Chemical Proteomics Strategies for Analyzing Protein Lipidation Reveal the Bacterial O-Mycoloylome.

Protein lipidation dynamically controls protein localization and function within cellular membranes. A unique form of protein O-fatty acylation in Corynebacterium, termed protein O-mycoloylation, involves the attachment of mycolic acids─unusually large and hydrophobic fatty acids─to serine residues of proteins in these organisms' outer mycomembrane. However, as with other forms of protein lipidation, the scope and functional consequences of protein O-mycoloylation are challenging to investigate due to the inherent difficulties of enriching and analyzing lipidated peptides. To facilitate the analysis of protein lipidation and enable the comprehensive profiling and site mapping of protein O-mycoloylation, we developed a chemical proteomics strategy integrating metabolic labeling, click chemistry, cleavable linkers, and a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing LC separation and complementary fragmentation methods tailored to the analysis of lipophilic, MS-labile O-acylated peptides. Using these tools in the model organism Corynebacterium glutamicum, we identified approximately 30 candidate O-mycoloylated proteins, including porins, mycoloyltransferases, secreted hydrolases, and other proteins with cell envelope-related functions─consistent with a role for O-mycoloylation in targeting proteins to the mycomembrane. Site mapping revealed that many of the proteins contained multiple spatially proximal modification sites, which occurred predominantly at serine residues surrounded by conformationally flexible peptide motifs. Overall, this study (i) discloses the putative protein O-mycoloylome for the first time, (ii) yields new insights into the undercharacterized proteome of the mycomembrane, which is a hallmark of important pathogens (e.g., Corynebacterium diphtheriae, Mycobacterium tuberculosis), and (iii) provides generally applicable chemical strategies for the proteomic analysis of protein lipidation.

Solution design to extend the pH range of the pH-responsive precipitation of a CspB fusion protein.

Cell surface protein B (CspB) from Corynebacterium glutamicum has been developed as a reversible pH-responsive tag for protein purification. CspB fusion proteins precipitate at acidic pH, after that they completely dissolve at neutral pH. This property has been used in a non-chromatographic protein purification method named pH-responsive Precipitation-Redissolution of CspB tag Purification (pPRCP). However, it is difficult to apply pPRCP to proteins that are unstable under acidic conditions. In an effort to shift the precipitation pH to a milder range, we investigated the solution conditions of CspB-fused Teriparatide (CspB50TEV-Teriparatide) during the process of pH-responsive precipitation using pPRCP. The purified CspB50TEV-Teriparatide in buffer without additives precipitated at pH 5.3. By contrast, CspB50TEV-Teriparatide in buffer with 0.5 M Na2SO4 precipitated at pH 6.6 because of the kosmotropic effect. Interestingly, the pH at which precipitation occurred was independent of the protein concentration. The precipitated CspB50TEV-Teriparatide was fully redissolved at above pH 8.0 in the presence or absence of salt. The discovery that proteins can be precipitated at a mild pH will allow pPRCP to be applied to acid-sensitive proteins.

Characterization of the Uncommon Lipid Families in Corynebacterium glutamicum by Mass Spectrometry.

This book chapter provides readers the step-by-step instruction for cell growth, lipid isolation, and lipid analysis to obtain the lipidome of Corynebacterium glutamicum (C. glutamicum) in the genus Corynebacterium, a biotechnologically important bacterium. We separate the lipid families by preparative HPLC with an analytical C-8 column, followed by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high-resolution mass measurement to define the structures of cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), α-D-mannopyranosyl-(1 â†’ 4)-α-D-glucuronyl diacylglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), and acyl trehalose monomycolate (acyl-TMM) whose structures have been previously mis-assigned or not defined by mass spectrometric means. We also define the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in the cell wall. The similarity of the lipidome to that in the Mycobacterium genera is consistent with the notion that Corynebacterium and Mycobacterium are gram-positive bacteria belonging to the suborder Corynebacterineae.

Corynebacterium glutamicum Mycoredoxin 3 protects against multiple oxidative stresses and displays thioredoxin-like activity.

