Identification of genes necessary for jinggangmycin biosynthesis from Streptomyces hygroscopicus 10-22

A series of large chromosomal deletions in Streptomyces hygroscopicus 10-22 were aligned on the physical map of the wild-type strain and the mutants were assessed for their ability to produce the aminocyclitol antibiotic 5102-I (jinggangmycin). Twenty-eight mutants were blocked for jinggangmycin production and all of them were found to lack a 300 kb AseI-F fragment of the wild-type chromosome. An ordered cosmid library of the 300 kb AseI-F fragment was made and one of the cosmids conferred jinggangmycin productivity to Streptomyces lividans ZX1. Three of the overlapping cosmids (18G7, 5H3 and 9A2) also hybridized to the valA gene of the validamycin pathway from S. hygroscopicus 5008 as a probe. This gene resembles acbC from Actinoplanes sp. 50/110, which encodes a C7-cyclitol synthase that catalyses the transformation of sedoheptulose 7-phosphate into 2-5-epi-valiolone for acarbose biosynthesis. The valA/acbC-homolog (orf1) of S. hygroscopicus 10-22 was shown to be essential for jinggangmycin biosynthesis as an engineered mutant with a specific in-frame deletion removing a 609 bp sequence internal to orf1 completely abolished jinggangmycin production and the corresponding knock-out mutant (JXH4) could be complemented for jinggangmycin production by the introduction of an orf1-containing construct. Concurrently, the identities of the genes common to S. hygroscopicus strains 10-22 and 5008 prompted a comparison of the chemical structures of jinggangmycin and validamycin, which led to a clear demonstration that they are identical.The first two authors contributed equally to this study.     

Genes, enzymes and coenzymes of queuosine biosynthesis in procaryotes

In almost all known tRNAs that are specific for Asp, Asn, His or Tyr the wobble position of the anticodon is occupied by the hypermodified tRNA nucleoside queuosine. This unusual deazaguanine derivative is synthesised only in eubacteria. The biosynthesis, as investigated in Escherichia coli, is accomplished in four steps involving many unprecedented enzymatic reactions.     

Mechanistic studies of Bacillus subtilis QueF, the nitrile oxidoreductase involved in queuosine biosynthesis

The enzyme QueF was recently identified as an enzyme involved in the biosynthesis of queuosine, a 7-deazaguanosine modified nucleoside found in bacterial and eukaryotic tRNA. QueF exhibits sequence homology to the type I GTP cyclohydrolases characterized by FolE, but contrary to the predictions based on sequence analysis the enzyme in fact catalyzes a mechanistically unrelated reaction, the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1), a late step in the queuosine pathway. The reduction of a nitrile is unprecedented in biology, and we report here characterization and mechanistic studies of the enzyme from Bacillus subtilis. The recombinant enzyme exhibits optimal activity at pH 7.5 and moderate ionic strength, and is not dependent on metal ions for catalytic activity. Steady-state kinetic analysis provided a kcat = 0.66 +/- 0.04 min-1, KM (preQ0) = 0.237 +/- 0.045 microM, and KM (NADPH) = 19.2 +/- 1.1 microM. Based on sequence analysis and homology modeling we predicted previously that Cys55 would be present in the active site and in proximity to the nitrile group of preQ0. Consistent with that prediction we observed that the enzyme was inactivated when preincubated with iodoacetamide, and protected from inactivation when preQ0 was present. Furthermore, titrations of the enzyme with preQ0 in the absence of NADPH were accompanied by the appearance of a new absorption band at 376 nm in the UV-vis spectrum consistent with the formation of an alpha,beta-unsaturated thioimide. Site-directed mutagenesis of Cys55 to Ala or Ser resulted in loss of catalytic activity and no absorption at 376 nm upon addition of preQ0. Based on our data we propose a chemical mechanism for the enzyme-catalyzed reaction, and a chemical rationale for the observation of covalent catalysis.     

