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. Received: 29 July 1997 / Accepted: 16 October 1997     

Mutations in the heavy chain of cytoplasmic dynein suppress the nudF nuclear migration mutation of Aspergillus nidulans

To identify proteins that interact directly or indirectly with the NUDF protein, which is required for nuclear migration in Aspergillus nidulans, we initiated a screen for extragenic suppressors of the heat-sensitive nudF6 mutation. Suppressor mutations in at least five genes, designated snfA–snfE, caused improved growth and nuclear migration at high temperatures compared to the nudF6 parent. Two snfC mutations mapped near the nudA gene, which encodes the cytoplasmic dynein heavy chain, and could be repaired by transformation with wild-type nudA DNA, demonstrating that they are mutations in nudA. The snfC mutations are bypass suppressors of nudF and genetic evidence indicated that NUDA and NUDF act in the same nuclear migration pathway. Taken together, our data suggests that NUDF affects nuclear migration by acting on the dynein motor system. Received: 4 January 1997 / Accepted: 26 February 1997     

Characterization of ptxS , a Pseudomonas aeruginosa gene which interferes with the effect of the exotoxin A positive regulatory gene, ptxR

The complicated process of exotoxin A production by Pseudomonas aeruginosa is controlled by several genes. We have recently described a toxA positive regulatory gene, ptxR. We also proposed the presence of another gene which is adjacent to ptxR and interferes with ptxR function on exotoxin A production. In the presence of a fragment that carries the putative gene, the enhancement in exotoxin A production by ptxR was reduced threefold. In this study, we describe the characterization of this gene. Nucleotide sequence analysis of the 2.1-kbp fragment at the 5′ end of ptxR revealed the presence of an open reading frame designated ptxS (the gene next to ptxR) which encodes a 37.4-kDa protein. The gene ptxS is transcribed in the opposite orientation to ptxR from the other DNA strand. The deduced amino acid sequence of ptxS exhibited a strong homology to several proteins of the GalR-LacI family of repressors. A putative helix-turn-helix DNA binding motif was identified at the amino-terminus region of PtxS. When PtxS was overexpressed in Escherichia coli using the T7 expression system, a single protein of 38-kDa molecular weight was detected. An isogenic mutant defective in ptxS was constructed in PAO1 using the gene replacement technique. The loss of ptxS resulted in a twofold increase in exotoxin A production compared to PAO1. The effect of ptxS on ptxR was examined using a ptxR-lacZ fusion. In the presence of ptxS, the level of β-galactosidase activity produced by the ptxR-lacZ fusion was significantly reduced. These results suggest that ptxS encodes a protein which negatively regulates ptxR expression in P. aeruginosa. Received: 29 September 1997 / Accepted: 22 December 1997     

The pilA gene of Xanthomonas campestris pv. citri is required for infection by the filamentous phage cf

 Host factors that are important for infection of Xanthomonas campestris pv. citri by the filamentous bacteriophage cf were investigated by transposon mutagenesis with Tn5tac1. A mutant, XT501, that was resistant to cf infection was recovered, showing that the gene inactivated by the transposon is required for infection by the phage but not for cf replication or assembly. A 1.7-kb SacI-ApaI DNA fragment from XT501 containing the bacterial DNA flanking one end of the transposon was cloned and shown to be required for cf infection. Nucleotide sequence analysis of the 1.7-kb fragment reveals the presence of an ORF that encodes a protein of 146 amino acids. This protein shows 42% identity to the type 4 prepilin encoded by the pilA genes of other bacteria. The pilA gene of X. campestris pv. citri is thus essential for infection by the bacteriophage cf. Received: 30 November 1998 / Accepted: 21 April 1999     

Cloning and characterization of the rpoH gene of Rhodobacter capsulatus

By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified a new σ factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34 kDa with strong similarity to the RpoH (σ ³²) factors from other bacterial species. It was not possible to inactivate the R. capsulatusrpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5′ ends of the mRNAs were mapped to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease in oxygen tension. Western analysis using a σ factor-specific antibody revealed the accumulation of a protein of about 34 kDa after heat shock, and an increase in the amounts of a protein with the same size after reduction of oxygen tension in R. capsulatus cultures. Received: 16 March 1998 / Accepted: 28 July 1998     

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     

Doc and copia instability in an isogenic Drosophila melanogaster stock

A high degree of heterogeneity and an overall increase in number of insertion sites of the mobile elements Doc and copia were revealed in one substock of an isogenic Drosophila melanogaster stock, while in two other substocks the distribution of copia sites was highly homogenous, but that of Doc sites was again heterogenous. We therefore concluded that copia was unstable in one of the substocks and Doc was unstable in all. Doc instability presumably arose earlier than copia instability. Doc and copia transpositions were directly observed in experiments with one substock. An abundance of copia insertions was revealed in the X chromosome where insertions with deleterious effects are exposed to selection in hemizygous condition. The locations of many other mobile elements (mdg1, mdg2, mdg3, mdg4, 297, B104, H.M.S. Beagle, I, P, BS, FB) were found to be conserved in each substock and did not differ between them, indicating that these mobile elements were stable. This homogeneity is a strong argument against any possibility of inadvertent contamination.     

Characterization of an lrp -like ( IrpC ) gene from Bacillus subtilis

In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4 kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilislrp-likegene is a bona fide Lrp protein – the first one to be detected in gram-positive bacteria. When expressed in E.␣coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B. subtilis Lrp-like protein plays a role in the growth phase transition. Received: 28 January 1997 / Accepted: 18 April 1997     

Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA

In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5%. In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA. The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly different from that in mitochondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked. The primary sequence of ms RNA showed low homology with mitochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary structure of ms RNA was similar to that of cytoplasmic 5S rRNA. In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA. However, ms RNA was detected mainly in the mitochondrial 25 000 × g supernatant fraction which was devoid of ribosomes. It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA. Received: 17 May 1997 / Accepted: 26 August 1997     

Germ line transposition of the copia retrotransposon in Drosophila melanogaster is restricted to males by tissue-specific control of copia RNA levels

Germ line transposition rates of the retrotransposon copia were directly measured in males and females of an inbred Drosophila melanogaster line, 2b3, which is highly polymorphic for copia insertion sites. The elevated germ line transposition rate of copia in this line (3–8 × 10⁻³ per generation per element) is confined to males, with transposition in females being undetectable under the conditions of the experiment but at most 50-fold lower than the rate for males. To determine the molecular basis of this effect, copia RNA levels were measured in whole bodies and germ lines of male and female flies of both the unstable 2b3 line and a stable line, Oregon RC-iso, which shows normal rates of copia transposition. Both male and female 2b3 flies contain much more copia RNA than flies of the stable line. However, 2b3 male germinal tissues contain much higher levels of copia RNA than the equivalent female tissues. The highest copia expression is detected in maturing primary spermatocytes. Our data show that high rates of germ line copia transposition are restricted to males by tissue-specific control of RNA levels and suggest that transposition of copia only occurs in fly tissues containing more than a relatively high threshold level of copia RNA. Received: 8 October 1996 / Accepted: 7 January 1997