Glutaredoxins (Grxs) and thioredoxins (Trxs) play a critical role in resistance to oxidative conditions. However, physiological and biochemical roles of Mycoredoxin 3 (Mrx3) that shared a high amino acid sequence similarity to Grxs remain unknown in Corynebacterium glutamicum. Here we showed that mrx3 deletion strains of C. glutamicum was involved in the protection against oxidative stress. Recombinant Mrx3 not only catalytically reduced the disulfide bonds in ribonucleotide reductase (RNR), insulin and 5,5'-dithiobis-(2-nitro-benzoicacid) (DTNB), but also reduced the mixed disulphides between mycothiol (MSH) and substrate, which was exclusively linked to the thioredoxin reductase (TrxR) electron transfer pathway by a dithiol mechanism. Site-directed mutagenesis confirmed that the conserved Cys17 and Cys20 in Mrx3 were necessary to maintain its activity. The mrx3 deletion mutant showed decreased resistance to various stress, and these sensitive phenotypes were almost fully restored in the complementary strain. The physiological roles of Mrx3 in resistance to various stress were further supported by the induced expression of mrx3 under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. Thus, we presented the first evidence that Mrx3 protected against various oxidative stresses by acting as a disulfide oxidoreductase behaving like Trx.

Harnessing PGPR inoculation through exogenous foliar application of salicylic acid and microbial extracts for improving rice growth.

Plant-growth-promoting rhizobacteria (PGPR) should effectively colonize along the plant root to enhance the plant and soil health. The present investigation aims to improve the PGPR-mediated plant health benefits through above-ground foliar management. A green fluorescent protein-tagged PGPR strain, Pseudomonas chlororaphis (ZSB15-M2) was inoculated in a nonautoclaved agricultural soil before rice culturing. Salicylic acid and cell extracts of Corynebacterium glutamicum and Saccharomyces cerevisiae as a supply of hormonal and inducer compounds were applied on the foliage of the 10-days-old rice plants and subsequently observed the colonizing ability of ZSB15-M2. The cell extracts of Corynebacteria and yeast showed a 100-fold increase in the ZSB15-M2 population in the rhizosphere of rice, whereas salicylic acid had a 10-fold increase in relation to mock control. The rice root exudates collected after the spraying of salicylic acid and microbial extracts showed significantly enhanced release of total carbon, total protein, total sugar, total amino nitrogen, total nitrogen, and phenol content. In vitro assays revealed that these root exudates collected after exogenous spray of these chemicals enhanced the chemotactic motility and biofilm formation of ZSB15-M2 compared to the control plant's root exudate. Metabolomic analysis of root exudates collected from these rice plants by gas chromatography-mass spectrometry revealed that the Corynebacteria and yeast cell extracts enhanced the divergence of metabolites of rice root exudate. Further, due to these cumulative effects in the rice rhizosphere, the total chlorophyll, total protein, total nitrogen, and total phosphorus of rice were significantly improved. These observations provide insights into the rhizosphere functioning of rice plants as modulated by above-ground treatments with improved colonization of inoculant strains as well as the plant growth.

In vitro reconstitution and characterization of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum.

Pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) are critical enzymes in central carbon metabolism. In Corynebacterium glutamicum, an unusual hybrid complex consisting of CgE1p (thiamine diphosphate-dependent pyruvate dehydrogenase, AceE), CgE2 (dihydrolipoamide acetyltransferase, AceF), CgE3 (dihydrolipoamide dehydrogenase, Lpd), and CgE1o (thiamine diphosphate-dependent 2-oxoglutarate dehydrogenase, OdhA) has been suggested. Here, we elucidated that the PDH-ODH hybrid complex in C. glutamicum probably consists of six copies of CgE2 in its core, which is rather compact compared with PDH and ODH in other microorganisms that have twenty-four copies of E2. We found that CgE2 formed a stable complex with CgE3 (CgE2-E3 subcomplex) in vitro, hypothetically comprised of two CgE2 trimers and four CgE3 dimers. We also found that CgE1o exists mainly as a hexamer in solution and is ready to form an active ODH complex when mixed with the CgE2-E3 subcomplex. Our in vitro reconstituted system showed CgE1p- and CgE1o-dependent inhibition of ODH and PDH, respectively, actively supporting the formation of the hybrid complex, in which both CgE1p and CgE1o associate with a single CgE2-E3. In gel filtration chromatography, all the subunits of CgODH were eluted in the same fraction, whereas CgE1p was eluted separately from CgE2-E3, suggesting a weak association of CgE1p with CgE2 compared with that of CgE1o. This study revealed the unique molecular architecture of the hybrid complex from C. glutamicum and the compact-sized complex would provide an advantage to determine the whole structure of the unusual hybrid complex.

Dynamic Control of 4-Hydroxyisoleucine Biosynthesis by Modified l-Isoleucine Biosensor in Recombinant Corynebacterium glutamicum.

