rpoS Function Is Essential for bgl Silencing Caused by C-Terminally Truncated H-NS in Escherichia coli

From evolutionary and physiological viewpoints, the Escherichia coli bgl operon is intriguing because its expression is silent (Bgl(-) phenotype), at least under several laboratory conditions. H-NS, a nucleoid protein, is known as a DNA-binding protein involved in bgl silencing. However, we previously found that bgl expression is still silent in a certain subset of hns mutations, each of which results in a defect in its DNA-binding ability. Based on this fact, we proposed a model in which a postulated DNA-binding protein(s) has an adapter function by interacting with both the cis-acting element of the bgl promoter and the mutated H-NS. To identify such a presumed adapter molecule, we attempted to isolate mutants exhibiting the Bgl(+) phenotype in the background of hns60, encoding the mutant H-NS protein lacking the DNA-binding domain by random insertion mutagenesis with the mini-Tn10cam transposon. These isolated mutations were mapped to five loci on the chromosome. Among these loci, three appeared to be leuO, hns, and bglJ, which were previously characterized, while the other two were novel. Genetic analysis revealed that the two insertions are within the rpoS gene and in front of the lrhA gene, respectively. The former encodes the stationary-phase-specific sigma factor, sigma(S), and the latter encodes a LysR-like DNA-binding protein. It was found that sigma(S) is defective in both types of mutant cells. These results showed that the rpoS function is involved in the mechanism underlying bgl silencing, at least in the hns60 background used in this study. We also examined whether the H-NS homolog StpA has such an adapter function, as was previously proposed. Our results did not support the idea that StpA has an adapter function in the genetic background used.     

The leuO Gene Product Has a Latent Ability To Relieve bgl Silencing in Escherichia coli

The Escherichia coli bgl operon is of interest, since its expression is silent (phenotypically Bgl⁻), at least under standard laboratory conditions. Here we attempted to identify a trans-acting factor(s) that is presumably relevant to the regulation of bgl by a random insertion mutagenesis with mini-Tn10. These collected mutations, conferring the phenotype of Bgl⁺, were localized in three loci on the genetic map, two of which appeared to be hns and bglJ, which were previously implicated as the factors affecting the Bgl phenotype. The other locus at 1 to 2 min on the genetic map appeared to be a new one. In this case, the insertion mutation was found to be just in front of the leuO gene encoding a putative LysR-like DNA-binding protein. Genetic analyses revealed that overproduction of LeuO in the wild-type cells causes the phenotype of Bgl⁺. A leuO deletion mutant was also characterized in terms of expression of bgl. From these results, the possible function of LeuO in bgl expression will be discussed from an evolutionary and/or ecological point of view.     

Role of DNA regions flanking the tryptophan promoter of Escherichia coli. I. Insertion of synthetic oligonucleotides

To examine the effect of altering the nucleotide sequence near the promoter on its activity, pKO-1 vector derivatives have been constructed which allow insertion of DNA fragments at specified sites upstream or downstream from the trp promoter. Oligonucleotides that might be expected to alter the melting properties, or have a tendency to form a distinctive nonstandard structure were introduced. These oligonucleotides had the repeating dinucleotide sequences GC, AT or AG. Sequence analysis of the inserts and studies of the relative galactokinase expression from the altered plasmids indicated that changes upstream from the trp promoter at -39 or beyond had little effect on trp promoter activity, whereas changes at +2 or farther downstream produced up to two-fold increases in gene expression, as compared to the control plasmid.