Targeted gene inactivation for the elucidation of deoxysugar biosynthesis in the erythromycin producer Saccharopolyspora erythraea

The production of erythromycin A by Saccharopolysporaerythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-α-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV?), ORF17 (eryCIV?) and ORF7 (eryBII?) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates.     

Analysis of seven genes from the eryAI –eryK region of the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA, and lying between eryA and the gene eryK, which is known to encode the C-12 hydroxylase, has been sequenced and shown to contain seven additional open reading frames (ORFs 13–19). On the basis of sequence similarities, roles are proposed for several of these ORFs in the biosynthesis of the deoxysugar mycarose and the deoxyaminosugar desosamine. A chromosomal mutant carrying a deletion in ORF15 has been constructed and shown to accumulate 3-O-mycarosyl-erythronolide B, as expected for an eryC mutant. Similarly, a chromosomal mutant carrying a deletion in ORF16 has been constructed and shown to accumulate erythronolide B, as expected for an eryB mutant.     

Analysis of eryBI , eryBIII and eryBVII from the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic β-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A␣mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis. Received: 13 August 1997 / Accepted: 27 November 1997     

Analysis of eryBI, eryBIII and eryBVII from the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic β-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A?mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis.     

A screen for upstream components of the yeast protein kinase C signal transduction pathway identifies the product of the SLG1 gene

We employed the constitutive BCK1-20 allele of the gene for the MAP kinase kinase kinase (MAPKKK) in the yeast Pkc signal transduction pathway to develop a genetic screen for mutants in genes encoding upstream components. Transposon mutagenesis yielded a mutant that was completely dependent on the active allele in the absence of osmotic stabilization. The transposon had integrated at the yeast SLG1 (HCS77) locus. This gene encodes a putative membrane protein. Haploid slg1 deletion strains are sensitive to caffeine, as expected for mutants in the Pkc pathway, as well as a variety of other drugs. The response to elevated temperatures and the dependence on osmotic stabilization depends on the genetic background. Thus, in the strain used for mutagenesis, disruption of SLG1 causes the cells to become non-viable in the absence of osmotic stabilization at both 30° C and 37° C. In a different genetic background this phenotype was not observed. Sensitivity of the haploid deletion mutants to caffeine can be partially suppressed by overexpression of genes for other components of the Pkc pathway, such as PKC1, SLT2, ROM2, and STE20. In addition, a SLG1-lacZ reporter construct shows higher expression in the presence of caffeine or magnesium chloride in a wild-type diploid background. Received: 2 December 1997 / Accepted: 15 December 1997     

Xanthomonas campestris pv. campestris lpsI and lpsJ genes encoding putative proteins with sequence similarity to the α- and β-subunits of 3-oxoacid CoA-transferases are involved in LPS biosynthesis

A 2.1-kb SmaI-EcoRI DNA fragment upstream of the xanA and xanB genes of Xanthomonas campestris pv. campestris carries two ORFs encoding putative proteins with sequence similarities to the α- and β-subunits of 3-oxoacid-CoA transferases. The two ORFs were termed lpsI and lpsJ because strains carrying appropriate mutations showed an autoagglutination phenotype and because lipopolysaccharides of these mutant strains were altered according to silver-stained polyacrylamide gels. The monosaccharide composition of the exopolysaccharide xanthan produced by lpsI and lpsJ mutants remained unchanged. Received: 29 March 1997 / Accepted: 21 July 1997     

Exopolysaccharide (EPS) synthesis in Bradyrhizobium japonicum : sequence, operon structure and mutational analysis of an exo gene cluster

The nucleotide sequence of a 8330-bp DNA fragment from Bradyrhizobium japonicum 110spc4 was determined. Sequence analysis revealed that six ORFs were present and the deduced amino acid sequences were homologous to enzymes involved in exopolysaccharide (EPS) biosynthesis. The genes appear to be organized into at least four different operons. One gene was found to be homologous to exoB, which encodes a UDP-galactose 4′-epimerase. Other ORFs were homologous to UDP-hexose transferases and one ORF showed similarity to Sinorhizobium (Rhizobium) meliloti ExoP, which has been suggested to be involved in EPS chain-length determination. A set of deletion and insertion mutants was constructed and the resulting B. japonicum strains were tested for their symbiotic traits. Deletion mutant ΔP22, which lacks the C-terminal part of ExoP, the UDP-hexose transferase ExoT and the N-terminal part of ExoB, shows a delayed nodulation phenotype and induces symptoms of plant defense reactions; its EPS does not contain galactose and no high molecular weight fraction is synthesized. In contrast, insertion mutant EH3, which expresses an exoP gene product that is truncated in its putative periplasmic domain, produced an EPS containing both HMW and LMW fractions. However, the interaction of EH3 with soybeans was severely perturbed. As a rule, only the initial steps of nodule formation were observed. Received: 2 January 1998 / Accepted: 24 March 1998     

Anthracyclines: isolation of overproducing strains by the selection and genetic recombination of putative regulatory mutants of Streptomycespeucetius var. caesius

In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium at 6 days of fermentation. Received: 14 April 1997 / Received revision: 19 June 1997 / Accepted: 4 July 1997     

trans activation of the Escherichia coliato structural genes by a regulatory protein from Bacillus megaterium : potential use in polyhydroxyalkanoate production

A Bacillus megaterium genomic fragment, which encoded an activator homologous to σ⁵⁴ regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B.␣megaterium screened for β-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties by recombinant E. coli strains. Received: 20 October 1997 / Received revision: 18 February 1998 / Accepted: 23 February 1998     