4-Hydroxyisoleucine (4-HIL), a promising drug for treating diabetes, can be synthesized from the self-produced l-isoleucine (Ile) by expressing the Ile dioxygenase gene ido in Corynebacterium glutamicum. However, the requirement of three substrates, Ile, α-ketoglutarate (α-KG), and O2, makes such de novo biosynthesis difficult to be fulfilled effectively under static engineering conditions. In this study, dynamic control of 4-HIL biosynthesis by the Ile biosensor Lrp-PbrnFE was researched. The native PbrnFE promoter of natural Ile biosensor was still weak even under Ile induction. Through tetA dual genetic selection, several modified stronger PbrnFEN promoters were obtained from the synthetic library of the Ile biosensor. Dynamic regulation of ido expression by modified Ile biosensors increased the 4-HIL titer from 24.7 mM to 28.9-74.4 mM. The best strain ST04 produced even a little more 4-HIL than the static strain SN02 overexpressing ido by the strong PtacM promoter (69.7 mM). Further dynamic modulation of α-KG supply in ST04 by expressing different PbrnFEN-controlled odhI decreased the 4-HIL production but increased the l-glutamate or Ile accumulation. However, synergistic modulation of α-KG supply and O2 supply in ST04 by different combinations of PbrnFEN-odhI and PbrnFEN-vgb improved the 4-HIL production significantly, and the highest titer (135.3 mM) was obtained in ST17 strain regulating all the three genes by PbrnFE7. This titer was higher than those of all the static metabolic engineered C. glutamicum strains ever constructed. Therefore, dynamic regulation by modified Ile biosensor is a predominant strategy for enhancing 4-HIL de novo biosynthesis in C. glutamicum.

Unveiling the biodiversity of lipid species in Corynebacteria- characterization of the uncommon lipid families in C. glutamicum and pathogen C. striatum by mass spectrometry.

Uncommon lipids in biotechnologically important Corynebacterium glutamicum and pathogen Corynebacterium striatum in genus Corynebacterium are isolated and identified by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high resolution mass measurement. We redefined several lipid structures that were previously mis-assigned or not defined, including cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), (α-d-mannopyranosyl)-(1 â†’ 4)-(α-D-glucuronyl diacyglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), acyl trehalose monomycolate (acyl-TMM). We also report the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in which many isomeric structures are present. The LIT MSn approaches afford identification of the functional group, the fatty acid substituents and their regiospecificity in the molecules, revealing the biodiversities of the lipid species in two Corynebacterium strains that have played very different and important roles in human nutrition and health.

Structural features of the glutamate-binding protein from Corynebacterium glutamicum.

L-glutamate (Glu) is the major excitatory transmitter in mammalian brain. Inadequate concentration of Glu in the brain correlates to mood disorder. In industry, Glu is used as a flavour enhancer in food and in foodstuff processing. A high concentration of Glu has several effects on human health such as hypersensitive effects, headache and stomach pain. The presence of Glu in food can be detected by different analytical methods based on chromatography, or capillary electrophoresis or amperometric techniques. We have isolated and characterized a glutamate-binding protein (GluB) from the Gram-positive bacteria Corynebacterium glutamicum. Together with GluC protein, GluD protein and the cytoplasmic protein GluA, GluB permits the transport of Glu in/out of cell. In this study, we have investigated the binding features of GluB as well as the effect of temperature on its structure both in the absence and in the presence of Glu. The results have showed that GluB has a high affinity and selectivity versus Glu (nanomolar range) and the presence of the ligand induces a higher thermal stability of the protein structure.

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum.

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.

PII Signal Transduction Protein GlnK Alleviates Feedback Inhibition of N-Acetyl-l-Glutamate Kinase by l-Arginine in Corynebacterium glutamicum.

