Reduced OxyR positively regulates the prodigiosin biosynthesis in Serratia marcescens FS14.

OxyR, a LysR family transcriptional regulator, plays vital roles in bacterial oxidative stress response. In this study, we found that the deletion of oxyR not only inhibited the antioxidant capacity of S. marcescens FS14, but also decreased the production of prodigiosin. Further study revealed that OxyR activated the prodigiosin biosynthesis at the transcriptional level. Complementary results showed that not only the wild-type OxyR but also the reduced form OxyRC199S could activate the prodigiosin biosynthesis. We further demonstrated that reduced form of wild type OxyR could bind to the promoter of pig gene cluster, and identified the binding sites which is different from oxidized OxyR binding sites in E. coli. Our results demonstrated that OxyR in FS14 uses oxidized form to regulate the expression of the antioxidant related genes and utilizes reduced form to activate prodigiosin production. Further in silico analysis suggested that the activation of prodigiosin biosynthesis by reduced OxyR should be general in S. marcesencs. To our knowledge, this is the first report to show that OxyR uses the reduced form to activate the gene's expression, therefore, our results provide a novel regulation mechanism of OxyR.

Structural basis for substrate binding and catalytic mechanism of the key enzyme VioD in the violacein synthesis pathway.

Violacein is a pigment synthesized by gram-negative bacteria with various biological activities such as antimicrobial, antiviral, and anticancer activities. VioD is a key oxygenase converting protodeoxyviolaceinic acid to protoviolaceinic acid in violacein biosynthesis. To elucidate the catalytic mechanism of VioD, here, we resolved two crystal structures of VioD, a binary complex structure containing VioD and a FAD and a ternary complex structure composed of VioD, a FAD and a 2-ethyl-1-hexanol (EHN). Structural analysis revealed a deep funnel like binding pocket with wide entrance, this pocket is positively charged. The EHN is located at the deep bottom of the binding pocket near isoalloxazine ring. Further docking simulation help us to propose the mechanism of the hydroxylation of the substrate catalyzed by VioD. Bioinformatic analysis suggested and emphasized the importance of the conserved residues involved in substrate binding. Our results provide a structural basis for the catalytic mechanism of VioD.

Crystal structure of prodigiosin binding protein PgbP, a GNAT family protein, in Serratia marcescens FS14.

Acetylation is a conserved modification catalyzed by acetyltransferases that play prominent roles in a large number of biological processes. Members of the general control non-repressible 5 (GCN5)-N-acetyltransferase (GNAT) protein superfamily are widespread in all kingdoms of life and are characterized by highly conserved catalytic fold, and can acetylate a wide range of substrates. Although the structures and functions of numerous eukaryotic GNATs have been identified thus far, many GNATs in microorganisms remain structurally and functionally undescribed. Here, we determined the crystal structure of the putative GCN5-N-acetyltransferase PgbP in complex with CoA in Serratia marcescens FS14. Structural analysis revealed that the PgbP dimer has two cavities, each of which binds a CoA molecule via conserved motifs of the GNAT family. In addition, the biochemical studies showed that PgbP is a prodigiosin-binding protein with high thermal stability. To our knowledge, this is the first view of GNAT binding to secondary metabolites and it is also the first report of prodigiosin binding protein. Molecular docking and mutation experiments indicated that prodigiosin binds to the substrate binding site of PgbP. The structure-function analyses presented here broaden our understanding of the multifunctionality of GNAT family members and may infer the mechanism of the multiple biological activities of prodigiosin.

Electroreductive Nickel-Catalyzed Allylation of Aryl Chlorides.

Herein, we report a facile and efficient allylation via electrochemical promoted Ni-catalyzed reductive carbon-carbon bond formation. Readily available (hetero)aryl chlorides with a variety of allylic sulfones are used as electrophiles in this electroreductive coupling. This Ni-catalyzed modular approach displays generally good functional group tolerance and broad substrate scope. This reaction allows a series of allylic compounds to be created including several structurally complex natural products and pharmaceutical motifs.