Molecular cloning and sequence analysis of interleukin 16 from nonhuman primates and from the mouse

 Interleukin 16 (IL-16) is synthesized as a 67 000 M _r precursor (pro-IL-16), but only a carboxy terminal part of 12 000–14 000 M _r is secreted by CD8(⁺) lymphocytes. This lymphokine binds to CD4 and has been shown to induce migration, affect the activation state of T cells, and inhibit immunodeficiency virus replication. It has been suggested that CD8(⁺) cell-derived soluble factors play a pivotal role in protecting natural-host nonhuman primates from developing immunodeficiency following SIV infection. In a first attempt to address this question, we cloned and sequenced the IL-16 cDNA from different primates. Here we report the pro-IL-16 sequence from chimpanzees, African green monkeys (AGM), rhesus macaques, and cynomolgus macaques. In order to compare and analyze structural motifs possibly involved in processing, intracellular targeting, or secretion, we extended our study to the New World monkeys saimiri and aotus and to the mouse. Alignments of deduced amino acids reveal that the human protein shares 99% similarity to that of chimpanzees, approximately 95% to rhesus, cynomolgus and AGM, about 90% to aotus and saimiri, and 77.5% to the mouse. Phylogenetic analyses revealed the expected evolutionary groupings. Received: 27 August 1997 / Revised: 7 October 1997     

Chromosomal deletions in Streptomyces griseus that remove the afsA locus

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA ⁻ mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome. Received: 29 July 1996 / Accepted: 27 August 1996     

Trifluoroleucine resistance and regulation of alpha-isopropyl malate synthase in Saccharomyces cerevisiae

Seven spontaneous Saccharomyces cerevisiae mutants that express dominant resistance to 5,5,5-trifluoro-DL-leucine have been characterised at the molecular level. The gene responsible for the resistance was cloned from one of the mutants (FSC2.4). Determination of its nucleotide sequence showed that it was an allele of LEU4 (LEU4-1), the gene that encodes α-isopropyl malate synthase I (α-IPM synthase I), and that the mutation involved a codon deletion localised close to the 3′ end of the LEU4 ORF. Six different point mutations – four transitions and two transversions – were found in the remaining mutants. α-IPM synthase activity was found to be insensitive to feedback inhibition by leucine in five of the strains. In the other two the enzyme was resistant to Zn²⁺-mediated inactivation by Coenzyme A, a previously postulated control mechanism in energy metabolism; as far as we know, this represents the first direct in vivo evidence for this mechanism. The seven mutations define a region, the R-region, involved in both leucine feedback inhibition and in Zn²⁺-mediated inactivation by CoA. Deletion experiments involving the R-region showed that it is also necessary for enzyme activity. Received: 30 September 1998 / Accepted: 20 October 1998     

A mutation in the gene for trigger factor suppresses the defect in cell division in the divE42 mutant of Escherichia coli K12

A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division. One of the suppressor mutants, designated TR4, had a novel phenotype: it was able to grow at 42° C but not at 32° C. The Kohara genomic library was screened for complementing clones. Clone 148 was able to complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148. DNA sequencing revealed that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation codon for this enzyme. When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32° C but not at 42° C, an indication that the intergenic suppressor mutation was recessive to the wild-type allele. A model is proposed that accounts for the phenotypes of the divE42 mutant and the TR4 suppressor mutant. Received: 3 March 1998 / Accepted: 14 July 1998     

Molecular phylogeny and systematics of drosophila retrotransposons and retroviruses

Full classification of Drosophila melanogaster retrotransposons with long terminal repeats (LTR-retrotransposons) has been recomposed, and their evolutional analysis in sequenced genomes of different species of drosophila and other arthropods has been carried out. D. melanogaster LTR-retrotransposons are divided into three groups: gypsy (one, two, or three open reading frames (ORFs)), copia (one ORF), and BEL (one ORF). The gypsy group is divided into three subgroups. Subgroup I is underrepresented by retrotransposons-retroviruses with three ORFs and their derivatives, which have lost the env gene (ORF3). Subgroup II is underrepresented by retrotransposons with two ORFs, and subgroup III is underrepresented by retrotransposons with one ORF. A comparative analysis of homologs of gypsy group LTR-retrotransposons evidences that subgroups I and II are represented only in the genomes of Lepidoptera and Diptera. The gypsy group of LTR-retrotransposons with one and two ORFs is found in almost all genomes of arthropods. Most of the families of D. melanogaster gypsy group LTR-retrotransposons have close homologs in the genomes of other species of drosophila. A degree of identity of retrotransposons sequences is correlated with a degree of relation between species of drosophila, indicating vertical transmission of retrotransposons. Obvious cases of horizontal transfer of some mobile elements have been detected including retrotransposons without the env gene. Homologs of distinct ORFs of retrotransposons—genes gag and env—have been found. Gene-homolog of the gag gene—Grp (CG5680)—is under purifying selection, so it has an important function in drosophila genome.     

Cloning of the Escherichia coli endo-1,4-D-glucanase gene and identification of its product

A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in the cosmid vector pLAFR3 in E. coli DH5α. A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated molecular weight of 41,700 Da. BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues. The predicted amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens. Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8. The apparent molecular mass of the protein, when expressed in E. coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space. The enzyme is optimally active at pH 7 and a temperature of 40° C. Received: 6 February 1998 / Accepted: 6 November 1998