant Phgt was also unable to glycosylate Dictyostelium HO-Skp1, it could hydrolyze UDP-GlcNAc and modify a synthetic hydroxypeptide from Dictyostelium Skp1. Pythium encodes two highly similar Skp1 isoforms, but only Skp1A was efficiently hydroxylated and glycosylated in vitro. While kinetic analysis revealed no evidence for processive processing of Skp1, the physical linkage of the two activities implies dedication to Skp1 in vivo. These findings indicate a widespread occurrence of the Skp1 modification pathway across protist phylogeny, suggest that both Gnt1 and PhyA are specific for Skp1 and indicate that the second Skp1 provides a bypass mechanism for O2-regulation in Pythium and other protists that conserve this gene.

Feedback regulation of small RNA processing by the cleavage product.

Many bacterial small RNAs (sRNAs) are processed resulting in variants with roles potentially distinct from the primary sRNAs. In Enterobacteriaceae sRNA GlmZ activates expression of glmS by base-pairing when the levels of glucosamine-6-phosphate (GlcN6P) are low. GlmS synthesizes GlcN6P, which is required for cell envelope biosynthesis. When dispensable, GlmZ is cleaved by RNase E in the base-pairing sequence. Processing requires protein RapZ, which binds GlmZ and recruits RNase E by interaction. Cleavage is counteracted by the homologous sRNA GlmY, which accumulates upon GlcN6P scarcity and sequesters RapZ. Here, we report a novel role for a processed sRNA. We observed that processing of GlmZ is never complete in vivo. Even upon RapZ overproduction, a fraction of GlmZ remains full-length, while the 5' cleavage product (GlmZ*) accumulates. GlmZ* retains all elements required for RapZ binding. Accordingly, GlmZ* can displace full-length GlmZ from RapZ and counteract processing in vitro. To mimic GlmZ* in vivo, sRNA chimeras were employed consisting of foreign 3' ends including a terminator fused to the 3' end of GlmZ*. In vitro, these chimeras perform indistinguishable from GlmZ*. Expression of the chimeras in vivo inhibited processing of endogenous GlmZ, causing moderate upregulation of GlmS synthesis. Hence, accumulation of GlmZ* prevents complete GlmZ turnover. This mechanism may serve to adjust a robust glmS basal expression level that is buffered against fluctuations in RapZ availability.

Combinatorial Fine-Tuning of GNA1 and GlmS Expression by 5'-Terminus Fusion Engineering Leads to Overproduction of N-Acetylglucosamine in Bacillus subtilis.

Glucosamine-6-phosphate N-acetyltransferase (GNA1) that catalyzes acetyl transfer from acetyl-coenzyme A to glucosamine-6-phosphate (GlcN-6P), and glutamine-fructose-6-phosphate aminotransferase (GlmS) that catalyzes the formation of GlcN-6P from fructose-6-phosphate (Fru-6P), are two key enzymes in Bacillus subtilis for the bioproduction of N-acetylglucosamine (GlcNAc), a nutraceutical that has various applications in healthcare. In this study, the expression of GNA1 and GlmS is fine-tuned by 5'-terminus fusion engineering to improve GlcNAc production. Specifically, the expression level of GNA1 is enhanced at the translational level via fusion of an epitope tag to the 5'-terminus of GNA1 gene and ribosome binding site (RBS) sequence engineering. Next, enhanced expression of GlmS is achieved at the transcriptional and translational levels by fusing an mRNA stabilizer to the 5'-terminus of GlmS gene. Under the control of GNA1 (fusion with cMyc tag and with the optimum RBS M-Rm) and GlmS (fusion with mRNA stabilizer ΔermC+14/7A), the GlcNAc titer and yield in the shake flask increase to 18.5 g L-1 and 0.37 g GlcNAc/g glucose, which are 2.9-fold and 2.3-fold that of the control, respectively. This synthetic pathway fine-tuning method at the transcriptional and translational levels by combinatorial modulation of regulatory elements, including epitope tag, RBS sequence, and mRNA stabilizer, might represent a general and effective approach for the construction of microbial cell factories.

The aldehyde dehydrogenase AldA contributes to the hypochlorite defense and is redox-controlled by protein S-bacillithiolation in Staphylococcus aureus.

