Analysis of gene islands involved in methanopterin-linked C1 transfer reactions reveals new functions and provides evolutionary insights

In this study, the occurrence and chromosomal clustering of genes encoding C(1) transfer reactions linked to tetrahydromethanopterin (H(4)MPT) were analyzed in a variety of proteobacteria and in representatives of the Planctomycetes via genomic analysis or via partial sequencing by cosmid walking. Although a tendency for clustering was found common for the genes of interest, significant variations in gene order and the degree of clustering were uncovered both between and within different groups of Proteobacteria and between Proteobacteria and Planctomycetes. Phylogenetic analyses suggested that the evolution of genes encoding H(4)MPT-linked reactions in Proteobacteria involved lateral transfers within Proteobacteria and possibly between Proteobacteria and other phyla. Gene cluster comparisons revealed a number of novel genes potentially involved in the C(1) transfer reactions, and these were analyzed by mutation and expression analyses. Four genes, a homolog of pabB, and three genes conserved between methanogenic Archaea and Bacteria possessing H(4)MPT-linked functions, orfY, orf1, and afpA were shown to be involved in formaldehyde oxidation/detoxification, as judged by specific mutant phenotypes. In particular, pabB contributes to the biosynthesis of para-aminobenzoic acid, a precursor of both tetrahydrofolate and H(4)MPT, and afpA apparently encodes a novel dihydromethanopterin reductase, based on mutant complementation experiments.     

Characterization of the formyltransferase from Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate-reducing archaea. Here we describe the purification and characterization of formylmethanofuran-tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylmethanofuran (formylMFR) and tetrahydromethanopterin (H4MPT) from N5-formylH4MPT and methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activity with MFR and H4MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent Km for formylMFR = 50 microM; apparent Km for H4MPT = 30 microM). The enzyme is encoded by the ffsA gene and exhibits a sequence identity of approximately 40% with Ftr from methanogenic and sulfate-reducing archaea. The 32-kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit. Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR. Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active-site cofactors molybdenum, molybdopterin and a [4Fe-4S] cluster. Cytochrome c was found to be spontaneously reduced by H4MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.     

Biochemical characterization of a dihydromethanopterin reductase involved in tetrahydromethanopterin biosynthesis in Methylobacterium extorquens AM1

During growth on one-carbon (C1) compounds, the aerobic alpha-proteobacterium Methylobacterium extorquens AM1 synthesizes the tetrahydromethanopterin (H4MPT) derivative dephospho-H4MPT as a C1 carrier in addition to tetrahydrofolate. The enzymes involved in dephospho-H4MPT biosynthesis have not been identified in bacteria. In archaea, the final step in the proposed pathway of H4MPT biosynthesis is the reduction of dihydromethanopterin (H2MPT) to H4MPT, a reaction analogous to the reaction of the bacterial dihydrofolate reductase. A gene encoding a dihydrofolate reductase homolog has previously been reported for M. extorquens and assigned as the putative H2MPT reductase gene (dmrA). In the present work, we describe the biochemical characterization of H2MPT reductase (DmrA), which is encoded by dmrA. The gene was expressed with a six-histidine tag in Escherichia coli, and the recombinant protein was purified by nickel affinity chromatography and gel filtration. Purified DmrA catalyzed the NAD(P)H-dependent reduction of H2MPT with a specific activity of 2.8 micromol of NADPH oxidized per min per mg of protein at 30 degrees C and pH 5.3. Dihydrofolate was not a substrate for DmrA at the physiological pH of 6.8. While the existence of an H2MPT reductase has been proposed previously, this is the first biochemical evidence for such an enzyme in any organism, including archaea. Curiously, no DmrA homologs have been identified in the genomes of known methanogenic archaea, suggesting that bacteria and archaea produce two evolutionarily distinct forms of dihydromethanopterin reductase. This may be a consequence of different electron donors, NAD(P)H versus reduced F420, used, respectively, in bacteria and methanogenic archaea.     

