Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli

Abstract

The Escherichia coli genefirA, previously reported to code for a small, histonelike DNA-binding protein, has been cloned and found to reside immediately downstream from skp, a gene previously identified as the firA locus. firA encodes a 36-kDa protein. The mutantfirA200(Ts) allele was also cloned and shown to contain three mutations, each mutation giving rise to a single amino acid change. Partially purified wild-type FirA It is well documented that selection for rifampin resistance in Escherichia coli gives mutations in rpoB, the gene encoding the P subunit of RNA polymerase (13,16,27). Suppressors of the rifampin resistance phenotype are worth studying because of the insight they might provide into the components of the transcription machinery. Several loci which can suppress this rifampin resistance have been identified and have been termed fir mutants (rif spelled backwards): firA (19) in the 4-min region, firB (26) in the 90-min region, and firC (3). Among these, firA has been the most studied. The firA 200(Ts) allele has been shown to cause temperature sensitivity for growth and to reverse the rifampin resistance of rifampin-resistant rpoB mutants (21). firA was also reported to confer temperature sensitivity to in vitro RNA synthesis by RNA polymerase partially purified from a firA200(Ts) strain and has been hypothesized to interact with RNA polymerase in vivo (22). A recombinant lambda phage containing a 6.7-kb insert was shown to complement the firA200(Ts) phenotypes (20) In this study, we report the cloning of thefirA and firA200 genes and show thatfirA is located immediately downstream from the previously reported skp gene. We also discuss data which indicate that the FirA protein coimmunoprecipitates with RNA polymerase holoenzyme. This study is part of our larger interest in better defining the role, if any, of firA in transcription.MATERIALS AND METHODS Strains and media. All strains used are listed in Table 1. Strain RL25-1 was obtained from R. Menzel (Bristol-MyersSquibb Co.), and KK2186 (31) was a gift from R. Zagursky (Du Pont Co.). JCR20, containing the authentic firA200(Ts) allele, was kindly supplied by J. Coleman, University of Louisiana, and was obtained by crossing a P1 lysate of the authentic RL25 into MF6R (Rif' dapD) (7) and selecting for * Corresponding author.prototrophs. The library of TnJOd-Cam transposon insertions was made (A. Segall, National Institutes of Health) by the reference method (9). Cells were grown in LB medium alone or LB medium containing 150 ,ug of ampicillin per ml or 150 or 50 ,ug of rifampin per ml. For maintenance of pMS421, spectinomycin was used at 50 p,g/ml. ID15 was isolated as a spontaneous rifampin-resistant colony on LB plates containing 150 pug of rifampin per ml. Plates were incubated at 42 and 43°C in water-jacketed incubators.Strain construction. P1 transduction was performed as previously described (24)

Mutants carrying conditionally lethal mutations in outer membrane genes omsA and firA (ssc) are phenotypically similar, and omsA is allelic to firA

“…This protein has a very conservative structure among the members of the family Enterobacteriaceae (15,18,20,22,43,46), carries a signal sequence that is characteristic of secreted proteins in its precursor form (18,20,21,43), can be found in isolated OM (18,22), and has an unknown function. Only three nucleotides separate the hlpA stop and firA initiation codons (5,16). We have previously shown by using minicells and by complementation assays that the firA gene of S. typhimurium is expressed in the absence of the hipA promoter, i.e., in a plasmid which lacks the 5' terminus of hlpA and where the vector-derived lac promoter is opposite in orientation to firA (17).…”

“…The study on the ssc-1 mutation led us to discover and sequence a novel gene of S. typhimurium, the ssc gene (16). The sequence of its analog in E. coli, the firA gene, is also known (5). On the other hand, the gene mutated in the omsA mutant was not identified by Tsuruoka et al (39), but their preliminary evidence suggested that the omsA mutation maps at approximately 4 min on the E. coli chromosome.…”

Membrane protein folding on the example of outer membrane protein A of Escherichia coli

“…The skp gene maps at the 4-min region on the chromosome and is located only four bases upstream of genes that encode proteins involved in lipid A biosynthesis [54][55][56], which is an essential component of LPS. Since LPS is also a major component of the outer membrane of E. coli, several studies have addressed its role in the folding of OMPs.…”

The Ssc protein of enteric bacteria has significant homology to the acyltransferase Lpxa of lipid A biosynthesis, and to three acetyltransferases

“…We found that all these proteins possess a 41 amino acid-long highly conserved region. An earlier report mentioned that the E. coli analogue, FirA, has homology with LacA and CysE (as well as to LpxA), but provided no data [6]. LacA, CysE and NodL have previously been shown to be remarkably homologous with each other and to display a common I-G-A-G-S-[L.I,V,M]-V motif, named as the 'the acetyltransferase motif.…”

“…The Sahnonella typhimuriurn mutant SH7622, which carries the mutant allele sscl, is extremely sensitive to numerous hydrophobic antibiotics at growth-permissive temperatures and does not grow at 42°C [4,5]. S. typhimurium ssc and its Escherichia cell analogue firA are highly homologous (identity, 88%) as are the corresponding proteins Ssc and FirA (identity, 96%) [4,6]. We are currently trying to identify the function of Ssc.…”