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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. [ABSTRACT FROM AUTHOR]

N‐Heterocyclic amines have been known as an important class of nitrogen‐containing molecules with a wide range of applications. Described here is a mild and efficient electrochemical desulfurizative amination of readily accessible heteroaromatic thiols without necessity of metal catalyst. The key to success for this reaction is the in‐situ generation of a hypervalent iodine reagent as an organocatalyst from iodoarene by anodic oxidation. This mild desulfurizative amination presents ample substrate scope and good functional group tolerance without the use of extra stoichiometric chemical oxidants. As only electrons serve as the oxidation reagents, this method offers a straightforward and sustainable manner towards versatile N‐heterocyclic amines, including late‐stage functionalization of pharmaceutically relevant molecules. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A high incidence of restenosis has been reported at the site of inflammation following angioplasty and stent implantation. The anti-proliferative drug paclitaxel (PTX) could help to reduce inflammation and restenosis; however, it has poor water solubility and serious adverse side effects at high doses. Given the presence of metabolic acidosis at the site of inflammation, we hypothesized that nanoparticles that are responsive to low pH could precisely release the loaded drug at the target site. We successfully constructed pH-responsive poly(D, L-lactic-co-glycolic acid) (PLGA) nanoparticles loaded with PTX and NaHCO3 as a pH-sensitive therapeutic agent (PTX-NaHCO3-PLGA NPs). The NPs exhibited remarkable pH sensitivity and a good safety profile both in vitro in rat vascular smooth muscle cells and in vivo in Sprague Dawley rats after tail vein injection. In the rat model, the PTX-NaHCO3-PLGA NPs treatment group showed suppressed intimal proliferation following balloon-induced carotid artery injury compared with that of the saline-treated control. Overall, these results demonstrate that our newly developed pH-responsive nanodrug delivery platform has the potential to effectively inhibit restenosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Myroilysin is a novel bacterial member of M12A metalloproteases family with an uncommon "cysteine switch" activation mechanism and a unique "cap" structure. However, activation of pro-myroilysin is elusive. Here, mature myroilysin was obtained for structure determination by treating pro-myroilysin with trypsin. The structure of mature myroilysin showed that the active-site zinc ion of the mature protein is coordinated by three histidine residues, a water molecule, and a tyrosine residue (Tyr208) in the conserved Met-turn motif (SIMHY). The "cap" structure moves away from the active-site to leave the active cleft open; the newly formed N-terminus is deeply buried in myroilysin, and Glu151 forms a salt bridge directly with the first amino acid residue (Gly38), whereas they are far from each other in the pro-myroilysin. The mutation of Tyr208 indicates that Tyr208 plays an important role in activity of myroilysin. The proteolytic activity and thermostability of mutant E151A decreased dramatically, implying that Glu151 is not only important for catalysis, but also crucial for structural stability in myroilysin. Structural comparison also reveals differences existed between myroilysin and astacin. Our biochemical and structural data provide new insights into the activation of myroilysin and functional involvement of crucial residues Tyr208 and Glu151. [ABSTRACT FROM AUTHOR]

Enolase is an evolutionarily conserved enzyme involved in the processes of glycolysis and gluconeogenesis. Mycoplasma hyopneumoniae belongs to Mycoplasma , whose species are wall-less and among the smallest self-replicating bacteria, and is an important colonizing respiratory pathogen in the pig industry worldwide. Mycoplasma hyopneumoniae enolase (Mhp Eno) expression is significantly increased after infection and was previously found to be a virulence factor candidate. Our studies show that Mhp Eno is a cell surface-localized protein that can adhere to swine tracheal epithelial cells (STECs). Adhesion to STECs can be specifically inhibited by an Mhp Eno antibody. Mhp Eno can recognize and interact with plasminogen with high affinity. Here, the first crystal structure of the mycoplasmal enolase from Mycoplasma hyopneumoniae was determined. The structure showed unique features of Mhp Eno in the S3/H1, H6/S6, H7/H8, and H13 regions. All of these regions were longer than those of other enolases and were exposed on the Mhp Eno surface, making them accessible to host molecules. These results show that Mhp Eno has specific structural characteristics and acts as a multifunctional adhesin on the Mycoplasma hyopneumoniae cell surface. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACT 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/β-fold containing a central four-stranded antiparallel β-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. [ABSTRACT FROM AUTHOR]

