The TRP1 gene of Phytophthora parasitica encoding indole-3-glycerolphosphate synthase-N-(5'-phosphoribosyl)anthranilate isomerase: structure and evolutionary distance from homologous fungal genes.

The TRP1 gene was isolated from the genome of Phytophthora parasitica. It encodes bifunctional enzyme of the tryptophan biosynthetic pathway indole-3-glycerolphosphate synthase-N-(5'-phosphoribosyl)anthranilate isomerase (IGPS-PRAI). The gene was localized and sequenced using random in vitro insertions of omega interposon. The domain structure of the protein product was found to be similar to that of enteric bacteria but different from the structure of homologous enzymes in fungi. Two introns in the IGPS domain were found. This is unique in eukaryotic IGPS-encoding genes so far sequenced. Comparative analysis of the primary structure of IGPS and PRAI domains [neighbor-joining method of Saitou and Nei, Mol. Biol. Evol. 44 (1987) 406-425] confirmed a large phylogenetic distance of TRP1 from corresponding fungal genes. In the resulting distance tree Phytophthora sequences are located outside of the cluster which encompasses all known homologous proteins from fungi indicating that the lineage of oomycetes took a separate course of development before speciation within the fungal line of descent began. Two of the oligopeptide insertions engineered into the F domain of the protein product did not abolish the enzymatic activity of the protein.

The immI region of Salmonella phage P22. 1. Characterisation of the mutants P22 cir4-1, P22 cir5-1 and P22 cir6-1.

Salmonella phage mutants P22 cir4-1, P22 cir5-1 and P22 cir6-1 at 37 degrees C form more or less clear plaques. The mutants complement each other and clear mutations of the immC region (Prell et al. 1983). The mutants exhibit a strongly reduced frequency of lysogenisation, but form stable prophages. The low frequency of lysogenisation of P22 cir5-1 and of P22 cir6-1 is suppressed by an additional ant- mutation. Similarly, about 50% of turbid plaque revertants of both mutants carry ant- suppressor mutations. This suggests interference by the cir5-1 and cir6-1 mutations with the expression of gene ant. In contrast, the cir4-1 mutation seems not to interfere with ant expression, the latent periods of P22 cir4-1 and P22 cir5-1 are reduced and extended, respectively. The geneticly related Salmonella phage L carries a gene able to complement P22 cir4-1.

The immI region of Salmonella phage P22. 2. The influence of the cir mutations on the regulation of antirepressor synthesis.

The effects were studied of three clear plaque mutations of phage P22 (cir4-1, cir5-1 and cir6-1) on antirepressor synthesis. The mutant site cir4-1 has no influence on the expression of gene ant. The cir5-1 and cir6-1 mutations prevent the repression of early ant synthesis shortly after infection: P22 cir5-1 exhibits a strong ant-overproduction because it renders the cir5 repressor protein defective for turning off early ant expression (Harvey et al. 1981). P22 cir6-1 exhibits only a low level of constitutive ant synthesis insensitive to cir5-directed repression. Complementation experiments between P22 wild type or mutant in the cir5 or cir6 sites reveal the following phenotypes of the cir6-1 mutation: (1) The cir6-1 site behaves as a weak promotor site insensitive to cir5-directed repression of ant synthesis. (2) In the P22 cir5-1 cir6-1 double recombinant the cir6-1 site is cis dominant preventing ant overproduction as observed in simple P22 cir5-1 infections. (3) Some of the deficient phenotypes of P22 cir6-1 can be complemented by co-infecting P22 mutants carrying a cir6+ allele. These results are explained by the following model: The active cir5 repressor protein is a dimer (or oligomer) and the cir5-1 and cir6-1 mutations map in different domains influencing the repressing as well as the dimer forming activities of this protein. Which particular cir6-1 phenotype is observed depends on the experimental conditions employed, and on the promotor site (either pANT or cir6-1) from which ant expression procedes.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of mitochondrial DNA of the oomycetous fungus Phytophthora infestans.

