Meiru Si

Type VI Secretion System Transports Zn2+ to Combat Multiple Stresses and Host Immunity

Type VI secretion systems (T6SSs) are widespread multi-component machineries that translocate effectors into either eukaryotic or prokaryotic cells, for virulence or for interbacterial competition. Herein, we report that the T6SS-4 from Yersinia pseudotuberculosis displays an unexpected function in the transportation of Zn2+ to combat diverse stresses and host immunity. Environmental insults such as oxidative stress induce the expression of T6SS-4 via OxyR, the transcriptional factor that also regulates many oxidative response genes. Zinc transportation is achieved by T6SS-4-mediated translocation of a novel Zn2+-binding protein substrate YezP (YPK_3549), which has the capacity to rescue the sensitivity to oxidative stress exhibited by T6SS-4 mutants when added to extracellular milieu. Disruption of the classic zinc transporter ZnuABC together with T6SS-4 or yezP results in mutants that almost completely lost virulence against mice, further highlighting the importance of T6SS-4 in resistance to host immunity. These results assigned an unconventional role to T6SSs, which will lay the foundation for studying novel mechanisms of metal ion uptake by bacteria and the role of this process in their resistance to host immunity and survival in harmful environments.

Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailandensis

Significance As an essential micronutrient, Gram-negative bacteria must concentrate Mn2+ into the cytosol via active transport systems to meet cellular demands. Whereas inner membrane Mn2+ transporters have been characterized, an active transporter for translocation of Mn2+ across the outer membrane has not been described. Here we report a Mn2+-scavenging pathway consisting of a newly identified TonB-dependent outer membrane manganese transporter, MnoT, and a type VI secretion system (T6SS)-secreted Mn2+-binding protein, TseM. Traditionally, T6SS is recognized as a contact-dependent nanomachine to inject effectors into eukaryotic or prokaryotic cells for virulence or for interbacterial competition. The contact-independent functions of T6SS for metal acquisition and bacteria–bacteria competition, reported here, suggest that T6SS may have been retrofitted by some bacteria to gain additional adaptive functions during evolution. Type VI secretion system (T6SS) is a versatile protein export machinery widely distributed in Gram-negative bacteria. Known to translocate protein substrates to eukaryotic and prokaryotic target cells to cause cellular damage, the T6SS has been primarily recognized as a contact-dependent bacterial weapon for microbe–host and microbial interspecies competition. Here we report contact-independent functions of the T6SS for metal acquisition, bacteria competition, and resistance to oxidative stress. We demonstrate that the T6SS-4 in Burkholderia thailandensis is critical for survival under oxidative stress and is regulated by OxyR, a conserved oxidative stress regulator. The T6SS-4 is important for intracellular accumulation of manganese (Mn2+) under oxidative stress. Next, we identified a T6SS-4–dependent Mn2+-binding effector TseM, and its interacting partner MnoT, a Mn2+-specific TonB-dependent outer membrane transporter. Similar to the T6SS-4 genes, expression of mnoT is regulated by OxyR and is induced under oxidative stress and low Mn2+ conditions. Both TseM and MnoT are required for efficient uptake of Mn2+ across the outer membrane under Mn2+-limited and -oxidative stress conditions. The TseM–MnoT-mediated active Mn2+ transport system is also involved in contact-independent bacteria–bacteria competition and bacterial virulence. This finding provides a perspective for understanding the mechanisms of metal ion uptake and the roles of T6SS in bacteria–bacteria competition.

Corynebacterium glutamicum methionine sulfoxide reductase A uses both mycoredoxin and thioredoxin for regeneration and oxidative stress resistance

ABSTRACT Oxidation of methionine leads to the formation of the S and R diastereomers of methionine sulfoxide (MetO), which can be reversed by the actions of two structurally unrelated classes of methionine sulfoxide reductase (Msr), MsrA and MsrB, respectively. Although MsrAs have long been demonstrated in numerous bacteria, their physiological and biochemical functions remain largely unknown in Actinomycetes. Here, we report that a Corynebacterium glutamicum methionine sulfoxide reductase A (CgMsrA) that belongs to the 3-Cys family of MsrAs plays important roles in oxidative stress resistance. Deletion of the msrA gene in C. glutamicum resulted in decrease of cell viability, increase of ROS production, and increase of protein carbonylation levels under various stress conditions. The physiological roles of CgMsrA in resistance to oxidative stresses were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic-function (ECF) sigma factor SigH. Activity assays performed with various regeneration pathways showed that CgMsrA can reduce MetO via both the thioredoxin/thioredoxin reductase (Trx/TrxR) and mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) pathways. Site-directed mutagenesis confirmed that Cys56 is the peroxidatic cysteine that is oxidized to sulfenic acid, while Cys204 and Cys213 are the resolving Cys residues that form an intramolecular disulfide bond. Mrx1 reduces the sulfenic acid intermediate via the formation of an S-mycothiolated MsrA intermediate (MsrA-SSM) which is then recycled by mycoredoxin and the second molecule of mycothiol, similarly to the glutathione/glutaredoxin/glutathione reductase (GSH/Grx/GR) system. However, Trx reduces the Cys204-Cys213 disulfide bond in CgMsrA produced during MetO reduction via the formation of a transient intermolecular disulfide bond between Trx and CgMsrA. While both the Trx/TrxR and Mrx1/Mtr/MSH pathways are operative in reducing CgMsrA under stress conditions in vivo, the Trx/TrxR pathway alone is sufficient to reduce CgMsrA under normal conditions. Based on these results, a catalytic model for the reduction of CgMsrA by Mrx1 and Trx is proposed.