S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs) in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens. [ABSTRACT FROM AUTHOR]

Violacein, a natural purple secondary metabolite, is sequentially biosynthesized by five enzymes in the following pathway: VioA-VioB-VioE-VioD-VioC. VioD, a flavin-dependent oxygenase, catalyzes the hydroxylation of the intermediate product prodeoxyviolaceinic acid (PVA) at the 5-position of one indole ring to yield proviolacein. In vitro biochemical data have revealed this process, but the catalytic mechanism still remains largely unclear. Here, the cloning, expression, purification, crystallization and diffraction of VioD are reported. Crystals of VioD diffracted to 1.7 Å resolution and belonged to space group P31, with unit-cell parameters a = b = 90.0, c = 94.5 Å, α = β = 90, γ = 120°. Solvent-content calculation and molecular-replacement results suggest the presence of two molecules of VioD in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Prodigiosin, a member of the prodiginines, is a tripyrrole red pigment synthesized by Serratia and some other microbes. A bifurcated biosynthesis pathway of prodigiosin has been proposed in Serratia in which 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. The first step for the synthesis of MBC is the activation of L-proline by PigI, but its catalytic mechanism has remained elusive. To elucidate its mechanism, recombinant PigI was purified and crystallized. Crystals obtained by the sitting-drop method belonged to space group P1 and diffracted to 2.0 Å resolution, with unit-cell parameters a = 51.2, b = 62.8, c = 91.3 Å, α = 105.1, β = 90.1, γ = 92.2°. Matthews coefficient analysis suggested two molecules in the asymmetric unit, with a VM of 2.6 Å3 Da−1 and a solvent content of 52.69%. [ABSTRACT FROM AUTHOR]

Highlights: [•] The first crystal structure of catalytic domain of PigE was revealed. [•] The substrate binding pocket is different from other homologs. [•] A long alpha helix 17 near the substrate binding site is observed. [Copyright &y& Elsevier]

Proteorhodopsins (PRs), members of the microbial rhodopsin superfamily of seven-transmembrane-helix proteins that use retinal chromophores, comprise the largest subfamily of rhodopsins, yet very little structural information is available. PRs are ubiquitous throughout the biosphere and their genes have been sequenced in numerous species of bacteria. They have been shown to exhibit ion-pumping activity like their archaeal homolog bacteriorhodopsin (BR). Here, the first crystal structure of a proteorhodopsin, that of a blue-light-absorbing proteorhodopsin (BPR) isolated from the Mediterranean Sea at a depth of 12 m ( Med12BPR), is reported. Six molecules of Med12BPR form a doughnut-shaped C 6 hexameric ring, unlike BR, which forms a trimer. Furthermore, the structures of two mutants of a related BPR isolated from the Pacific Ocean near Hawaii at a depth of 75 m ( HOT75BPR), which show a C 5 pentameric arrangement, are reported. In all three structures the retinal polyene chain is shifted towards helix C when compared with other microbial rhodopsins, and the putative proton-release group in BPR differs significantly from those of BR and xanthorhodopsin (XR). The most striking feature of proteorhodopsin is the position of the conserved active-site histidine (His75, also found in XR), which forms a hydrogen bond to the proton acceptor from the same molecule (Asp97) and also to Trp34 of a neighboring protomer. Trp34 may function by stabilizing His75 in a conformation that favors a deprotonated Asp97 in the dark state, and suggests cooperative behavior between protomers when the protein is in an oligomeric form. Mutation-induced alterations in proton transfers in the BPR photocycle in Escherichia coli cells provide evidence for a similar cross-protomer interaction of BPR in living cells and a functional role of the inter-protomer Trp34-His75 interaction in ion transport. Finally, Wat402, a key molecule responsible for proton translocation between the Schiff base and the proton acceptor in BR, appears to be absent in PR, suggesting that the ion-transfer mechanism may differ between PR and BR. [ABSTRACT FROM AUTHOR]