PII signal transduction proteins are ubiquitous and highly conserved in bacteria, archaea, and plants and play key roles in controlling nitrogen metabolism. However, research on biological functions and regulatory targets of PII proteins remains limited. Here, we illustrated experimentally that the PII protein Corynebacterium glutamicum GlnK (CgGlnK) increased l-arginine yield when glnK was overexpressed in Corynebacterium glutamicum Data showed that CgGlnK regulated l-arginine biosynthesis by upregulating the expression of genes of the l-arginine metabolic pathway and interacting with N-acetyl-l-glutamate kinase (CgNAGK), the rate-limiting enzyme in l-arginine biosynthesis. Further assays indicated that CgGlnK contributed to alleviation of the feedback inhibition of CgNAGK caused by l-arginine. In silico analysis of the binding interface of CgGlnK-CgNAGK suggested that the B and T loops of CgGlnK mainly interacted with C and N domains of CgNAGK. Moreover, F11, R47, and K85 of CgGlnK were identified as crucial binding sites that interact with CgNAGK via hydrophobic interaction and H bonds, and these interactions probably had a positive effect on maintaining the stability of the complex. Collectively, this study reveals PII-NAGK interaction in nonphotosynthetic microorganisms and further provides insights into the regulatory mechanism of PII on amino acid biosynthesis in corynebacteria.IMPORTANCE Corynebacteria are safe industrial producers of diverse amino acids, including l-glutamic acid and l-arginine. In this study, we showed that PII protein GlnK played an important role in l-glutamic acid and l-arginine biosynthesis in C. glutamicum Through clarifying the molecular mechanism of CgGlnK in l-arginine biosynthesis, the novel interaction between CgGlnK and CgNAGK was revealed. The alleviation of l-arginine inhibition of CgNAGK reached approximately 48.21% by CgGlnK addition, and the semi-inhibition constant of CgNAGK increased 1.4-fold. Furthermore, overexpression of glnK in a high-yield l-arginine-producing strain and fermentation of the recombinant strain in a 5-liter bioreactor led to a remarkably increased production of l-arginine, 49.978 g/liter, which was about 22.61% higher than that of the initial strain. In conclusion, this study provides a new strategy for modifying amino acid biosynthesis in C. glutamicum.

Solution NMR structure of CGL2373, a polyketide cyclase-like protein from Corynebacterium glutamicum.

Protein CGL2373 from Corynebacterium glutamicum was previously proposed to be a member of the polyketide_cyc2 family, based on amino-acid sequence and secondary structure features derived from NMR chemical shift assignments. We report here the solution NMR structure of CGL2373, which contains three α-helices and one antiparallel ß-sheet and adopts a helix-grip fold. This structure shows moderate similarities to the representative polyketide cyclases, TcmN, WhiE, and ZhuI. Nevertheless, unlike the structures of these homologs, CGL2373 structure looks like a half-open shell with a much larger pocket, and key residues in the representative polyketide cyclases for binding substrate and catalyzing aromatic ring formation are replaced with different residues in CGL2373. Also, the gene cluster where the CGL2373-encoding gene is located in C. glutamicum contains additional genes encoding nucleoside diphosphate kinase, folylpolyglutamate synthase, and valine-tRNA ligase, different from the typical gene cluster encoding polyketide cyclase in Streptomyces. Thus, although CGL2373 is structurally a polyketide cyclase-like protein, the function of CGL2373 may differ from the known polyketide cyclases and needs to be further investigated. The solution structure of CGL2373 lays a foundation for in silico ligand screening and binding site identifying in future functional study.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance.

MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)-uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882-ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to ß-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR-uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Double deletion of murA and murB induced temperature sensitivity in Corynebacterium glutamicum.

Currently, the mechanism of temperature-sensitive production of glutamate in Corynebacterium glutamicum has not been clarified. We first found the murA and murB genes were potentially related to temperature-sensitive secretion of glutamate, which were not existed in a temperature-sensitive mutant. When replenishing murA or/and murB in the mutant, the temperature sensitivity was weakened. While, their knockout in a wild-type strain resulted in temperature-sensitive secretion of glutamate. Peptidoglycan analysis showed that deletion of murA and murB decreased the peptidoglycan synthesis. Comparative metabolomics analysis suggested that the variation in cell wall structure resulted in decreased overall cellular metabolism but increased carbon flow to glutamate synthesis, which was a typical metabolism pattern in industrial temperature-sensitive producing strains. This study clarifies the mechanism between murA and murB deletion and the temperature-sensitive secretion of glutamate in C. glutamcium, and provides a reference for the metabolic engineering of cell wall to obtain increased bioproduction of chemicals.