Multicopy expression of sigma factor RpoH reduces prodigiosin biosynthesis in Serratia marcescens FS14.

Regulation of prodigiosin biosynthesis is received wide attention due to the antimicrobial, immunosuppressive and anticancer activities of prodigiosin. Here, we constructed a transposon mutant library in S. marcescens FS14 to identify genes involved in the regulation of prodigiosin biosynthesis. 62 strains with apparently different colors were obtained. Identification of the transposon insertion sites revealed that they are classified into three groups: the coding region of cyaA and two component system eepS/R and the promoter region of rpoH. Since the effect of cyaA and eepS/R genes on prodigiosin was extensively investigated in Serratia marcescens, we chose the mutant of rpoH for further investigation. Further deletion mutation of rpoH gene showed no effect on prodigiosin production suggesting that the effect on prodigiosin production caused by transposon insertion is not due to the deletion of RpoH. We further demonstrated that multicopy expression of RpoH reduced prodigiosin biosynthesis indicating that transposon insertion caused RpoH enhanced expression. Previous results indicate that RpoS is the sigma factor for transcription of pig gene cluster in FS14, to test whether the enhanced expression of RpoH prevents prodigiosin by competing with RpoS, we found that multicopy expression of RpoS could alleviate the prodigiosin production inhibition by enhanced RpoH. We proposed that multicopy expressed RpoH competes with RpoS for core RNA polymerase (RNAP) resulting in decreased transcription of pig gene cluster and prodigiosin production reduction. We also demonstrated that RpoH is not directly involved in prodigiosin biosynthesis. Our results suggest that manipulating the transcription level of sigma factors may be applied to regulate the production of secondary metabolites.

Rupture of sinus of Valsalva aneurysm: a case report in a child.

BACKGROUND: Sinus of Valsalva aneurysm (SVA) is a rare congenital disease that can cause severe clinical presentations when the aneurysm ruptures. Here, we report a rare case of a noncoronary sinus of Valsalva aneurysm with rupture into the right atrium. CASE PRESENTATION: A 14-year-old Chinese female patient presented viral myocarditis with acute heart failure at the local hospital, and she was finally diagnosed with a noncoronary sinus Valsalva aneurysm with rupture into the right atrium by digital subtraction angiography with cardiac catheterization angiography and echocardiography at our hospital (Children's Hospital of Chongqing Medical University). Percutaneous closure intervention was performed shortly after her diagnosis, and the patient showed good functional recovery. CONCLUSIONS: We report a case of ruptured sinus of Valsalva aneurysm successfully treated by percutaneous closure, which is an excellent alternative treatment.

Two component system CpxR/A regulates the prodigiosin biosynthesis by negative control in Serratia marcescens FS14.

Prodigiosin is a tripyrrole red secondary metabolite synthesized by many microorganisms, including Serratia marcescens. In this study, we found that the deletion of the gene of sensor kinase CpxA dramatically decreased the prodigiosin production, while the deletion of the gene of the response regulator CpxR or both genes of CpxRA has no effect on prodigiosin production, the kinase function of CpxA is not essential for its regulation on prodigiosin production while the phosphorylation site of CpxR is required. We further demonstrated that the CpxA regulates the prodigiosin biosynthesis at the transcriptional level and the phosphatase activity of CpxA plays vital roles in the regulation of prodigiosin biosynthesis. Finally, we proposed that CpxR/A regulates the prodigiosin biosynthesis by negative control and the phosphorylation level of CpxR may determine the positive or negative control of the genes it regulated.

RpoS Activates the Prodigionsin Production by Activating the Transcription of the RpoS-Dependent Pig Gene Cluster in Serratia marcescens FS14.