Staphylococcus aureus produces bacillithiol (BSH) as major low molecular weight (LMW) thiol which functions in thiol-protection and redox-regulation by protein S-bacillithiolation under hypochlorite stress. The aldehyde dehydrogenase AldA was identified as S-bacillithiolated at its active site Cys279 under NaOCl stress in S. aureus. Here, we have studied the expression, function, redox regulation and structural changes of AldA of S. aureus. Transcription of aldA was previously shown to be regulated by the alternative sigma factor SigmaB. Northern blot analysis revealed SigmaB-independent induction of aldA transcription under formaldehyde, methylglyoxal, diamide and NaOCl stress. Deletion of aldA resulted in a NaOCl-sensitive phenotype in survival assays, suggesting an important role of AldA in the NaOCl stress defense. Purified AldA showed broad substrate specificity for oxidation of several aldehydes, including formaldehyde, methylglyoxal, acetaldehyde and glycol aldehyde. Thus, AldA could be involved in detoxification of aldehyde substrates that are elevated under NaOCl stress. Kinetic activity assays revealed that AldA is irreversibly inhibited under H2O2 treatment in vitro due to overoxidation of Cys279 in the absence of BSH. Pre-treatment of AldA with BSH prior to H2O2 exposure resulted in reversible AldA inactivation due to S-bacillithiolation as revealed by activity assays and BSH-specific Western blot analysis. Using molecular docking and molecular dynamic simulation, we further show that BSH occupies two different positions in the AldA active site depending on the AldA activation state. In conclusion, we show here that AldA is an important target for S-bacillithiolation in S. aureus that is up-regulated under NaOCl stress and functions in protection under hypochlorite stress.

Real-Time Imaging of the Bacillithiol Redox Potential in the Human Pathogen Staphylococcus aureus Using a Genetically Encoded Bacilliredoxin-Fused Redox Biosensor.

AIMS: Bacillithiol (BSH) is utilized as a major thiol-redox buffer in the human pathogen Staphylococcus aureus. Under oxidative stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolation, which can be reversed by bacilliredoxins (Brx). In eukaryotes, glutaredoxin-fused roGFP2 biosensors have been applied for dynamic live imaging of the glutathione redox potential. Here, we have constructed a genetically encoded bacilliredoxin-fused redox biosensor (Brx-roGFP2) to monitor dynamic changes in the BSH redox potential in S. aureus. RESULTS: The Brx-roGFP2 biosensor showed a specific and rapid response to low levels of bacillithiol disulfide (BSSB) in vitro that required the active-site Cys of Brx. Dynamic live imaging in two methicillin-resistant S. aureus (MRSA) USA300 and COL strains revealed fast and dynamic responses of the Brx-roGFP2 biosensor under hypochlorite and hydrogen peroxide (H2O2) stress and constitutive oxidation of the probe in different BSH-deficient mutants. Furthermore, we found that the Brx-roGFP2 expression level and the dynamic range are higher in S. aureus COL compared with the USA300 strain. In phagocytosis assays with THP-1 macrophages, the biosensor was 87% oxidized in S. aureus COL. However, no changes in the BSH redox potential were measured after treatment with different antibiotics classes, indicating that antibiotics do not cause oxidative stress in S. aureus. Conclusion and Innovation: This Brx-roGFP2 biosensor catalyzes specific equilibration between the BSH and roGFP2 redox couples and can be applied for dynamic live imaging of redox changes in S. aureus and other BSH-producing Firmicutes. Antioxid. Redox Signal. 26, 835-848.

In vitro evolution of coenzyme-independent variants from the glmS ribozyme structural scaffold.

Uniquely among known natural ribozymes that cleave RNA sequence-specifically, the glmS ribozyme-riboswitch employs a small molecule, glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. In vitro selection was employed to search for coenzyme-independent variants of this ribozyme. In addition to shedding light on the catalytic mechanism of the ribozyme, such variants could resemble the evolutionary ancestors of the modern, GlcN6P-regulated ribozyme-riboswitch. A mutant pool was constructed such that the secondary structure elements, which define the triply-pseudoknotted global fold of the ribozyme, was preserved. A stringent selection scheme that relies on thiol-mercury affinity chromatography for separating active and inactive sequences ultimately yielded a triple mutant with a cleavage rate exceeding 3min(-1) that only requires divalent cations for activity. Mutational analysis demonstrated that a point reversion of the variant toward the wild-type sequence was sufficient to partially restore GlcN6P-dependence, suggesting that coenzyme dependence can be readily be acquired by RNAs that adopt the glmS ribozyme fold. The methods employed to perform this selection experiment are described in detail in this review.

Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress.