Characterization of the expression and regulation of genes necessary for myo-inositol biosynthesis and transport in the seminiferous epithelium

In many mammals, the concentration of myo-inositol in the fluid of the seminiferous tubules is dramatically higher than levels found in serum. Two enzymes involved in myo-inositol synthesis: myo-inositol-1-phosphate synthase (ISYNA1) and myo-inositol monophosphatase-1 (IMPA1), are known to have high activity in the testes. ISYNA1 is an isomerase that catalyzes the conversion of glucose-6-phoshate to myo-inositol-1-phosphate. IMPA1 then hydrolyzes the phosphate group to produce myo-inositol. Although no physiological role for the high concentration of myo-inositol has yet to be elucidated, it has been suggested that it could be involved in osmoregulation. Previous research on these enzymes in the testis has focused on enzyme activity. The objective of this study was to evaluate the expression of these genes and the myo-inositol transporter, Slc5a3, within the testis. Using Northern blot analyses, we found that all three genes, Impa1, Isyna1, and Slc5a3 are expressed in Sertoli cells. Isyna1 is highly expressed in two types of germ cells, pachytene spermatocytes and round spermatids. IMPA1 was expressed in round spermatids. Slc5a3 expression is upregulated when Sertoli cells are treated with 0.1 mM dibutyryl cAMP. When Sertoli cells were cultured in a hypertonic medium, there was an increase in the expression of Isyna1 and Slc5a3. We postulate that this upregulation is a result of the capability of the Sertoli cell to sense and then react to a change in osmolarity by increasing the transport and production of the osmolyte myo-inositol.     

Identification of aminotransferase genes for biosynthesis of aminoglycoside antibiotics from soil DNA

Aminoglycoside has been known as a clinically important antibiotic for a long time, but genetic information for the biosynthesis of aminoglycoside is still insufficient. In this study, we tried to clone aminoglycoside-biosynthetic genes from soil DNA for accumulation of genetic information. We chose the genes encoding L-glutamine:(2-deoxy-)scyllo-inosose aminotransferase as the target, because it is specific for all types of aminoglycoside biosynthesis. By degenerate PCR, we obtained 33 individual clones that were homologous with aminotransferase genes in aminoglycoside biosynthesis. Phylogenetic analysis and alignment of these genes showed that horizontal gene transfer has occurred in the soil. Among these, several quite interesting genes were obtained. Some genes probably originated from non-actinomycetes, and some were far from the known homologs. These genes can be useful markers for the isolation of entire gene clusters and originating organisms.     

Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway

Background

20-hydroxyecdysone (20E) and its receptor complex ecdysone receptor (EcR) and ultraspiracle (USP) play a crucial role in controlling development, metamorphosis, reproduction and diapause. The ligand-receptor complex 20E-EcR/USP directly activates a small set of early-response genes and a much larger set of late-response genes. However, ecdysone-responsive genes have not been previously characterized in the context of insect chitin biosynthesis.

Principal Findings

Here, we show that injection-based RNA interference (RNAi) directed towards a common region of the two isoforms of SeEcR in a lepidopteron insect Spodoptera exigua was effective, with phenotypes including a high mortality prior to pupation and developmental defects. After gene specific RNAi, chitin contents in the cuticle of an abnormal larva significantly decreased. The expression levels of five genes in the chitin biosynthesis pathway, SeTre-1, SeG6PI, SeUAP, SeCHSA and SeCHSB, were significantly reduced, while there was no difference in the expression of SeTre-2 prior to 72 hr after injection of EcR dsRNA. Meanwhile, injection of 20E in vivo induced the expression of the five genes mentioned above. Moreover, the SeTre-1, SeG6PI, SeUAP and SeCHSB genes showed late responses to the hormone and the induction of SeTre-1, SeG6PI, SeUAP and SeCHSB genes by 20E were able to be inhibited by the protein synthesis inhibitor cycloheximide in vitro indicating these genes are 20E late-response genes.

Conclusions

We conclude that SeTre-1, SeG6PI, SeUAP and SeCHSB in the chitin biosynthesis pathway are 20E late-response genes and 20E and its specific receptors plays a key role in the regulation of chitin biosynthesis via inducing their expression.     