RpoS, an alternative sigma factor of RNA polymerase, regulates the expression of a great deal of genes involved in stationary-phase survival and stress response. To identify the function of RpoS homologue in Serratia marcescens FS14, in-frame deletion mutant of rpoS was constructed. It was found that RpoS activates the biosynthesis of prodigiosin in FS14 which is just opposite to what was observed in Serratia sp. ATCC 39006. We also demonstrated that RpoS positively regulates the prodigiosin production by activating the transcription of pig cluster in FS14, and the transcription of pig cluster is RpoS-dependent. Further study showed that the differences in the promoters of pig clusters in FS14 and 39006 lead to the different selection of the sigma factors and result in the different regulation mechanisms. The -10 element and the spacer region between -10 and -35 elements of the pig cluster in FS14 are vital for the RpoS recognition in FS14. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00952-4.

Pyrethroid Carboxylesterase PytH from Sphingobium faniae JZ-2: Structure and Catalytic Mechanism.

Carboxylesterase PytH, isolated from the pyrethroid-degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin, and bifenthrin. To elucidate the catalytic mechanism of PytH, we report here the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported α/ß-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230, and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low-activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrate binding; both lid and core domains are involved in substrate binding, and the lid domain-induced core domain movement may make the active center correctly positioned with substrates.IMPORTANCE Pyrethroid pesticides are widely applied in agriculture and household; however, extensive use of these pesticides also causes serious environmental and health problems. The hydrolysis of pyrethroids by carboxylesterases is the major pathway of microbial degradation of pyrethroids, but the structure of carboxylesterases and its catalytic mechanism are still unknown. Carboxylesterase PytH from Sphingobium faniae JZ-2 could effectively hydrolyze a wide range of pyrethroid pesticides. The crystal structures of PytH are solved in this study. This showed that PytH belongs to the α/ß-hydrolase fold proteins with typical catalytic Ser-His-Asp triad, though PytH has a low sequence identity (about 20%) with them. The special large hydrophobic binding pocket enabled PytH to bind bigger pyrethroid family substrates. Our structures shed light on the substrate selectivity and the future application of PytH and deepen our understanding of α/ß-hydrolase members.

Crystal structures of the kinase domain of PpkA, a key regulatory component of T6SS, reveal a general inhibitory mechanism.

The type VI secretion system (T6SS) is a versatile and widespread export system found in many Gram-negative bacteria that delivers effector proteins into target cells. The functions of T6SSs are tightly regulated by diverse mechanisms at multiple levels, including post-translational modification through threonine phosphorylation via the Ser/Thr protein kinase (STPK) PpkA. Here, we identified that PpkA is essential for T6SS secretion in Serratia marcescens since its deletion eliminated the secretion of haemolysin co-regulated protein, while the periplasmic and transmembrane portion of PpkA was found to be disposable for T6SS secretion. We further determined the crystal structure of the kinase domain of PpkA (PpkA-294). The structure of PpkA-294 was determined in its apo form to a 1.6 Šresolution as well as in complex with ATP to a 1.41 Šresolution and with an ATP analogue AMP-PCP to a 1.45 Šresolution. The residues in the activation loop of PpkA-294 were fully determined, and the N-terminus of the loop was folded into an unprecedented inhibitory helix, revealing that the PpkA kinase domain was in an auto-inhibitory state. The ternary MgATP-PpkA-294 complex was also inactive with nucleotide ribose and phosphates in unexpected and unproductive conformations. The αC-helix in the inactive PpkA-294 adopted a conformation towards the active site but with the conserved glutamate in the helix rotated away, which we suggest to be a general conformation for all STPK kinases in the inactive form. Structural comparison of PpkA with its eukaryotic homologues reinforced the universal regulation mechanism of protein kinases.

Myroilysin Is a New Bacterial Member of the M12A Family of Metzincin Metallopeptidases and Is Activated by a Cysteine Switch Mechanism.

