A comparative study of variation in codon 33 of the rpoS gene in Escherichia coli K12 stocks: implications for the synthesis of σs

Abstract

The Escherichia coli rpoS gene encodes an RNA polymerase sigma factor (sigma S or sigma(S)) required for the expression of stationary-phase genes. In the first published rpoS sequence from E. coli K-12 codon 33 is given as CAG. However, several subsequent independent studies found the amber codon TAG at this position ( rpoSAm). Besides this amber codon, other codons such as TAT have also been found at this location in rpoS. Comparative genome analysis now leads us to propose TAG as the parental codon 33 in rpoS in E. coli K-12. Five different stocks of the strain W3110, which differ in the levels of sigma(S) protein they express, were investigated. We sequenced the rpoS gene from these, and found a T at nucleotide position 97 in four out of the five stocks and a G at position 99 in three out of the five. W1485, a parental strain of W3110, and W3350, a derivative of W3110, are also rpoSAm mutants. Such rpoSAm mutants would be expected to show no RpoS activity. The retention of partial or intermediate sigma(S) activity by suppressor-free rpoSAm mutants is therefore puzzling. We propose that a functional, N-terminally truncated, sigma(S) (Delta1-53sigma(S)) can be translated from a Secondary Translation Initiation Region (STIR) located downstream of the amber codon 33. It has recently been reported that a fragment of RpoS (Delta1-53sigma(S)) that lacks the first 53 amino acids is functional when synthesized in vivo. Taken together, our results support the hypothesis that the original codon 33 of the rpoS gene in E. coli K-12 strains is the amber codon TAG.

MicroReview: Growth versus maintenance: a trade‐off dictated by RNA polymerase availability and sigma factor competition?

“…One of the most common down-mutations in rpoS is the TAG amber codon at position 33, which, in the first published rpoS sequence of E. coli K-12 is given as CAG. However, comparative genome analysis lead Subbarayan and Sarkar (2004) to propose that TAG is actually the parental codon 33 in rpoS and that a functional, N-terminally truncated, s s can be translated from a secondary translation initiation region located downstream of codon 33. This would explain why s s activity is retained in suppressor-free strains carrying the amber 33 codon.…”

“…This would explain why s s activity is retained in suppressor-free strains carrying the amber 33 codon. Indeed, a fragment of s s (rpoSD1-53) lacking the first 53 amino acids is functional in vivo (Subbarayan and Sarkar, 2004) as has previously been shown for a fragment lacking the postulated N-terminal subregion 1.1 (Rajkumari and Gowrishankar, 2002;Gowrishankar et al, 2003). Thus, uncertainties surrounding the ancestral sequence of the rpoS gene, the genetic relationships between different K-12 strains, and the strong selective pressures these strains have encountered in the laboratory, make it difficult to draw conclusions about how common down-mutations (or up-mutations) in rpoS are in the real world outside the laboratory.…”

The acid-resistance pathways of Shigella flexneri 2457T

“…The truncation means that the protein is missing the end of the fourth domain of the conserved s 70 family structure, which is involved in -35 promoter recognition of target genes (Lonetto et al, 1992). The loss of the fourth-domain-encoding region of RpoS in E. coli is sufficient to render the protein effectively inactive (Ohnuma et al, 2000;Subbarayan & Sarkar, 2004). In an attempt to determine whether the 2457T RpoS is functional, a catalase assay was performed.…”

“…The qualitative catalase activity was estimated by dropping 10 ml 30 % H 2 O 2 onto individual colonies grown overnight on LB plates at 37 uC (Subbarayan & Sarkar, 2004). The rapidity and degree of bubbling were measured for each strain.…”

“…To determine the activity of the truncated RpoS of SFL1001, it is possible to measure the activity of RpoS using a rapid semi-quantitative catalase assay, in which an RpoS mutant will display a reduced level of bubbling, although some bubbling will still be observed as the cell possesses a second hydrogen peroxidase (HPI) that is not regulated by RpoS (Subbarayan & Sarkar, 2004). E. coli MG1655, which possesses a full-sized (330 aa) RpoS, had a rapid, vigorous bubbling phenotype, while S. flexneri 2457T, which contains a truncated (255 aa) RpoS, displayed reduced oxygen production, with slow bubbling observed.…”