Rod A. Kelln

Regulation of Expression of the 2-Deoxy-d-Ribose Utilization Regulon, deoQKPX, from Salmonella enterica Serovar Typhimurium

Salmonella enterica, in contrast to Escherichia coli K12, can use 2-deoxy-D-ribose as the sole carbon source. The genetic determinants for this capacity in S. enterica serovar Typhimurium include four genes, of which three, deoK, deoP, and deoX, constitute an operon. The fourth, deoQ, is transcribed in the opposite direction. The deoK gene encodes deoxyribokinase. In silico analyses indicated that deoP encodes a permease and deoQ encodes a regulatory protein of the deoR family. The deoX gene product showed no match to known proteins in the databases. Deletion analyses showed that both a functional deoP gene and a functional deoX gene were required for optimal utilization of deoxyribose. Using gene fusion technology, we observed that deoQ and the deoKPX operon were transcribed from divergent promoters located in the 324-bp intercistronic region between deoQ and deoK. The deoKPX promoter was 10-fold stronger than the deoQ promoter, and expression was negatively regulated by DeoQ as well as by DeoR, the repressor of the deoxynucleoside catabolism operon. Transcription of deoKPX but not of deoQ was regulated by catabolite repression. Primer extension analysis identified the transcriptional start points of both promoters and showed that induction by deoxyribose occurred at the level of transcription initiation. Gel retardation experiments with purified DeoQ illustrated that it binds independently to tandem operator sites within the deoQ and deoK promoter regions with K(d) values of 54 and 2.4 nM, respectively.

Regulation of pyrC expression in Salmonella typhimurium: Identification of a regulatory region

SummaryDeletion analysis of a plasmid carrying the entire pyrC gene of Salmonella typhimurium served to localize the regulatory region within a 120 base pair DNA fragment comprising the promoter-leader region and the first 10 codons of pyrC. A region of dyad symmetry is present in the leader DNA and may result in the formation of a stable hairpin in the transcript with part of the Shine-Dalgarno sequence included in the stem. Four independently-isolated regulatory mutants, overexpressing pyrC, were found to have point mutations within the symmetry region and, significantly, the mutations occurred in sequences pertaining to either side of the stem of the putative hairpin of the transcript. All four mutations would decrease the stability of the hairpin, suggesting that pyrC expression is controlled at the level of translation. Additional evidence for translational control was provided by the finding that synthesis of galactokinase mediated from a pyrC-galK transcriptional fusion is not regulated by pyrimidines. The importance of the symmetry region in the leader was further emphasized by showing that pyrC expression is strongly affected when this region is deleted, inverted, or structured as a tandem duplication.

Construction and use of pyr::lac fusion strains to study regulation of pyrimidine biosynthesis in Salmonella typhimurium.

SummaryThe technique developed by Rosenfeld and Brenchley [J Bacteriol 144, 848–851 (1980)] has been used to introduce Mu d1 (Aprlac) into Salmonella typhimurium for purposes of constructing pyr::lac fusion strains. A stable pyrB::lac fusion mutant was subsequently derived and used for the genetic characterization of the pyrB gene. The direction of transcription of pyrB was determined to be counterclockwise on the S. typhimurium linkage map and argI was shown to be located clockwise of pyrB. Mutants altered in the regulation of expression of pyrB were isolated and two of the isolates chosen for further study were tentatively categorized as promoter or operator mutants.

Construction of plasmid-free derivatives ofSalmonella typhimurium LT2 using temperature-sensitive mutants of pKZ1 for displacement of the resident plasmid, pSLT

SummaryReplication (or partitioning) temperature-sensitive mutants of pKZ1 were isolated and shown to exhibit incompatibility with the resident plasmid (pSLT) ofSalmonella typhimurium LT2. Following displacement of pSLT, the mutant plasmids were effectively eliminated from the cell population by passage at 42° C, yielding plasmid-free isolates.

Control of arg gene expression in salmonella typhimurium by the arginine repressor from Escherichia coli K-12

SummaryThe regulation of synthesis of arg enzymes in Salmonella typhimurium by the arginine repressor of Escherichia coli K-12 has been reevaluated using a strain of S. typhimurium in which the argR gene was rendered nonfunctional by inserting the translocatable tetracyclineresistance element Tn10 into the argR gene. In contrast to previous studies, the introduction of the argR+ allelle of E. coli on an F-prime factor to the argR::Tn10 S. typhimurium strain reduced the synthesis of arg enzymes to essentially wild-type levels. The elevated levels of arg enzymes observed in other hybrid merodiploids may have been the consequence of the formation of hybrid repressor molecules. The readily scoreable phenotype of tetracycline resistance facilitated establishing linkage of cod and argR (0.6% cotransduction) by P22 phage-mediated transduction.

A mutation in Salmonella typhimurium imparting conditional resistance to 5-fluorouracil and a bioenergetic defect: Mapping of cad

SummaryThe position of the genetic locus allelic with the cad-2 mutation has been located between units 14 and 15 of the linkage map of S. typhimurium. Fine structure mapping established the gene order as cad flrB nag. The genetic evidence coupled with biochemical evidence indicates that this cad locus is homologous to the ubiF gene of Escherichia coli.

Arginine Regulon Control in a Salmonella typhimurium- Escherichia coli Hybrid Merodiploid

SummaryThe regulation of synthesis of arg enzymes was studied in a hybrid merodiploid in which an episome of Escherichia coli carrying the argR+ allele was transferred to a Salmonella typhimurium argR strain. The arg enzyme levels of the hybrid merodiploid were compared to that found in argR and argR+ haploids of S. typhimurium. The results showed that repression of synthesis of arg enzymes was effected through the introduction of the E. coli argR+ allele but significant quantitative differences of arg enzyme levels in the argR+ haploid and the hybrid merodiploid were observed.