Proteases play important roles in all living organisms and also have important industrial applications. Family M12A metalloproteases, mainly found throughout the animal kingdom, belong to the metzincin protease family and are synthesized as inactive precursors. So far, only flavastacin and myroilysin, isolated from bacteria, were reported to be M12A proteases, whereas the classification of myroilysin is still unclear due to the lack of structural information. Here, we report the crystal structures of pro-myroilysin from bacterium Myroides sp. cslb8. The catalytic zinc ion of pro-myroilysin, at the bottom of a deep active site, is coordinated by three histidine residues in the conserved motif HEXXHXXGXXH; the cysteine residue in the pro-peptide coordinates the catalytic zinc ion and inhibits myroilysin activity. Structure comparisons revealed that myroilysin shares high similarity with the members of the M12A, M10A, and M10B families of metalloproteases. However, a unique "cap" structure tops the active site cleft in the structure of pro-myroilysin, and this "cap" structure does not exist in the above structure-reported subfamilies. Further structure-based sequence analysis revealed that myroilysin appears to belong to the M12A family, but pro-myroilysin uses a "cysteine switch" activation mechanism with a unique segment, including the conserved cysteine residue, whereas other reported M12A family proteases use an "aspartate switch" activation mechanism. Thus, our results suggest that myroilysin is a new bacterial member of the M12A family with an exceptional cysteine switch activation mechanism. Our results shed new light on the classification of the M12A family and may suggest a divergent evolution of the M12 family.

Differential roles for ArcA and ArcB homologues in swarming motility in Serratia marcescens FS14.

ArcAB is a two-component regulatory system that can help bacteria respond to and survive in a changing environment. To identify the function of ArcAB homologues in Serratia marcescens FS14, in-frame deletion mutants of the arcA, arcB and arcAB genes were constructed. Surprisingly, ArcB affects the motility of FS14, but ArcA does not. These results are the reverse of those found in Escherichia coli. Further studies demonstrated that ArcB could promote bacterial motility by activating the synthesis of flagella and particularly by activating the expression of the biosurfactant serrawettin W1. Our results suggest that ArcB may regulate FS14 motility by interacting with an unidentified response regulator other than ArcA. The regulation of ArcAB may be bacterial strain-specific, and the same regulatory system may participate in different mechanisms to adapt to different environments.

Crystal Structure and Function of PqqF Protein in the Pyrroloquinoline Quinone Biosynthetic Pathway.

Pyrroloquinoline quinone (PQQ) has received considerable attention due to its numerous important physiological functions. PqqA is a precursor peptide of PQQ with two conserved residues: glutamate and tyrosine. After linkage of the Cγ of glutamate and Cϵ of tyrosine by PqqE, these two residues are hypothesized to be cleaved from PqqA by PqqF. The linked glutamate and tyrosine residues are then used to synthesize PQQ. Here, we demonstrated that the pqqF gene is essential for PQQ biosynthesis as deletion of it eliminated the inhibition of prodigiosin production by glucose. We further determined the crystal structure of PqqF, which has a closed clamshell-like shape. The PqqF consists of two halves composed of an N- and a C-terminal lobe. The PqqF-N and PqqF-C lobes form a chamber with the volume of the cavity of ∼9400 Å(3) The PqqF structure conforms to the general structure of inverzincins. Compared with the most thoroughly characterized inverzincin insulin-degrading enzyme, the size of PqqF chamber is markedly smaller, which may define the specificity for its substrate PqqA. Furthermore, the 14-amino acid-residue-long tag formed by the N-terminal tag from expression vector precisely protrudes into the counterpart active site; this N-terminal tag occupies the active site and stabilizes the closed, inactive conformation. His-48, His-52, Glu-129 and His-14 from the N-terminal tag coordinate with the zinc ion. Glu-51 acts as a base catalyst. The observed histidine residue-mediated inhibition may be applicable for the design of a peptide for the inhibition of M16 metalloproteases.

Crystal structure of the sensor domain of BaeS from Serratia marcescens FS14.

The sensor histidine kinases of two-component signal-transduction systems (TCSs) are essential for bacteria to adapt to variable environmental conditions. The two-component regulatory system BaeS/R increases multidrug and metal resistance in Salmonella and Escherichia coli. In this study, we report the X-ray structure of the periplasmic sensor domain of BaeS from Serratia marcescens FS14. The BaeS sensor domain (34-160) adopts a mixed α/ß-fold containing a central four-stranded antiparallel ß-sheet flanked by a long N-terminal α-helix and additional loops and a short C-terminal α-helix on each side. Structural comparisons revealed that it belongs to the PDC family with a remarkable difference in the orientation of the helix α2. In the BaeS sensor domain, this helix is situated perpendicular to the long helix α1 and holds helix α1 in the middle with the beta sheet, whereas in other PDC domains, helix α2 is parallel to helix α1. Because the helices α1 and α2 is involved in the dimeric interface, this difference implies that BaeS uses a different dimeric interface compared with other PDC domains. Proteins 2017; 85:1784-1790. © 2017 Wiley Periodicals, Inc.