Novel dephosphotetrahydromethanopterin biosynthesis genes discovered via mutagenesis in Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 was used to explore the genetics of dephosphotetrahydromethanopterin (dH(4)MPT) biosynthesis. Strains with mutations in eight "archaeal-type" genes linked on the chromosome of M. extorquens AM1 were analyzed for the ability to synthesize dH(4)MPT, and six were found to be dH(4)MPT negative. Putative functions of these genes in dH(4)MPT biosynthesis are discussed.     

The adenovirus E4orf6 protein inhibits DNA double strand break repair and radiosensitizes human tumor cells in an E1B-55K-independent manner

The adenoviral protein E4orf6 has been shown to inhibit both in vitro V(D)J recombination and adenoviral DNA concatenation, two processes that rely on cellular DNA double strand break repair (DSBR) proteins. Most of the known activities of E4orf6 during adenoviral infection require its interaction with another adenoviral protein, E1B-55K. Here we report that E4orf6, stably expressed in RKO human colorectal carcinoma cells or transiently expressed by adenoviral vector in U251 human glioblastoma cells, inhibits DSBR and induces significant radiosensitization in the absence of E1B-55K. Expression of a mutant form of E4orf6 (L245P) failed to radiosensitize RKO cells. E4orf6 reduced DSBR capacity in transfected and infected cells, as measured by sublethal DNA damage repair assay and phosphorylated H2AX (gamma-H2AX) levels, respectively. Consistent with the inhibitory effect of E4orf6 on DSBR, expression of wild-type but not mutant E4orf6 reduced recovery of a transfected, replicating reporter plasmid (pSP189) in 293 cells but did not increase the mutation frequency measured in the reporter plasmid. The kinase activity of DNA-PKcs (the DNA-dependent protein kinase catalytic subunit) toward heterologous substrates was not affected by expression of E4orf6; however, autophosphorylation of DNA-PKcs at Thr-2609 following ionizing radiation was prolonged in the presence of E4orf6 when compared with control-infected cells. Our results demonstrate for the first time that E4orf6 expression hinders the cellular DNA repair process in mammalian cells in the absence of E1B-55K or other adenoviral genes and suggest that viral-mediated delivery of E4orf6, combined with localized external beam radiation, could be a useful approach for the treatment of radioresistant solid tumors such as glioblastomas.     

Novel methylotrophy genes of Methylobacterium extorquens AM1 identified by using transposon mutagenesis including a putative dihydromethanopterin reductase

Ten novel methylotrophy genes of the facultative methylotroph Methylobacterium extorquens AM1 were identified from a transposon mutagenesis screen. One of these genes encodes a product having identity with dihydrofolate reductase (DHFR). This mutant has a C(1)-defective and methanol-sensitive phenotype that has previously only been observed for strains defective in tetrahydromethanopterin (H(4)MPT)-dependent formaldehyde oxidation. These results suggest that this gene, dmrA, may encode dihydromethanopterin reductase, an activity analogous to that of DHFR that is required for the final step of H(4)MPT biosynthesis.     

Application of a colorimetric assay to identify putative ribofuranosylaminobenzene 5′-phosphate synthase genes expressed with activity in<Emphasis Type="Italic">Escherichia coli</Emphasis>

Tetrahydromethanopterin (H₄MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5′-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H₄MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies inEscherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase fromArchaeoglobus fulgidus was produced inE. coli and purified to homogeneity. The production of active RFAP synthase fromMethanothermobacter thermautotrophicus was achieved by coexpression of the geneMTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase. Florida Agricultural Experiment Station Journal Series no. R-09353 Published: March 4, 2003     