Identification of genes specifying proline biosynthesis in marine bacteriumAlteromonas haloplanktis

Summary A gene bank of total DNA of a marine bacterium,Alteromonas haloplanktis, was constructed on pBR322. Two hybrid plasmids pUS2010 and pUS2011 carrying inserts of 8.2 and 5.7 kb, respectively, were isolated that complemented theproBA deletion inE.coli CSH26. Restriction map of the inserts showed that both plasmids in common carried a 5.7 kb fragment. This restriction fragment thus contains both the genes involved in proline biosynthesis inA.haloplanktis and could be expressed inE.coli.     

Identification of gamma irradiated Brachypodium mutants with altered genes responsible for lignin biosynthesis

Brachypodium distachyon (Brachypodium) is a novel model plant for structural and functional genomic studies of Poaceae. Brachypodium has many favorable features, such as small size, small genome, short life cycle, and easy handling. Bioethanol, as renewable resource, has been widely studied as a replacement for fossil fuels. Lignin is involved with the efficiency of energy feedstock. It is generally accepted that bioethanol production is negatively affected by lignin content. Brachypodium was irradiated with gamma irradiation, at doses of 50, 100, 150, 200, and 250 Gy, and 25 M₂ plants that showed the least staining with phloroglucinol were selected. Nucleotide alteration within genes that contribute to the lignin biosynthesis pathway was analyzed. In total, 4 INDELs and 249 SNPs which included 2 additional nonsense mutations, a mutation at the start codon, and a mutation at the 3′ splicing site were identified in the M₂ lines. The transition/transversion rate was 7.59, and single nucleotide substitutions were found every 1,143 bp. As biological resources, the M₂ populations generated in this work will contribute to functional genomics of Brachypodium and to the breeding of grass crops.     

Identification of genes for mycothiol biosynthesis in Streptomyces coelicolor A3(2)

Mycothiol is a low molecular weight thiol compound produced by a number of actinomycetes, and has been suggested to serve both anti-oxidative and detoxifying roles. To investigate the metabolism and the role of mycothiol in Streptomyces coelicolor, the biosynthetic genes (mshA, B, C, and D) were predicted based on sequence homology with the mycobacterial genes and confirmed experimentally. Disruption of the mshA, C, and D genes by PCR targeting mutagenesis resulted in no synthesis of mycothiol, whereas the mshB mutation reduced its level to about 10% of the wild type. The results indicate that the mshA, C, and D genes encode non-redundant biosynthetic enzymes, whereas the enzymatic activity of MshB (acetylase) is shared by at least one other gene product, most likely the mca gene product (amidase).     

Identification of additional genes required for O-antigen biosynthesis in Vibrio cholerae O1.

The cloning and expression of the genes encoding the Vibrio cholerae O1 lipopolysaccharide O antigen in a heterologous host have been described previously (P. A. Manning, M. W. Heuzenroeder, J. Yeadon, D. I. Leavesley, P. R. Reeves, and D. Rowley, Infect. Immun. 53:272-277, 1986). It was thus assumed that all the genes required for O-antigen expression were located on a 20-kb SacI restriction fragment. We present evidence for a number of other as yet undescribed genes that are essential for O-antigen biosynthesis in V. cholerae O1 and that these genes are somehow complemented in Escherichia coli K-12. The two genes termed Vibrio cholerae rfbV and rfbU are transcribed in the opposite orientation from the rest of the rfb operon, whereas the galE dehydratase and rfbP (Salmonella enterica) homologs, designated ORF35x7 and rfbW, respectively, are transcribed in the same orientation. The evidence presented here, using chromosomal insertion mutants, clearly shows that the three genes now designated rfbV, rfbU, and rfbW appear to be accessory rfb genes and are essential for O-antigen biosynthesis in V. cholerae but that ORF35x7 is not.     