Proteorhodopsins ( PRs), light-driven proton pumps, constitute the largest family of the microbial rhodopsins. PRs are widely distributed in the oceanic environment and freshwater, but no bacteria with PRs have been isolated from freshwater so far. To facilitate isolation of the bacteria with PR genes, we constructed a vector system that can be used to clone potential PR genes and render color changes when overexpressed in Escherichia coli. Using this method, we successfully isolated a strain with PR gene from freshwater and identified it as Exiguobacterium sp. JL-3. The full length PR gene was then cloned using the SEFA PCR method. Protein sequence alignment showed that JL-3_ PR shares high sequence identity (84-89%) with the PRs from Exiguobacterium strains, but low sequence identity (< 38%) with other PRs. Surprisingly, we could not detect any proton-pumping activity in the native JL-3 cells and protoplasts, but the recombinant JL-3_ PR do pump protons when overexpressed in E. coli. Sequence analysis further revealed that the PRs from Exiguobacterium had an unusual lysine as the proton donor instead of the typical acidic residue. These data suggest that JL-3_ PR is a sensory PR rather than a proton pump. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Oxaloacetate decarboxylase catalyses the decarboxylation of oxaloacetate to pyruvate and CO2. Recently, the Corynebacterium glutamicum gene product Cg1458 was determined to be a soluble oxaloacetate decarboxylase. To elucidate the mechanism of oxaloacetate decarboxylation by Cg1458, recombinant Cg1458 was purified and crystallized. The best crystal was grown from 0.2 M MgCl2, 0.1 M Bis-Tris pH 6.0, 25%( w/ v) polyethylene glycol 3350 using the hanging-drop method. The crystals belonged to space group P43212, with unit-cell parameters a = b = 124.1, c = 73.6 Å. The crystals are most likely to contain a dimer in the asymmetric unit, with a VM value of 2.27 Å3 Da−1. A full data set was collected at 1.9 Å resolution using synchrotron radiation on beamline BL17U of SSRF, Shanghai, China. Structure-solution attempts by molecular replacement were successful with PDB entries or as the template. [ABSTRACT FROM AUTHOR]

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. • The crystal structure of PgbP-CoA complex was determined. • PgbP is a prodigiosin-binding protein and prodigiosin is bound in the substrate-binding pocket. • PgbP is a GNAT family protein with high thermostability. [ABSTRACT FROM AUTHOR]

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. • CpxR/A activates the prodigiosin (PG) biosynthesis at the transcriptional level. • Kinase activity of CpxA is not necessary for the PG biosynthesis. • Phosphorylation of CpxR is vital for the regulation of PG biosynthesis. • Phosphatase activity of CpxA plays vital role in the PG biosynthesis regulation. [ABSTRACT FROM AUTHOR]

Abstract Type VI secretion system (T6SS), as a macromolecular system, is commonly found in Gram-negative bacteria and responsible for exporting effectors. T6SS consists of 13 core proteins. TssL is a component of the membrane complex and plays a pivotal role in T6SS. Here, we report the crystal structure of the C-terminal periplasmic domain of TssL (TssL Cter) from Serratia marcescens FS14. The TssL Cter (310–503) contain a five-stranded anti-parallel β-sheet flanked by five α-helices and a short N-terminal helix. Structural comparisons revealed that it belongs to the OmpA-like family with a remarked difference in the conformation of the loop3–5. In OmpA-like family, the corresponding loop is located close to loop2–3, forming a cavity with a small opening together with the longest α5, whereas in TssL Cter , loop3–5 flipped away from this cavity region. In addition, significant differences in the peptidoglycan (PG) binding site suggest that big conformational change must take place to accomplish the PG binding for TssL Cter. Furthermore, a long flexible loop between helices α1 and α2 is unique in TssL. TssL would have a big conformational change during the delivery of the Hcp needle and effectors. So we speculate that the long flexible endows TssL the adaptation of its evolutionary new function. [ABSTRACT FROM AUTHOR]