Mitochondrial DNA (mtDNA) of Phytophthora infestans has been isolated and preliminarily characterized. It has a low GC content of about 22.4% which is distinctly different from that of nuclear DNA (51 %). This property has been used to separate both DNA species in the presence of 4',6-diamidine-2-phenylindole (DAPI) in CsCl density gradients. The use of cetyl triammonium bromide (CTAB) for extraction of DNA significantly reduced its degradation. The base distribution of the mtDNA shows a limited intramolecular heterogeneity. The molecule contains 36.2 ± 0.3 kb as revealed by endonuclease digestion and seems to be circular as shown by restriction mapping. No differences were found in restriction patterns between mtDNAs from various pathotypes.

P22 antirepressor protein prevents in vivo recA-dependent proteolysis of P22 repressor.

A method was developed to demonstrate recA-dependent P22-repressor breakdown in vivo by SDS-polyacrylamide electrophoresis of unfractionated extracts of phage-infected, lysogenic Salmonella typhimurium strains TA1530 rec+ and TA1530 recA1-. The antirepressor of P22 is not cleaved by recA protein. Under conditions of unregulated ant-overproduction (Harvey et al. 1981) antirepressor protects c2-repressor in vivo against proteolytic cleavage by recA protein.

Repression of ant synthesis early in the lytic cycle of phage P22.

Using SDS-polyacrylamide gel electrophoresis to study the early expression of P22 genes we show that early expression of the ant-gene (imm I region) is turned off after 6-8 min, independent of the 'late' acting mnt-repressor. A semi-clear mutant called cir5 is defective for this early ant turn-off. The mutation cir5 maps in the imm I region of P22 between genes mnt and ant. P22 cir5 mutants are defective for a repressor which acts in trans to regulate early ant synthesis. There appears to be no absolute requirement of the cir5 allele for the establishment of lysogeny. The overproduction of ant in the P22 cir5 mutant leads to a marked increase in abortive infections, killing the infected cells. The cir5-phenotype can be suppressed by an ant- mutation.

Kinetics of c2-repressor synthesis in a regulatory defective P22 mutant.

Phage P22 defective in gene 24 and harbouring the Oc mutation k5 in OR exhibits a strongly increased c2-repressor synthesis after infection of non-lysogenic S. typhimurium. The repressor synthesis depends strictly on an intact c1 gene. The kinetics of its synthesis, as monitored by polyacrylamide gel electrophoresis, is the same as with P22 c+, namely a turn off 8-10 min after infection. - After infection of P22-lysogenic bacteria with either P22 24- k5 or P22 24- k5 c1, much lower amounts of repressor are synthesized but again with the same kinetics. These results suggest a cro-like function acting at PRE and PRM of P22. The possible reason for the c2 overproduction is discussed.

Kinetics of P22 early gene expression suggests a cro-like regulatory function.

Analysis of phage-specified protein synthesis after phage infection of UV-irradiated cells shows a turn-off of early gene expression, a regulatory event that is independent of the known P22 regulatory functions. This supports the suggestion of a cro lambda-like function in P22. We have identified the products of genes 18 and int as contributing to the complex 40,000 dalton band in our SDS-polyacrylamide gels. Both gene products appear to be subject to regulation by the cro-like function of P22. Proteins of 33,000, 29,000, 27,000, 25,000, and 24,000 MW, specified by as yet unidentified P22 genes of the early leftward operon, are regulated by the same function. Our data suggest that the cro-like function is expressed from the early rightward operon.

Ant-mediated inactivation of Salmonella phage L-specified repression at OR of prophage L.

Ant product of phage P22 inactivates repression of prophage L at the right-hand operator OR and allows for transactivation of prophage gene 12. The transactivation efficiency observed with a series of phage and prophage recombinants, using single superinfection of a lysogenic bacterium, is about the same as that recently observed at OL of prophage L. This finding is in contrast to the failure to demonstrate derepression at OR of prophage L in an experimental system employing double superinfection (Prell, 1978a). The reasons for the differing results are discussed and it is shown that derepression by the ant product in trans at OR of the prophage is not modified to any significant degree by the immunity specificity (L or p22) of the prophage or of the superinfecting phage.