Bacillus methanolicus MGA3 is a model facultative methylotroph of interest for fundamental research and biotechnological applications. Previous research uncovered a number of pathways potentially involved in one-carbon substrate utilization. Here, we applied dynamic (13) C labeling to elucidate which of these pathways operate during growth on methanol and to uncover potentially new ones. B. methanolicus MGA3 uses the assimilatory and dissimilatory ribulose monophosphate (RuMP) cycles for conversion of the central but toxic intermediate formaldehyde. Additionally, the operation of two cofactor-dependent formaldehyde oxidation pathways with distinct roles was revealed. One is dependent on tri- and tetraglutamylated tetrahydrofolate (THF) and is involved in formaldehyde oxidation during growth on methanol. A second pathway was discovered that is dependent on bacillithiol, a thiol cofactor present also in other Bacilli where it is known to function in redox-homeostasis. We show that bacillithiol-dependent formaldehyde oxidation is activated upon an upshift in formaldehyde induced by a substrate switch from mannitol to methanol. The genes and the corresponding enzymes involved in the biosynthesis of bacillithiol were identified by heterologous production of bacillithiol in Escherichia coli. The presented results indicate metabolic plasticity of the methylotroph allowing acclimation to fluctuating intracellular formaldehyde concentrations.

Inhibition of PKC-Induced COX-2 and IL-8 Expression in Human Breast Cancer Cells by Glucosamine.

Breast cancer is a common cancer leading to many deaths among females. Cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) are two highly expressed inflammatory mediators to be induced by the protein kinase C (PKC) signaling via various inflammatory stimuli and both contribute significantly to cancer metastasis/progression. Glucosamine has been shown to act as an anti-inflammation molecule. The aim of this study was to clarify the role and acting mechanism of glucosamine during the PKC-regulation of COX-2/IL-8 expression and the associated impact on breast cancer. In MCF-7 breast cancer cells, glucosamine effectively suppresses the PKC induction of COX-2 and IL-8 promoter activity, mRNA and protein levels, as well as the production of prostaglandin E(2) (PGE(2)) and IL-8. Glucosamine is able to promote COX-2 protein degradation in a calpain-dependent manner and IL-8 protein degradation in calpain-dependent and proteasome-dependent manners. The MAPK and NF-κB pathways are involved in PKC-induced COX-2 expression, but only the NF-κB pathway is involved in PKC-induced IL-8 expression. Glucosamine attenuates PKC-mediated IκBα phosphorylation, nuclear NF-κB translocation, and NF-κB reporter activation. Both PGE(2) and IL-8 promote cell proliferation and IL-8 induces cell migration; thus, glucosamine appears to suppress PKC-induced cell proliferation and migration. Furthermore, glucosamine significantly inhibits the growth of breast cancer xenografts and this is accompanied by a reduction in COX-2 and IL-8 expression. In conclusion, glucosamine seems to attenuate the inflammatory response in vitro and in vivo and this occurs, at least in part by targeting to the NF-κB signaling pathway, resulting in an inhibition of breast cancer cell growth.

Basal transcription is regulated by lipopolysaccharide and glucosamine via the regulation of DNA binding of RNA polymerase II in RAW264.7 cells.

AIMS: The objective of this study is to investigate glucosamine (GlcN) as a transcriptional regulator of iNOS and other genes in association with the dynamic O-GlcNAcylation of RNA polymerase II (RNAPII). MAIN METHODS: The LPS- and/or GlcN-stimulated transcriptional activities of various Gal4-binding site/TATA-box-containing reporter constructs were measured. KEY FINDINGS: Basal transcriptional activities of nuclear factor-κB (NF-κB) and nitric oxide synthase (iNOS) reporter plasmids are inhibited by GlcN in RAW264.7 cells. Furthermore, GlcN suppressed whereas lipopolysaccharide (LPS) stimulated the basal activity of Gal4-binding site/TATA-box-containing reporter constructs. LPS reduced the O-linked N-acetylglucosamine modification (O-GlcNAcylation) of RNAPII, but enhanced the binding of this enzyme to the iNOS promoter. In contrast, GlcN enhanced RNAPII O-GlcNAcylation, but inhibited iNOS promoter binding. Furthermore, the basal activities of reporter plasmids containing activator protein 1 (AP1), E2F, or cyclic AMP response element (CRE) binding sites were consistently inhibited by GlcN in a dose-dependent manner. However, GlcN did not inhibit the phorbol 12-myristate 13-acetate- (PMA-) or forskolin-induced transcriptional activities of AP1 and CRE. The transcriptional activity of transforming growth factor alpha (TGF-α) was slightly increased by both LPS and GlcN. SIGNIFICANCE: In conclusion, our data demonstrate that LPS activates, whereas GlcN suppresses, basal activities of transcription through the regulation of RNAPII O-GlcNAcylation and DNA binding.

Importance of bacillithiol in the oxidative stress response of Staphylococcus aureus.