Identification of two late acyltransferase genes responsible for lipid A biosynthesis in Moraxella catarrhalis

Lipid A is a biological component of the lipo-oligosaccharide of a human pathogen, Moraxella catarrhalis. No other acyltransferases except for UDP-GlcNAc acyltransferase, responsible for lipid A biosynthesis in M. catarrhalis, have been identified. By bioinformatics, two late acyltransferase genes, lpxX and lpxL, responsible for lipid A biosynthesis were identified, and knockout mutants of each gene in M. catarrhalis strain O35E were constructed and named O35ElpxX and O35ElpxL. Structural analysis of lipid A from the parental strain and derived mutants showed that O35ElpxX lacked two decanoic acids (C10:0), whereas O35ElpxL lacked one dodecanoic (lauric) acid (C12:0), suggesting that lpxX encoded decanoyl transferase and lpxL encoded dodecanoyl transferase. Phenotypic analysis revealed that both mutants were similar to the parental strain in their toxicity in vitro. However, O35ElpxX was sensitive to the bactericidal activity of normal human serum and hydrophobic reagents. It had a reduced growth rate in broth and an accelerated bacterial clearance at 3 h (P < 0.01) or 6 h (P < 0.05) after an aerosol challenge in a murine model of bacterial pulmonary clearance. O35ElpxL presented similar patterns to those of the parental strain, except that it was slightly sensitive to the hydrophobic reagents. These results indicate that these two genes, particularly lpxX, encoding late acyltransferases responsible for incorporation of the acyloxyacyl-linked secondary acyl chains into lipid A, are important for the biological activities of M. catarrhalis.     

RNA turnover in cultured hamster embryo cells: Identification of modified nucleoside end products

Fourteen methylated nucleosides (N-2-dimethylGuo, N-2-methylGuo, N-1-methylGuo, N-5-methylUrd, N-3-methylUrd, N-1-methylAdo, N-3-methylCyd, N-5-methylCyd, N-1-methyllno, 2′-0methyl-Cyd, 2′-0-methylUrd, 2′-0-methylGuo, 2′-0-methyllno, and thymidine) and one methylated base (m⁷Gua) have been identified as normal excretion products of cultured hamster embryo cells. The methylated nucleosides are excreted in the culture media subsequent to RNA turnover. The excretion pattern of the base-methylated nucleosides was determined by continuous labeling of serum-stimulated quiescent hamster embryo cells with [³H-CH₃]methionine and measurement of radioactivity in the excreted nucleosides between 23 and 81^1/2 hours after the label was added. These nucleosides accumulate exponentially until a maximum level is reached after 60 hours. These maximum levels were maintained for at least an additional 20 hours.     

Identification and characterization of the genes involved in glycosylation pathways of mycobacterial glycopeptidolipid biosynthesis

Glycopeptidolipids (GPLs) are major components present on the outer layers of the cell walls of several nontuberculous mycobacteria. GPLs are antigenic molecules and have variant oligosaccharides in mycobacteria such as Mycobacterium avium. In this study, we identified four genes (gtf1, gtf2, gtf3, and gtf4) in the genome of Mycobacterium smegmatis. These genes were independently inactivated by homologous recombination in M. smegmatis, and the structures of GPLs from each gene disruptant were analyzed. Thin-layer chromatography, gas chromatography-mass spectrometry, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses revealed that the mutants Deltagtf1 and Deltagtf2 accumulated the fatty acyl-tetrapeptide core having O-methyl-rhamnose and 6-deoxy-talose as sugar residues, respectively. The mutant Deltagtf4 possessed the same GPLs as the wild type, whereas the mutant Deltagtf3 lacked two minor GPLs, consisting of 3-O-methyl-rhamnose attached to O-methyl-rhamnose of the fatty acyl-tetrapeptide core. These results indicate that the gtf1 and gtf2 genes are responsible for the early glycosylation steps of GPL biosynthesis and the gtf3 gene is involved in transferring a rhamnose residue not to 6-deoxy-talose but to an O-methyl-rhamnose residue. Moreover, a complementation experiment showed that M. avium gtfA and gtfB, which are deduced glycosyltransferase genes of GPL biosynthesis, restore complete GPL production in the mutants Deltagtf1 and Deltagtf2, respectively. Our findings propose that both M. smegmatis and M. avium have the common glycosylation pathway in the early steps of GPL biosynthesis but differ at the later stages.