Prodigiosin, a tripyrrole red pigment is 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. PigI, PigG and PigA have been shown to be involved in the first steps of MBC biosynthesis (proline incorporation). The crystal structure of PigG was resolved to elucidate its function and mechanism. PigG, an acyl carrier protein (ACP), features the ACP architecture:, a helical bundle fold containing three major helices and a minor distorted helix together with a conserved “S” motif. An in-frame deletion mutation of the pigG gene abolished the synthesis of prodigiosin in Serratia marcescens FS14. The production of prodigiosin was fully restored by complementation of intact pigG ; however the S36A mutant was not able to restore function in the in-frame deletion pigG mutant, indicating that PigG and the conserved serine residue (S36) of PigG are essential for the synthesis of prodigiosin. [ABSTRACT FROM AUTHOR]

A serralysin family protease (Serralysin-like protease B, SPB) with unique V-shaped thermostable property was isolated and identified from the Serratia marcescens FS14 by biochemical and molecular biological methods. It is the first time to report the isolation of a native serralysin family protease directly from Serratia species except the well-studied serralysin. SPB has an optimum pH at 8.0 and an optimum temperature at 37 °C. It shows high proteolytic activities after pretreated at 4–50 °C for 10 min respectively and almost no detectable activity after pretreated at 60 °C. Surprisingly, increasing activities were observed after pretreated at 70–90 °C respectively. Further study revealed that the reason behind this phenomenon may be the self-digestion property of SPB with an optimum temperature around 60 °C. This self-digestion property may expand the SPB future application in industry. The bioassay using the healthy cotton bollworm Helicoverpa armigera larvae demonstrated that the serralysin and SPB from FS14 are toxic to the H. armigera larvae. This result implied that FS14 strain and/or the SPB and serralysin in FS14 might have a potential application in insect control. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Background: Coarctation of the aorta (CoA) is a common congenital cardiovascular malformation, and improvements in the diagnostic process for surgical decision-making are important. We sought to compare the diagnostic accuracy of transthoracic echocardiography (TTE) with computed tomographic angiography (CTA) to diagnose CoA., Methods: We retrospectively reviewed 197 cases of CoA diagnosed by TTE and CTA and confirmed at surgery from July 2009 to August 2019., Results: The surgical findings confirmed that 19 patients (9.6%) had isolated CoA and 178 (90.4%) had CoA combined with other congenital cardiovascular malformations. The diagnostic accuracy of CoA by CTA was significantly higher than that of TTE ( χ 2  = 6.52, p  = 0.01). In contrast, the diagnostic accuracy of TTE for associated cardiovascular malformations of CoA was significantly higher than that of CTA ( χ 2  = 15.36, p  < 0.0001). Infants and young children had more preductal type of CoA, and PDA was the most frequent cardiovascular lesion associated with CoA. The pressure gradient was significantly decreased after the first operation, similar at 6 months, 1 year, and 3 years follow-ups by TTE., Conclusions: CTA is more accurate as a clinical tool for diagnosing CoA; however, TTE with color Doppler can better identify associated congenital cardiovascular malformations. Therefore, combining TTE and CTA would benefit clinical evaluation and management in patients suspected of CoA. TTE was valuable for post-operation follow-up and clinical management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Gong, Zhang, Feng, Zhu, Deng, Ran, Li, Kong, Sun and Ji.)

Objective: This study aimed to explore the safety and efficacy of simultaneous interventional therapy for compound congenital heart disease (CCHD) in children., Methods: In total, 155 children with CCHD who received simultaneous interventional therapy at the Children's Hospital of Chongqing Medical University between January 2007 and December 2021 were included in study. Data on clinical manifestations, transthoracic echocardiography, electrocardiogram, and follow-up were retrospectively analyzed., Results: The most common type of CCHD was atrial septal defect (ASD) combined with ventricular septal defect (VSD), accounting for 32.3% of the patients. Simultaneous interventional therapy was successfully administered to 151 children (97.4%). The pulmonary gradient of patients with pulmonary stenosis decreased from 47.3 ± 21.9 mmHg to 15.2 ± 12.2 mmHg ( P  < 0.05) immediately after the procedure. One patient had failed PBPV as he had residual PS >40 mmHg post procedure. The right ventricular dimension and left ventricular end-diastolic dimension significantly decreased in the first month after the procedure in patients with ASD combined with VSD. Twenty-five (16.1%) patients had mild residual shunt, which spontaneously disappeared in more than half of these patients 6 months after the procedure. The major adverse events were minimal ( n  = 4, 2.58%), including one patient requiring drug treatment for complete atrioventricular block and three patients receiving surgical treatment because of cardiac erosion, anterior tricuspid valve chordae rupture, and hemolysis, respectively., Conclusions: ASD combined with VSD is the most common type of CCHD in children, and simultaneous interventional therapy for CCHD in children is safe and effective with satisfactory results. Ventricular remodeling can be reversed in patients with ASD combined with VSD 1 month after the procedure. Most adverse events associated with interventional therapy are mild and manageable., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Ran, Feng, Li, Yi, Zhu and Ji.)