In Staphylococcus aureus, the low-molecular-weight thiol called bacillithiol (BSH), together with cognate S-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH in S. aureus, we constructed mutants with the deletion of bshA (sa1291), which encodes the glycosyltransferase that catalyzes the first step of BSH biosynthesis, and fosB (sa2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in several S. aureus strains, including clinical isolates. Mutation of fosB or bshA caused a 16- to 60-fold reduction in fosfomycin resistance in these S. aureus strains. High-pressure liquid chromatography analysis, which quantified thiol extracts, revealed some variability in the amounts of BSH present across S. aureus strains. Deletion of fosB led to a decrease in BSH levels. The fosB and bshA mutants of strain COL and a USA300 isolate, upon further characterization, were found to be sensitive to H2O2 and exhibited decreased NADPH levels compared with those in the isogenic parents. Microarray analyses of COL and the isogenic bshA mutant revealed increased expression of genes involved in staphyloxanthin synthesis in the bshA mutant relative to that in COL under thiol stress conditions. However, the bshA mutant of COL demonstrated decreased survival compared to that of the parent in human whole-blood survival assays; likewise, the naturally BSH-deficient strain SH1000 survived less well than its BSH-producing isogenic counterpart. Thus, the survival of S. aureus under oxidative stress is facilitated by BSH, possibly via a FosB-mediated mechanism, independently of its capability to produce staphyloxanthin.

An in vitro evolved glmS ribozyme has the wild-type fold but loses coenzyme dependence.

Uniquely among known ribozymes, the glmS ribozyme-riboswitch requires a small-molecule coenzyme, glucosamine-6-phosphate (GlcN6P). Although consistent with its gene-regulatory function, the use of GlcN6P is unexpected because all of the other characterized self-cleaving ribozymes use RNA functional groups or divalent cations for catalysis. To determine what active site features make this ribozyme reliant on GlcN6P and to evaluate whether it might have evolved from a coenzyme-independent ancestor, we isolated a GlcN6P-independent variant through in vitro selection. Three active site mutations suffice to generate a highly reactive RNA that adopts the wild-type fold but uses divalent cations for catalysis and is insensitive to GlcN6P. Biochemical and crystallographic comparisons of wild-type and mutant ribozymes show that a handful of functional groups fine-tune the RNA to be either coenzyme or cation dependent. These results indicate that a few mutations can confer new biochemical activities on structured RNAs. Thus, families of structurally related ribozymes with divergent function may exist.

Deoxysugar pathway interchange for erythromycin analogues heterologously produced through Escherichia coli.

The overall erythromycin biosynthetic pathway can be sub-divided into macrocyclic polyketide formation and polyketide tailoring to produce the final bioactive molecule. In this study, the native deoxysugar tailoring reactions were exchanged for the purpose of demonstrating the production of alternative final erythromycin compounds. Both the d-desosamine and l-mycarose deoxysugar pathways were replaced with the alternative d-mycaminose and d-olivose pathways to produce new erythromycin analogues through the Escherichia coli heterologous system. Both analogues exhibited bioactivity against multiple antibiotic-resistant Bacillus subtilis strains. Besides demonstrating an intrinsic flexibility for the biosynthetic system to accommodate alternative tailoring pathways, the results offer an initial attempt to leverage the E. coli platform for erythromycin analogue production.

Distribution and infection-related functions of bacillithiol in Staphylococcus aureus.

Bacillithiol (Cys-GlcN-malate, BSH) serves as a major low molecular weight thiol in low GC Gram-positive bacteria including Bacillus species and a variety of Staphylococcus aureus strains. These bacteria do not produce glutathione (GSH). In this study, HPLC analyses were used to determine BSH levels in different S. aureus strains. Furthermore, the role of BSH in the resistance against oxidants and antibiotics and its function in virulence was investigated. We and others (Newton, G.L., Fahey, R.C., Rawat, M., 2012. Microbiology 158, 1117-1126) found that BSH is not produced by members of the S. aureus NCTC8325 lineage, such as strains 8325-4 and SH1000. Using bioinformatics we show that the BSH-biosynthetic gene bshC is disrupted by an 8-bp duplication in S. aureus NCTC8325. The functional bshC-gene from BSH-producing S. aureus Newman (NWMN_1087) was expressed in S. aureus 8325-4 to reconstitute BSH-synthesis. Comparison of the BSH-producing and BSH-minus strains revealed higher resistance of the BSH-producing strain against the antibiotic fosfomycin and the oxidant hypochlorite but not against hydrogen peroxide or diamide. In addition, a higher bacterial load of the BSH-producing strain was detected in human upper-airway epithelial cells and murine macrophages. This indicates a potential role of BSH in protection of S. aureus during infection.