Crystal Structure and Biochemical Characterization of Ketol-Acid Reductoisomerase from Corynebacterium glutamicum.

l-Valine belongs to the branched-chain amino acids (BCAAs) and is an essential amino acid that is crucial for all living organisms. l-Valine is industrially produced by the nonpathogenic bacterium Corynebacterium glutamicum and is synthesized by the BCAA biosynthetic pathway. Ketol-acid reductoisomerase (KARI) is the second enzyme in the BCAA pathway and catalyzes the conversion of (S)-2-acetolactate into (R)-2,3-dihydroxy-isovalerate, or the conversion of (S)-2-aceto-2-hydroxybutyrate into (R)-2,3-dihydroxy-3-methylvalerate. To elucidate the enzymatic properties of KARI from C. glutamicum (CgKARI), we successfully produced CgKARI protein and determined its crystal structure in complex with NADP+ and two Mg2+ ions. Based on the complex structure, docking simulations, and site-directed mutagenesis experiments, we revealed that CgKARI belongs to Class I KARI and identified key residues involved in stabilization of the substrate, metal ions, and cofactor. Furthermore, we confirmed the difference in the binding of metal ions that depended on the conformational change.

The 138th residue of acetohydroxyacid synthase in Corynebacterium glutamicum is important for the substrate binding specificity.

Corynebacterium glutamicum acetohydroxyacid synthase (AHAS), composed of two subunits IlvB and IlvN, catalyzes the first reaction in the biosynthetic pathway of branched-chain amino acids. It either condenses two pyruvates to yield acetolactate, leading to the biosynthesis of L-valine and L-leucine, or condenses pyruvate with 2-ketobutyrate to yield acetohydroxybutyrate, leading to L-isoleucine biosynthesis. However, the mechanism for the substrate specificity of C. glutamicum AHAS remains unknown. In this study, AHASs from an L-valine-producing C. glutamicum VWB-1 and an L-isoleucine-producing C. glutamicum IWJ001 were analyzed. The amino acid sequence of IlvN from both strains are the same, but the 138th and 404th residues of IlvB from the two strains are different; they are alanine and valine in IWJ001 (IlvB138A404V), but valine and alanine in VWB-1 (IlvB138V404A). When IlvB138A404V and IlvB138V404A were overexpressed in wild type C. glutamicum ATCC14067 and its △alr△aceE△ilvA△leuA mutant YTW-104, the latter led to much more L-valine production than the former. AHAS activity studies also showed that the 138th valine was important for binding the 2nd substrate pyruvate but not the 404th alanine. YTW-104/pJYW4-ilvB138V404A-ilvNCE could produce 25.93 g/L L-valine. The results indicate that the 138th valine of IlvB in AHAS could play an important role, leading to the increased L-valine biosynthesis in C. glutamicum.

Identification of Surf1 as an assembly factor of the cytochrome bc1-aa3 supercomplex of Actinobacteria.

Respiration in aerobic Actinobacteria involves a cytochrome bc1-aa3 supercomplex with a diheme cytochrome c1, first isolated from Corynebacterium glutamicum. Synthesis of a functional cytochrome c oxidase requires incorporation of CuA, CuB, heme a, and heme a3. In contrast to eukaryotes and α-proteobacteria, this process is poorly understood in Actinobacteria. Here, we analyzed the role of a Surf1 homolog of C. glutamicum in the formation of a functional bc1-aa3 supercomplex. Deletion of the surf1 gene (cg2460) in C. glutamicum caused a growth defect and cytochrome spectra revealed reduced levels of cytochrome c and a and an increased level of cytochrome d. Membranes of the Δsurf1 strain had lost the ability to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine, suggesting that Surf1 is essential for the formation of a functional cytochrome aa3 oxidase. In contrast to the wild type, a bc1-aa3 supercomplex could not be purified from solubilized membranes of the Δsurf1 mutant. A transcriptome comparison revealed that the genes of the SigC regulon including those for cytochrome bd oxidase were upregulated in the Δsurf1 strain as well as the copper deprivation-inducible gene ctiP. Complementation studies showed that the Surf1 homologs of Corynebacterium diphtheriae, Mycobacterium smegmatis and Mycobacterium tuberculosis could at least partially abolish the growth defect of the C. glutamicum Δsurf1 mutant, suggesting that Surf1 is a conserved assembly factor for actinobacterial cytochrome aa3 oxidase.

Structures of two ArsR As(III)-responsive transcriptional repressors: Implications for the mechanism of derepression.