Vascular endothelial growth factor (VEGF) is a potent agonist of angiogenesis that induces proliferation and differentiation of endothelial progenitor cells (EPCs) after vascular injury. Previous studies have suggested that stromal cell-derived factor 1-alpha (SDF-1α) and VEGF have a synergistic effect on vascular stenosis. The aim of the present study was to investigate whether VEGF and SDF-1α act synergistically in EPCs and vascular smooth muscle cells (VSMCs). In this study, EPCs were isolated from rat bone marrow and their morphology and function were studied. Subsequently, VEGF was delivered into EPCs using an adenoviral vector. Tube formation, migration, proliferation, and apoptosis of VEGF-overexpressing EPCs was analyzed. Then, EPCs were co-cultured with VSMCs in the presence or absence of SDF-1α, the migration, proliferation, apoptosis, and differentiation capacity of EPCs and VSMCs were analyzed respectively. The isolated EPCs showed typical morphological features, phagocytic capacity, and expressed surface proteins. While stable expression of VEGF remarkably enhanced tube formation, migration, and proliferation capacity of EPCs, apoptosis was decreased. Moreover, the proliferation, migration, and differentiation capacity of EPCs in the co-cultured model was enhanced in the presence of SDF-1α, and apoptosis was decreased. However, these effects were reversed in VSMCs. Therefore, our results showed that VEGF and SDF-1α synergistically increased the migration, differentiation, and proliferation capabilities of EPCs, but not VSMCs. This study suggests a promising strategy to prevent vascular stenosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, He, Bi, Zhu, Deng, Ran and Ji.)

Prodigiosin, a red linear tripyrrole pigment, is a typical secondary metabolite with numerous biological functions, such as anticancer, antibacterial and immunosuppressant activities, and is synthesized through a bifurcated biosynthesis pathway from 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3- n -amylpyrrole (MAP). The last step in the biosynthetic pathway of MBC is catalysed by PigF, which transfers a methyl group to 4-hydroxy-2,20-bipyrrole-5-carbaldehyde (HBC) to form the final product MBC. However, the catalytic mechanism of PigF is still elusive. In this study, crystal structures of apo PigF and S -adenosylhomocysteine (SAH)-bound PigF were determined. PigF forms a homodimer and each monomer consists of two domains: a C-terminal catalytic domain and an N-terminal dimerization domain. Apo PigF adopts an open conformation, while the structure of the complex with the product SAH adopts a closed conformation. The binding of SAH induces dramatic conformational changes of PigF, suggesting an induced-fit substrate-binding mechanism. Further structural comparison suggests that this induced-fit substrate-recognition mechanism may generally exist in O -methyltransferases. Docking and mutation studies identified three key residues (His98, His247 and Asp248) that are crucial for enzyme activity. The essential function of His247 and Asp248 and structure analysis suggests that both residues are involved in activation of the HBC substrate of PigF. The invariance of Asp248 in PigF further confirmed its essential role. The invariance and essential role of His98 in PigF suggests that it is involved in correctly positioning the substrate. This study provides new insight into the catalytic mechanism of PigF, reveals an induced-fit substrate-recognition model for PigF and broadens the understanding of O -methyltransferases., (© Shenshen Qiu et al. 2022.)