橙色红曲菌As3.4384 orf7基因缺失株的构建及其功能分析

红曲菌能产生多种有益的次级代谢产物,但红曲菌也产生一种对人和哺乳动物肝和肾有毒害的毒素,即桔霉素。因此控制毒素的产生是保障红曲产品安全性所必须的。故对桔霉素的合成途径及相关的基因做深入了解。6个桔霉素合成相关的基因成簇位于21 kb的DNA片段上。克隆了一个新基因(orf7基因),其位于该基因簇的外侧。采用基因敲除技术,构建红曲菌orf7基因缺失菌株。并采用紫外分光光度法检测orf7基因缺失菌株的红曲色素产量,HPLC法检测其桔霉素产量。orf7缺失菌株产红曲色素能力与出发菌株As3.4384相比没有变化;产桔霉素培养13~19 d,桔霉素的产量与出发菌株As3.4384相比增加了 142.4%。从而证实orf7基因与桔霉素代谢相关。     

Adenovirus Type 5 E4 Open Reading Frame 4 Protein Induces Apoptosis in Transformed Cells

Adenovirus type 5 E4 open reading frame 4 (E4orf4) protein has been previously shown to counteract transactivation of the junB and c-fos genes by cyclic AMP plus E1A protein and to interact with protein phosphatase 2A (PP2A). Here, we show that the wild-type E4orf4 protein induces apoptosis in the E1A-expressing 293 cells, in NIH 3T3 cells transformed with v-Ras, and in the lung carcinoma cell line H1299. The induction of apoptosis is not accompanied by enhanced levels of p53 in 293 cells and occurs in the absence of p53 in H1299 cells, indicating involvement of a p53-independent pathway. A mutant E4orf4 protein that had lost the ability to induce apoptosis also lost its ability to bind PP2A. We suggest that E4orf4 antagonizes continuous signals to proliferate, like those given by E1A or v-Ras, and that the conflicting signals lead to the induction of cell death.     

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth.

spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis. Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth. We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth. In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation. However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth. The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon. The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H. The chs203 insertion mutation caused a 2- to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H. The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.     

Analyses of single-amino-acid substitution mutants of adenovirus type 5 E1B-55K protein

The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus dl309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.     

Characterization of two cryptic Helicobacter pylori plasmids: a putative source for horizontal gene transfer and gene shuffling

Many Helicobacter pylori isolates carry cryptic plasmids of extremely variable size. In this study we analyzed two H. pylori plasmids, pHel4 and pHel5, from H. pylori strains P8 and P29, respectively. Plasmid pHel4 consists of 10,970 bp, constituting 15 putative open reading frames (ORFs), whereas pHel5 consists of 18,291 bp, constituting 17 ORFs. The findings that both plasmids encode a conserved RepA protein and that both have an origin of replication containing an iteron place them in the group of theta plasmids. In pHel4, the products of the overlapping orf4C, orf4D, orf4E, and orf4F sequences are homologous to MobA, MobB, MobC, and MobD, encoded by colicinogenic plasmids, suggesting that pHel4 might be mobilizable. A further putative operon consists of orf4B and orf4A, the products of which are homologous to microcin C7 (MccC7) biosynthesis and secretion proteins MccB and MccC, respectively. Plasmid pHel5 carries putative genes encoding proteins with homology to an endonuclease and gene products of an H. pylori chromosomal plasticity zone. Both plasmids contain repeat sequences, such as the previously identified R2 repeat, which are considered preferred recombination sites. In pHel4, a new repeat sequence (R4 repeat), which seems to act as a hot spot for site-specific recombination, was identified. All H. pylori plasmids characterized so far have a modular structure. We suggest a model that explains the existing plasmids by insertions and deletions of genetic elements at the repeat sequences. A genetic exchange between plasmids and the bacterial chromosome, combined with plasmid mobilization, might add a novel mechanism to explain the high genetic macrodiversity within the H. pylori population.     

Roles for the E4 orf6, orf3, and E1B 55-Kilodalton Proteins in Cell Cycle-Independent Adenovirus Replication

Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.     