ArsR As(III)-responsive transcriptional repressors, members of the ArsR/SmtB family of metalloregulatory proteins, have been characterized biochemically but, to date, no As(III)-bound structure has been solved. Here we report two crystal structures of ArsR repressors from Acidithiobacillus ferrooxidans (AfArsR) and Corynebacterium glutamicum (CgArsR) in the As(III)-bound form. AfArsR crystallized in P21 space group and diffracted up to 1.86 Å. CgArsR crystallized in P212121 and diffracted up to 1.6 Å. AfArsR showed one As(III) bound in one subunit of the homodimer, while the CgArsR structure showed two As(III) bound with S3 coordination, one in each monomer. Previous studies indicated that in AfArsR As(III) binds to Cys95, Cys96 and Cys102 from the same monomer, while, in CgArsR, to Cys15, Cys16 from one monomer and Cys55 from the other monomer. The dimer interfaces of these structures showed distinct differences from other members of the ArsR/SmtB family of proteins, which potentially renders multiple options for evolving metal(loid) binding sites in this family of proteins. Also, CgArsR presents a new α2-N binding site, not the previously predicted α3-N site. Despite differences in the location of the binding cysteines in the primary sequences of these proteins, the two metal binding sites are almost congruent on their structures, an example of convergent evolution. Analyses of the electrostatic surface of the proteins at the DNA binding domain indicate that there two different modes of derepression in the ArsR/SmtB family of metalloregulatory proteins.

Bacterial Cell Wall Modification with a Glycolipid Substrate.

Despite the ubiquity and importance of glycans in biology, methods to probe their structures in cells are limited. Mammalian glycans can be modulated using metabolic incorporation, a process in which non-natural sugars are taken up by cells, converted to nucleotide-sugar intermediates, and incorporated into glycans via biosynthetic pathways. These studies have revealed that glycan intermediates can be shunted through multiple pathways, and this complexity can be heightened in bacteria, as they can catabolize diverse glycans. We sought to develop a strategy that probes structures recalcitrant to metabolic incorporation and that complements approaches focused on nucleotide sugars. We reasoned that lipid-linked glycans, which are intermediates directly used in glycan biosynthesis, would offer an alternative. We generated synthetic arabinofuranosyl phospholipids to test this strategy in Corynebacterium glutamicum and Mycobacterium smegmatis, organisms that serve as models of Mycobacterium tuberculosis. Using a C. glutamicum mutant that lacks arabinan, we identified synthetic glycosyl donors whose addition restores cell wall arabinan, demonstrating that non-natural glycolipids can serve as biosynthetic intermediates and function in chemical complementation. The addition of an isotopically labeled glycan substrate facilitated cell wall characterization by NMR. Structural analysis revealed that all five known arabinofuranosyl transferases could process the exogenous lipid-linked sugar donor, allowing for the full recovery of the cell envelope. The lipid-based probe could also rescue wild-type cells treated with an inhibitor of cell wall biosynthesis. Our data indicate that surrogates of natural lipid-linked glycans can intervene in the cell's traditional workflow, indicating that biosynthetic incorporation is a powerful strategy for probing glycan structure and function.

Equilibrium of the intracellular redox state for improving cell growth and L-lysine yield of Corynebacterium glutamicum by optimal cofactor swapping.

BACKGROUND: NAD(H/+) and NADP(H/+) are the most important redox cofactors in bacteria. However, the intracellular redox balance is in advantage of the cell growth and production of NAD(P)H-dependent products. RESULTS: In this paper, we rationally engineered glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and isocitrate dehydrogenase (IDH) to switch the nucleotide-cofactor specificity resulting in an increase in final titer [from 85.6 to 121.4 g L-1] and carbon yield [from 0.33 to 0.46 g (g glucose)-1] of L-lysine in strain RGI in fed-batch fermentation. To do this, we firstly analyzed the production performance of original strain JL-6, indicating that the imbalance of intracellular redox was the limiting factor for L-lysine production. Subsequently, we modified the native GAPDH and indicated that recombinant strain RG with nonnative NADP-GAPDH dramatically changed the intracellular levels of NADH and NADPH. However, L-lysine production did not significantly increase because cell growth was harmed at low NADH level. Lastly, the nonnative NAD-IDH was introduced in strain RG to increase the NADH availability and to equilibrate the intracellular redox. The resulted strain RGI showed the stable ratio of NADPH/NADH at about 1.00, which in turn improved cell growth (µmax. = 0.31 h-1) and L-lysine productivity (qLys, max. = 0.53 g g-1 h-1) as compared with strain RG (µmax. = 0.14 h-1 and qLys, max. = 0.42 g g-1 h-1). CONCLUSIONS: This is the first report of balancing the intracellular redox state by switching the nucleotide-cofactor specificity of GAPDH and IDH, thereby improving cell growth and L-lysine production.