Metabolic Flux in Both the Purine Mononucleotide and Histidine Biosynthetic Pathways Can Influence Synthesis of the Hydroxymethyl Pyrimidine Moiety of Thiamine in Salmonella enterica

ABSTRACT Together, the biosyntheses of histidine, purines, and thiamine pyrophosphate (TPP) contain examples of convergent, divergent, and regulatory pathway integration. Mutations in two purine biosynthetic genes (purI and purH) affect TPP biosynthesis due to flux through the purine and histidine pathways. The molecular genetic characterization of purI mutants and their respective pseudorevertants resulted in the conclusion that <1% of the wild-type activity of the PurI enzyme was sufficient for thiamine but not for purine synthesis. The respective pseudorevertants were found to be informational suppressors. In addition, it was shown that accumulation of the purine intermediate aminoimidazole carboxamide ribotide inhibits thiamine synthesis, specifically affecting the conversion of aminoimidazole ribotide to hydroxymethyl pyrimidine.

Intragenic Suppressors of an OmpF Assembly Mutant and Assessment of the Roles of Various OmpF Residues in Assembly through Informational Suppressors

ABSTRACT We employed two separate genetic approaches to examine the roles of various OmpF residues in assembly. In one approach, intragenic suppressors of a temperature-sensitive OmpF assembly mutant carrying a W214E substitution were sought at 42°C, or at 37°C in a genetic background lacking the periplasmic folding factor SurA. In the majority of cases (58 out of 61 revertants), the suppressors mapped either at the original site (position 214) or two residues downstream from it. In the remaining three revertants that were obtained in asurA background, an alteration of N230Y was located 16 residues away from the original site. The N230Y suppressor also corrected OmpF315 assembly at 42°C in asurA + background, indicating that the two different physiological environments imposed similar assembly constraints. The specificity of N230Y was tested against five different residues at position 214 of mature OmpF. Clear specificity was displayed, with maximum suppression observed for the original substitution at position 214 (E214) against which the N230Y suppressor was isolated, and no negative effect on OmpF assembly was noted when the wild-type W214 residue was present. The mechanism of suppression may involve compensation for a specific conformational defect. The second approach involved the application of informational suppressors (Su-tRNA) in combination with ompF amber mutations to generate variant OmpF proteins. In this approach we targeted the Y40, Q66, W214, and Y231 residues of mature OmpF and replaced them with S, Q, L, and Y through the action of Su-tRNAs. Thus, a total of 16 variant OmpF proteins were generated, of which three were identical to the parental protein, and two variants carrying W214Q and Y231Q substitutions were similar to assembly-defective proteins isolated previously (R. Misra, J. Bacteriol. 175:5049–5056, 1993). The results obtained from these analyses provided useful information regarding the compatibility of various alterations in OmpF assembly.

FRAMESHIFT SUPPRESSION IN SACCHAROMYCES CEREVISIAE. II. GENETIC PROPERTIES OF GROUP II SUPPRESSORS

ABSTRACT Suppressors of ICR-induced mutations that exhibit behavior similar to bacterial frameshift suppressors have been identified in the yeast Saccharomyces cerevisiae. The yeast suppressors have been divided into two groups. One of these groups (Group II: SUFI, SUF3, SUF4, SUF5 and SUF6) appears to include a set of informational suppressors in which the vehicle of suppression is glycyl-tRNA. Some of the genetic properties of Group II suppressors are described in this communication.—Corevertants of the Group II frameshift mutations his4-519 and leu2-3 have been characterized to determine the spectrum of reversion events induced by the frameshift mutagen ICR-170. Seventythree ICR-induced corevertants were analyzed. With the exception of one corevertant, which carried an allele of SUF1, all carried alleles of SUF3 or SUF5, SUF1, SUF3, SUF4 and SUF6 were represented among spontaneous and UV-induced corevertants. In the course of these experiments one of the suppressors was mapped. SUF5, the probable structural gene for tRNAGLY1, is located between ade2 and ade9 on chromosome XV.—SUF1, SUF4 and SUF6 have novel properties and comprise a distinct subset of suppressors. Although these suppressors show no genetic linkage to each other, they share several common features including lethality in haploid pairwise combinations, reduced tRNAGLY3 isoacceptor activity and increased efficiency of suppression in strains carrying the cytoplasmically inherited [PSI] element. In addition, strains carrying SUFI, SUF4 or SUF6 are phenotypically unstable and give rise to mitotic Suf+ segregants at high frequency. These segregants invariably contain a linked, second-site mutation that maps in or adjacent to the suppressor gene itself. Strains carrying any of these suppressors also give rise to mitotic segregants that exhibit enhanced efficiency of suppression; mutations responsible for this phenotype map at two loci, upf1 and upf2. These genes show no genetic linkage to any of the Group II suppressors.—Methods that permit positive selection for mutants with decreased or enhanced efficiency of suppression have been devised in order to examine large numbers of variants. The importance of these interacting mutants is underscored by their potential utility in studying suppressor function at the molecular level.

Genetic evidence for a polycistronic unit of transcription in the complex locus ‘14’ inPodospora anserinaII. Genetic analysis of informational suppressors

SUMMARYForty-one suppressors obtained after NG and EMS mutagenesis of two ‘polar’ mutants of segment ‘29’ inPodospora anserinawere genetically analysed. Three classes of suppressor could be distinguished on spectrum pattern criteria. One representative suppressor of each class was demonstrated to be non-gene specific. The class I suppressor was dominant and only suppressed polar mutants in segment ‘29’ and non-ICR-induced ones in genes where polarity cannot be determined. Class II and III suppressors were partially dominant and they suppressed polar, non-polar and even ICR-induced mutants. The difference between classes II and III seems to be only quantitative. According to whether class II and III suppressors are considered strongly or weakly allele-specific, two hypotheses are considered. First,tRNAs could be involved in all three classes of suppression: class I would be nonsense-specific and classes II and III would be nonsense-missense suppressors. Secondly,tRNA could be involved only in class I suppression, while ribosomal ambiguity could be responsible for class II and III suppression.