Sphingomonas gei sp. nov., isolated from roots of Geum aleppicum

A yellow-pigmented bacterium, designated strain ZFGT-11(T), was isolated from roots of Geum aleppicum Jacq. collected from Taibai Mountain in Shaanxi Province, north-west China, and was subjected to a taxonomic study by using a polyphasic approach. Cells of strain ZFGT-11(T) were Gram-stain-negative, strictly aerobic rods that were surrounded by a thick capsule and were motile by means of a single polar flagellum. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ZFGT-11(T) was a member of the genus Sphingomonas and was closely related to Sphingomonas naasensis KACC 16534(T) (97.6% similarity), Sphingomonas kyeonggiense JCM 18825(T) (96.8%), Sphingomonas asaccharolytica IFO 15499(T) (96.7%) and Sphingomonas leidyi DSM 4733(T) (96.6%). The predominant respiratory quinone was ubiquinone-10 (Q-10) and the major cellular fatty acids were summed feature 8 (comprising C(18 : 1)ω7c and/or C(18 : 1)ω6c), C(17 : 1)ω6c, C(14 : 0) 2-OH, C(16 : 0) and C(15 : 0) 2-OH. The major polyamine of strain ZFGT-11(T) was sym-homospermidine. Phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, sphingoglycolipid, two unidentified aminoglycolipids, two unidentified phospholipids and two unidentified lipids were detected in the polar lipid profile. The DNA G+C content was 66.8 mol%. DNA-DNA relatedness for strain ZFGT-11(T) with respect to its closest phylogenetic relative S. naasensis KACC 16534(T) was 26.2±4.8% (mean±SD). On the basis of data from the present polyphasic taxonomic study, strain ZFGT-11(T) is considered to represent a novel species of the genus Sphingomonas , for which the name Sphingomonas gei sp. nov. is proposed. The type strain is ZFGT-11(T) ( = CCTCC AB 2013306(T) = KCTC 32449(T) = LMG 27608(T)).

NrdH Redoxin Enhances Resistance to Multiple Oxidative Stresses by Acting as a Peroxidase Cofactor in Corynebacterium glutamicum

ABSTRACT NrdH redoxins are small protein disulfide oxidoreductases behaving like thioredoxins but sharing a high amino acid sequence similarity to glutaredoxins. Although NrdH redoxins are supposed to be another candidate in the antioxidant system, their physiological roles in oxidative stress remain unclear. In this study, we confirmed that the Corynebacterium glutamicum NrdH redoxin catalytically reduces the disulfides in the class Ib ribonucleotide reductases (RNR), insulin and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), by exclusively receiving electrons from thioredoxin reductase. Overexpression of NrdH increased the resistance of C. glutamicum to multiple oxidative stresses by reducing ROS accumulation. Accordingly, elevated expression of the nrdH gene was observed when the C. glutamicum wild-type strain was exposed to oxidative stress conditions. It was discovered that the NrdH-mediated resistance to oxidative stresses was largely dependent on the presence of the thiol peroxidase Prx, as the increased resistance to oxidative stresses mediated by overexpression of NrdH was largely abrogated in the prx mutant. Furthermore, we showed that NrdH facilitated the hydroperoxide reduction activity of Prx by directly targeting and serving as its electron donor. Thus, we present evidence that the NrdH redoxin can protect against the damaging effects of reactive oxygen species (ROS) induced by various exogenous oxidative stresses by acting as a peroxidase cofactor.

Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum

Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30C, and interestingly, it could utilize NAD+ and NADP+ as coenzymes with similar efficiency and showed no obvious difference toward NAD+ and NADP+. In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum.

Functional characterization of Corynebacterium glutamicum mycothiol S-conjugate amidase.