Cis- and trans-activity of P22 antirepressor protein against c-repression specified by the closely related Salmonella phages L and Px1.

The ant products of Salmonella phage P22, synthesized by its immI region, releases when acting in cis replication inhibition for phages P22 and L. When ant product acts trans on a coinfecting immunity sensitive phage (Thomas-Bertani-experiment as test for release of replication inhibition) full replication ensues only if both superinfecting phages are homologous in the specificities of their immC and immI regions. If these regions are heterologous, differing in immC, immI or in both, the replication of the phage expected to be complemented by ant is inhibited. This inhibition is observed in both L- and Px-lysogenic bacteria and can be released in case of ant- amber phage by action of ant in cis in su+ lysogenic bacteria.

Repressor synthesis in regulatory mutants of bacteriophage P22.

SDS-polyacrylamide gel analysis of P22-infected Salmonella has allowed identification of the c2 repressor (MW 31,000) and study of repressor synthesis in regulatory mutants of P22. Repressor is synthesized in reduced amounts or is absent in infections with P22, clNo.7, P22, c2 am08, P22 c3 am03, and P22 c3 am012, but is synthesized in markedly increased amounts in the virulent mutant, P22 virB3, and its component mutants, vx and k5. Higher levels of repressor are also found in the P22 cly 17 mutant.

Ant-mediated transactivation of early genes in Salmonella prophage P22 by superinfecting virulent P22 mutants.

The virulent mutants P22 virB vy and P22 vy mutants, both insensitive to mnt-repressor, transactivate the early genes of a P22 prophage. The transactivation of early P22 prophage genes depends strictly on the expression of gene ant ("antirepressor"-protein) by the superinfecting P22 mutant and therefore occurs by derepression.

The activity of ant product of the Salmonella phage P22 against the closely related but heteroimmune phage L.

P22 mutants defective in the early gene 24 are complemented by phage L in mixed infection. P22 12- and P22 23- mutants are not complemented by phage L. Gene function 24 of an L prophage is turned on by a superinfecting P22 24- mutant and complements the missing function of the defective P22 phage. Since this transactivation of prophage gene 24 depends on a functional gene ant in the superinfecting P22 mutant, it indicates derepression for leftward directed gene expression in prophage L. On the contrary neither the rightward directed expression of gene 12 nor of gene 23 in prophage L. can be turned on by superinfecting P22 24- 12- or P22 24- 23- mutants (and also not by P22 12- and P22 23-) to a degree sufficient for complementation of simultaneously superinfecting L virB 12- or L virB 23- mutants. The failure to detect release of repression for rightward directed gene expression of prophage L corresponds to the earlier observation (Prell, 1975) that P22 superinfecting L lysogens cannot release replication inhibition for simultaneously infecting phage L. The results are discussed with respect to the mechanism underlying the different action of P22 antirepressor in L and in P22 lysogens.

Regulation of gene expression in Salmonella phage P22. II. Regulation of expression of late functions.

Transactivation experiments were performed involving the genetically related Salmonella phages P22, L and Px1 in order to find out if more than one positively acting regulatory product is engaged in the expression of vegetative gene functions of each of these phages. The results obtained with Px1- and L-lysogenic cells superinfected with P22 suggest the following conclusions: 1. The expression of the early genes 12 and 23 and of the late gene 19 (lysozyme synthesis) is positively regulated by two different regulatory products, since P22 transactivates in prophage Px1 both early and late genes (Prell, 1973), in prophage L only late genes. 2. The transactivation by P22 of the lysozyme gene of prophage L takes place in the presence of L repressor. This conclusion is suggested, since the superinfecting P22 does not derepress early gene expression (see 1.), and is confirmed by demonstration of replication inhibition for L phage in L lysogenic cells doubly superinfected with L and P22 phages (Thomas-Bertani-experiment). 3. The late gene regulatory protein seems to be synthesized by gene 23, as transactivation experiments with both L- and Px1 prophages suggest. 4. The expression of gene 23 itself is turned on by an early regulatory product. The gene which codes for it is still unidentified. However its product seems to by highly specific, since it is active on Px1- but not on L-prophage.