Mycoplasmas are a group of prokaryotes without cell walls that have evolved through several rounds of degenerative evolution. With a low cell DNA G + C content and definitively long genetic lineages, mycoplasmas are thought to be in a state of rapid evolution. However, little associated evidence has been provided. Enolase is a key enzyme in glycolysis that is widely found in all species from the three domains, and it is evolutionarily conserved. In our previous studies, enolase acted as a virulence factor and participated in cell-surface adhesion in Mycoplasma hyopneumoniae . Furthermore, unique loop regions were first found in the crystal structure of Mhp Eno. Here, enolase structures from Mycoplasma pneumoniae and Mycoplasma bovis were determined. An extra helix 7 is specific and conservatively found in almost all mycoplasma enolases, as confirmed by crystal structures and sequence alignment. Particular motifs for helix 7, which is composed of F-K/G-K-L/F-K-X-A-I, have been proposed and could be regarded as molecular markers. To our surprise, the genetic distances between any two mycoplasma enolases were obviously longer than those between the two corresponding species themselves, indicating divergent evolution of mycoplasma enolases, whereas no horizontal gene transfer was detected in mycoplasma enolase genens. Furthermore, different evolutionary patterns were adopted by different loop regions of mycoplasma enolase. Enolases from different Mycoplasma species also showed different affinities for PLG and fibronectin. Our results indicate the rapid and divergent evolution of mycoplasma enolase and mycoplasmas. This study will also aid understanding the independent evolution of Mycoplasma species after separation from their common ancestor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Zhao, Gan, Feng, Cui, Xie, Hao, Zhang, Wang, Ran, Wang, Zhang, Li, Zhang, Pang, Xiong and Shao.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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/β-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/β-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/β-hydrolase members., (Copyright © 2020 American Society for Microbiology.)

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 Å resolution as well as in complex with ATP to a 1.41 Å resolution and with an ATP analogue AMP-PCP to a 1.45 Å resolution. 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., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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 HE XX H XX G XX H; 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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The HNH endonuclease superfamily usually contains a conserved HNH motif in the sequence, and the second subfamily of it uses N to replace the second H in the HNH motif. A bacterium with extracellular thermostable DNase was isolated and identified as Exiguobacterium sp. yc3. A 20 kDa putative DNase was later purified and the encoding gene of it was amplified and sequenced, the deduced amino acid sequence analysis showed that the protein belongs to the HNH endonuclease superfamily, and therefore it was named as EheA ( E: xiguobacterium H: NH E: ndonuclease). Characterization of the recombinant EheA confirmed that EheA is a DNase. By site-directed mutation method, H116, N141 and N156 were proved to be essential for the DNase activity. EheA is the first experimentally determined bacterial source endonuclease belonging to the second subfamily of HNH superfamily. Further bioinformatic analysis showed that EheA homologue genes are conserved in the Exiguobacterium species, which suggests their possible important functions for Exiguobacterium species. And as a thermostable DNase, EheA also has a promising future in many application fields., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Proteorhodopsins (PRs), seven-transmembrane chromoproteins with retinal as a chromophore, are light-driven proton pumps. To elucidate the light-driven proton-pumping mechanism of PRs, a pET28a vector containing the blue-light-absorbing proteorhodopsin (BPR) gene was constructed and the protein was overexpressed in Escherichia coli. The protein was purified by immobilized metal-ion affinity chromatography (IMAC). The purified BPR D97N mutant protein (BPR&#95;D97N) was crystallized using the vapour-diffusion method. Preliminary X-ray diffraction data analysis showed that the crystal belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 161.6, b = 168.6, c = 64.7 Å. A complete data set was collected to 3.3 Å resolution using synchrotron radiation on beamline X06 of the Swiss Light Source (SLS). Molecular replacement was unsuccessful. To solve the structure of BPR&#95;D97N by experimental phasing, selenomethionine-substituted protein crystals were prepared. These crystals diffracted to 3.0 Å resolution and a complete data set was collected on beamline BL17U of the Shanghai Synchrotron Radiation Facility (SSRF). Heavy-atom substructure determination and phasing by SAD clearly showed that the crystal contained five molecules in the asymmetric unit, with a V(M) of 3.26 Å(3) Da(-1) and a solvent content of 62.3%., (© 2012 International Union of Crystallography)