The present study focuses on the genetic and biochemical characterization of mycothiol S-conjugate amidase (Mca) of Corynebacterium glutamicum. Recombinant C. glutamicum Mca was heterologously expressed in Escherichia coli and purified to apparent homogeneity. The molecular weight of native Mca protein determined by gel filtration chromatography was 35 kDa, indicating that Mca exists as monomers in the purification condition. Mca showed amidase activity with mycothiol S-conjugate of monobromobimane (MSmB) in vivo while mca mutant lost the ability to cleave MSmB. In addition, Mca showed limited deacetylase activity with N-acetyl-D-glucosamine (GlcNAc) as substrate. Optimum pH for amidase activity was between 7.5 and 8.5, while the highest activity in the presence of Zn2+ confirmed Mca as a zinc metalloprotein. Amino acid residues conserved among Mca family members were located in C. glutamicum Mca and site-directed mutagenesis of these residues indicated that Asp14, Tyr137, His139 and Asp141 were important for activity. The mca deletion mutant showed decreased resistance to antibiotics, alkylating agents, oxidants and heavy metals, and these sensitive phenotypes were recovered in the complementary strain to a great extent. The physiological roles of Mca in resistance to various toxins were further supported by the induced expression of Mca in C. glutamicum under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH.

Overexpression of Mycothiol Disulfide Reductase Enhances Corynebacterium glutamicum Robustness by Modulating Cellular Redox Homeostasis and Antioxidant Proteins under Oxidative Stress.

Mycothiol (MSH) is the dominant low-molecular-weight thiol (LMWT) unique to high-(G+C)-content Gram-positive Actinobacteria, such as Corynebacterium glutamicum, and is oxidised into its disulfide form mycothiol disulfide (MSSM) under oxidative conditions. Mycothiol disulfide reductase (Mtr), an NADPH-dependent enzyme, reduces MSSM to MSH, thus maintaining intracellular redox homeostasis. In this study, a recombinant plasmid was constructed to overexpress Mtr in C. glutamicum using the expression vector pXMJ19-His6. Mtr-overexpressing C. glutamicum cells showed increased tolerance to ROS induced by oxidants, bactericidal antibiotics, alkylating agents, and heavy metals. The physiological roles of Mtr in resistance to oxidative stresses were corroborated by decreased ROS levels, reduced carbonylation damage, decreased loss of reduced protein thiols, and a massive increase in the levels of reversible protein thiols in Mtr-overexpressing cells exposed to stressful conditions. Moreover, overexpression of Mtr caused a marked increase in the ratio of reduced to oxidised mycothiol (MSH:MSSM), and significantly enhanced the activities of a variety of antioxidant enzymes, including mycothiol peroxidase (MPx), mycoredoxin 1 (Mrx1), thioredoxin 1 (Trx1), and methionine sulfoxide reductase A (MsrA). Taken together, these results indicate that the Mtr protein functions in C. glutamicum by protecting cells against oxidative stress.

Mycothiol protects Corynebacterium glutamicum against acid stress via maintaining intracellular pH homeostasis, scavenging ROS, and S-mycothiolating MetE

Mycothiol (MSH) plays a major role in protecting cells against oxidative stress and detoxification from a broad range of exogenous toxic agents. In the present study, we reveal that intracellular MSH contributes significantly to the adaptation to acidic conditions in the model organism Corynebacterium glutamicum. We present evidence that MSH confers C. glutamicum with the ability to adapt to acidic conditions by maintaining pHi homeostasis, scavenging reactive oxygen species (ROS), and protecting methionine synthesis by the S-mycothiolation modification of methionine synthase (MetE). The role of MSH in acid adaptation was further confirmed by improving the acid tolerance of C. glutamicum by overexpressing the key MSH synthesis gene mshA. Hence, our work provides insights into a previously unknown, but important, aspect of the C. glutamicum cellular response to acid stress. The results reported here may help to understand acid tolerance mechanisms in acid sensitive bacteria and may open a new avenue for improving acid resistance in industry strains for the production of bio-based chemicals from renewable biomass.

Ohr Protects Corynebacterium glutamicum against Organic Hydroperoxide Induced Oxidative Stress

Ohr, a bacterial protein encoded by the Organic Hydroperoxide Resistance (ohr) gene, plays a critical role in resistance to organic hydroperoxides. In the present study, we show that the Cys-based thiol-dependent Ohr of Corynebacterium glutamicum decomposes organic hydroperoxides more efficiently than hydrogen peroxide. Replacement of either of the two Cys residues of Ohr by a Ser residue resulted in drastic loss of activity. The electron donors supporting regeneration of the peroxidase activity of the oxidized Ohr of C. glutamicum were principally lipoylated proteins (LpdA and Lpd/SucB). A Δohr mutant exhibited significantly decreased resistance to organic hydroperoxides and marked accumulation of reactive oxygen species (ROS) in vivo; protein carbonylation was also enhanced notably. The resistance to hydrogen peroxide also decreased, but protein carbonylation did not rise to any great extent. Together, the results unequivocally show that Ohr is essential for mediation of organic hydroperoxide resistance by C. glutamicum.