Molecular cloning and expression of the ilvGEDAY genes from Salmonella typhimurium

The ilvGEDAY genes of Salmonella typhimurium were cloned in Escherichia coli K-12 by in vitro recombination techniques. A single species of recombinant plasmid, designated pDU1, was obtained by selecting for Valr Ampr transformants of strain SK1592. pDU1 was shown to contain a 14-kilobase EcoRI partial digestion product of the S. typhimurium chromosome inserted into the EcoRI site of the pVH2124 cloning vector. The ilvGEDAY genes were found to occupy a maximum length of 7.5 kilobases. Restriction endonuclease analysis of the S. typhimurium ilv gene cluster provided another demonstration of the gene order as well as established the location of ilv Y between ilvA and ilvC. The presence of a ribosomal ribonucleic acid operon on the pDU1 insert, about 3 kilobases from the 5' end of ilvG, was shown by Southern hybridization. The expression of the ilvGEDA operon from pDU1 was found to be elevated, reflecting the increased gene dosage of the multicopy plasmid. A polarity was observed with respect to ilvEDA expression which is discussed in terms of the possible translational effects of the two internal promoter sequences, one located proximal to ilvE and the other located proximal to ilvD.     

pyrR identical to pyrH in Salmonella typhimurium: control of expression of the pyr genes.

Mutants of Salmonella typhimurium showing constitutive synthesis of the pyrimidine biosynthetic enzymes coded for by the pyrA-F genes (G. A. O'Donavan and J. C. Gerhart, 1972) have been reinvestigated. The high rate of expression of the pyrB-F genes in these mutants as well as their pyrimidine excretion is shown to be due to mutations in the gene pyrH encoding uridine 5'-monophosphate kinase. Thus, the term pyrR used for these mutants should be replaced by the designation pyrH.     

Fis, a DNA nucleoid-associated protein, is involved in Salmonella typhimurium SPI-1 invasion gene expression

The ability of Salmonella enterica serovar Typhimurium to cause disease depends upon the co-ordinated expression of many genes located around the Salmonella chromosome. Specific pathogenicity loci, termed Salmonella pathogenicity islands, have been shown to be crucial for the invasion and survival of Salmonella within host cells. Salmonella pathogenicity island 1 (SPI-1) harbours the genes required for the stimulation of Salmonella uptake across the intestinal epithelia of the infected host. Regulation of SPI-1 genes is complex, as invasion gene expression responds to a number of different signals, presumably signals similar to those found within the environment of the intestinal tract. As a result of our continued studies of SPI-1 gene regulation, we have discovered that the nucleoid-binding protein Fis plays a pivotal role in the expression of HilA and InvF, two activators of SPI-1 genes. A S. typhimurium fis mutant demonstrates a two- to threefold reduction in hilA:Tn5lacZY and a 10-fold reduction in invF:Tn5lacZY expression, as well as a 50-fold decreased ability to invade HEp-2 tissue culture cells. This decreased expression of hilA and invF resulted in an altered secreted invasion protein profile in the fis mutant. Furthermore, the virulence of a S. typhimurium fis mutant is attenuated 100-fold when administered orally, but has wild-type virulence when administered intraperitoneally. Expression of hilA:Tn5lacZY and invF:Tn5lacZY in the fis mutant could be restored by introducing a plasmid containing the S. typhimurium fis gene or a plasmid containing hilD, a gene encoding an AraC-like regulator of Salmonella invasion genes.     

Cloning and sequence analysis of hydrogenase regulatory genes (hydHG) from Salmonella typhimurium.

The nucleotide sequence of the hydHG operon, comprised of chromosomal genes that regulate labile hydrogenase activity in Salmonella typhimurium, was compared with the reported hydHG sequence of Escherichia coli. Nucleotide sequence analysis of a 4.8 kb EcoRI fragment of Salmonella chromosomal DNA revealed that one of the open reading frames (ORF) encoded a protein of 441 amino acid residues. This large ORF was identified on a 2.7 kb Eco RI/HindIII fragment and coded for the complete hydG gene. The carboxy-terminus (626 bp) of the hydH gene also was present immediately upstream of hydG. Expression of the Salmonella hydG gene in a T7 promoter/polymerase system revealed the presence of a unique 45 kDa protein band. The incomplete hydH gene was not expressed. It is proposed that the labile hydrogenase activity in S. typhimurium may be regulated by the multiple component system.