Expansion of the clavulanic acid gene cluster: identification and in vivo functional analysis of three new genes required for biosynthesis of clavulanic acid by Streptomyces clavuligerus

Clavulanic acid is a potent inhibitor of beta-lactamase enzymes and is of demonstrated value in the treatment of infections by beta-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain Streptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219-236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11 (fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement and trans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9 (cad) in the clavulanic acid cluster. While the orf10 (cyp) and orf11 (fd) proteins show homologies to other known CYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed.     

Aerobic degradation of mercaptosuccinate by the gram-negative bacterium Variovorax paradoxus strain B4

The Gram-negative bacterium Variovorax paradoxus strain B4 was isolated from soil under mesophilic and aerobic conditions to elucidate the so far unknown catabolism of mercaptosuccinate (MS). During growth with MS this strain released significant amounts of sulfate into the medium. Tn5::mob-induced mutagenesis was successfully employed and yielded nine independent mutants incapable of using MS as a carbon source. In six of these mutants, Tn5::mob insertions were mapped in a putative gene encoding a molybdenum (Mo) cofactor biosynthesis protein (moeA). In two further mutants the Tn5::mob insertion was mapped in the gene coding for a putative molybdopterin (MPT) oxidoreductase. In contrast to the wild type, these eight mutants also showed no growth on taurine. In another mutant a gene putatively encoding a 3-hydroxyacyl-coenzyme A dehydrogenase (paaH2) was disrupted by transposon insertion. Upon subcellular fractionation of wild-type cells cultivated with MS as sole carbon and sulfur source, MPT oxidoreductase activity was detected in only the cytoplasmic fraction. Cells grown with succinate, taurine, or gluconate as a sole carbon source exhibited no activity or much lower activity. MPT oxidoreductase activity in the cytoplasmic fraction of the Tn5::mob-induced mutant Icr6 was 3-fold lower in comparison to the wild type. Therefore, a new pathway for MS catabolism in V. paradoxus strain B4 is proposed: (i) MPT oxidoreductase catalyzes the conversion of MS first into sulfinosuccinate (a putative organo-sulfur compound composed of succinate and a sulfino group) and then into sulfosuccinate by successive transfer of oxygen atoms, (ii) sulfosuccinate is cleaved into oxaloacetate and sulfite, and (iii) sulfite is oxidized to sulfate.     

Molecular cloning and identification of the laspartomycin biosynthetic gene cluster from Streptomyces viridochromogenes

The biosynthetic gene cluster for laspartomycins, a family of 11 amino acid peptide antibiotics, has been cloned and sequenced from Streptomyces viridochromogenes ATCC 29814. Annotation of a segment of 88912bp of S. viridochromogenes genomic sequence revealed the putative lpm cluster and its flanking regions which harbor 43 open reading frames. The lpm cluster, which spans approximately 60 kb, consists of 21 open reading frames. Those include four NRPS genes (lpmA/orf18, lpmB/orf25, lpmC/orf26 and lpmD/orf27), four genes (orfs 21, 22, 24 and 29) involved in the lipid tail biosynthesis and attachment, four regulatory genes (orfs 13, 19, 32 and 33) and three putative exporters or self-resistance genes (orfs 14, 20 and 30). In addition, the gene involved in the biosynthesis of the nonproteinogenic amino acid Pip was also identified in the lpm cluster while the genes necessary for the biosynthesis of the rare residue diaminopropionic acid (Dap) were found to reside elsewhere on the chromosome. Interestingly, the dabA, dabB and dabC genes predicted to code for the biosynthesis of the unusual amino acid diaminobutyric acid (Dab) are organized into the lpm cluster even though the Dab residue was not found in the laspartomycins. Disruption of the NRPS lpmC gene completely abolished laspartomycin production in the corresponding mutant strain. These findings will allow molecular engineering and combinatorial biosynthesis approaches to expand the structural diversity of the amphomycin-group peptide antibiotics including the laspartomycins and friulimicins.