Presence of an amino acid residue at position 619 required for the function of YidC in Rhodobacter sphaeroides.

YidC, the bacterial homologous protein of Oxa1 and Alb3, could insert membrane proteins into the membrane. Rhodobacter sphaeroides is a kind of photobacteria with abundant intracytoplasmic membranes. In this study, the functions of R. sphaeroides YidC and its C-terminus were investigated in the Escherichia coli YidC gene depletion strain FTL10. The results showed that RS_YidC could complement the growth of the strain FTL10, but the RS_YidC last 5 residues (619-623, KKRKP) deletion mutant could not. Interestingly, the site-directed RS_YidC mutants of any one or all of these 5 residues were still active. The deletion mutant of the last 4 residues and even the last 4 residues deletion mutant with substitution of the Ala or Glu for Lys619 still had sufficient activity to complement the growth of the strain FTL10. These results indicated that the length of the C-terminus of Rs_YidC is more important for its function than the amino acid composition or the charges of it, and the presence of an amino acid residue at position 619 is required for Rs_YidC function in E. coli. Our result also suggests that Rs_YidC may function differently as compared to its homologs.

Crystal structure of the catalytic domain of PigE: a transaminase involved in the biosynthesis of 2-methyl-3-n-amyl-pyrrole (MAP) from Serratia sp. FS14.

Prodigiosin, a tripyrrole red pigment synthesized by Serratia and some other microbes through a bifurcated biosynthesis pathway, MBC (4-methoxy-2,2'-bipyrrole-5-carbaldehyde) and MAP (2-methyl-3-n-amyl-pyrrole) are synthesized separately and then condensed by PigC to form prodigiosin. MAP is synthesized sequentially by PigD, PigE and PigB. PigE catalyzes the transamination of an amino group to the aldehyde group of 3-acetyloctanal, resulting in an aminoketone, which spontaneously cyclizes to form H2MAP. Here we report the crystal structure of the catalytic domain of PigE which involved in the biosynthesis of prodigiosin precursor MAP for the first time to a resolution of 2.3Å with a homodimer in the asymmetric unit. The monomer of PigE catalytic domain is composed of three domains with PLP as cofactor: a small N-terminal domain connecting the catalytic domain with the front part of PigE, a large PLP-binding domain and a C-terminal domain. The residues from both monomers build the PLP binding site at the interface of the dimer which resembles the other PLP-dependent enzymes. Structural comparison of PigE with Thermus thermophilus AcOAT showed a higher hydrophobic and smaller active site of PigE, these differences may be the reason for substrate specificity.

Crystal structures of Cg1458 reveal a catalytic lid domain and a common catalytic mechanism for the FAH family.

Cg1458 was recently characterized as a novel soluble oxaloacetate decarboxylase. However, sequence alignment identified that Cg1458 has no similarity with other oxaloacetate decarboxylases and instead belongs to the FAH (fumarylacetoacetate hydrolase) family. Differences in the function of Cg1458 and other FAH proteins may suggest a different catalytic mechanism. To help elucidate the catalytic mechanism of Cg1458, crystal structures of Cg1458 in both the open and closed conformations have been determined for the first time up to a resolution of 1.9 Å (1 Å=0.1 nm) and 2.0 Å respectively. Comparison of both structures and detailed biochemical studies confirmed the presence of a catalytic lid domain which is missing in the native enzyme structure. In this lid domain, a glutamic acid-histidine dyad was found to be critical in mediating enzymatic catalysis. On the basis of structural modelling and comparison, as well as large-scale sequence alignment studies, we further determined that the catalytic mechanism of Cg1458 is actually through a glutamic acid-histidine-water triad, and this catalytic triad is common among FAH family proteins that catalyse the cleavage of the C-C bond of the substrate. Two sequence motifs, HxxE and Hxx…xxE have been identified as the basis for this mechanism.

PqqF inhibits T6SS secretion by decreasing the pH in Serratia marcescens FS14.

Pyrroloquinoline quinone (PQQ) is a redox cofactor with numerous important physiological functions, and the type VI secretion system (T6SS) is commonly found in Gram-negative bacteria and plays important roles in physiological metabolism of the bacteria. In this study, we found that the deletion of pqqF enhanced the secretion of Hcp-1 in Serratia marcesens FS14 in M9 medium. Transcriptional analysis showed that the deletion of pqqF almost had no effect on the expression of T6SS-1. Further study revealed that the increased secretion of Hcp-1 was altered by the pH changes of the culture medium through the reaction catalyzed by the glucose dehydrogenases in FS14. Finally, we demonstrated that decreased pH of culture medium has similar inhibition effects as PQQ induced on the secretion of T6SS-1. This regulation mode on T6SS by pH in FS14 is different from previously reported in other bacteria. Therefore, our results suggest a novel pH regulation mode of T6SS in S. marcesens FS14, and would broaden our knowledge on the regulation of T6SS secretion.

Structural insight into subunit F of respiratory chain complex I from Xanthomonas oryzae pv. oryzae inhibition by parthenolide.

BACKGROUND: Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious diseases of rice, and there is a lack of bactericides for controlling this disease. We previously found parthenolide (PTL) is a potential lead for developing bactericides against Xoo, and subunit F of respiratory chain complex I (NuoF) is an important target protein of PTL. However, the binding modes of PTL with NuoF need further elucidation. RESULTS: In this study, we obtained the crystal structure of Xoo NuoEF (complex of subunit E and F of respiratory chain complex I) with a resolution of 2.36 Å, which is the first report on the protein structure of NuoEF in plant-pathogenic bacteria. The possible binding sites of PTL with NuoF (Cys105 and Cys187) were predicted with molecular docking and mutated into alanine using a base mismatch method. The mutated proteins were expressed in Escherichia coli and purified with affinity chromatography. The binding abilities of PTL with mutated proteins were investigated via pull-down assay and BIAcore analysis, which revealed that double mutation of Cys105 and Cys187 in NuoF severely affected the binding ability of PTL with NuoF. In addition, the binding modes were further simulated with combined quantum mechanical/molecular mechanical calculations, and the results indicated that PTL may have a stronger binding with Cys105 than Cys187. CONCLUSION: NuoEF protein structure of Xoo was resolved, and Cys105 and Cys187 in NuoF are important binding sites of PTL. This study further clarified the action mechanism of PTL against Xoo, and will promote the innovation of bactericides targeting Xoo complex I. © 2024 Society of Chemical Industry.

Molecular Mechanism of Fusarium Fungus Inhibition by Phenazine-1-carboxamide.

Fusarium head blight caused by Fusarium graminearum is a devastating disease in wheat that seriously endangers food security and human health. Previous studies have found that the secondary metabolite phenazine-1-carboxamide produced by biocontrol bacteria inhibited F. graminearum by binding to and inhibiting the activity of histone acetyltransferase Gcn5 (FgGcn5). However, the detailed mechanism of this inhibition remains unknown. Our structural and biochemical studies revealed that phenazine-1-carboxamide (PCN) binds to the histone acetyltransferase (HAT) domain of FgGcn5 at its cosubstrate acetyl-CoA binding site, thus competitively inhibiting the histone acetylation function of the enzyme. Alanine substitution of the residues in the binding site shared by PCN and acetyl-CoA not only decreased the histone acetylation level of the enzyme but also dramatically impacted the development, mycotoxin synthesis, and virulence of the strain. Taken together, our study elucidated a competitive inhibition mechanism of Fusarium fungus by PCN and provided a structural template for designing more potent phenazine-based fungicides.