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

Abstract

We have previously identified the gene (the ssc gene) defective in the thermosensitive and antibiotic-supersusceptible outer membrane permeability mutant SS-C of Salmonella typhimurium and shown that this gene is analogous to the Escherichia coli gene firA (L. Hirvas, P. Koski, and M. Vaara, EMBO J. 10:1017-1023, 1991). Others have tentatively implicated firA in a different function, mRNA synthesis. Here we report that the defect in the thermosensitive outer membrane omsA mutant of E. coli (T. Tsuruoka, M. Ito, S. Tomioka, A. Hirata, and M. Matsuhashi, J. Bacteriol. 170:5229-5235, 1988) is due to a mutation in firA; this mutation changed codon 271 from serine to asparagine. The omsA-induced phenotype was completely reverted by plasmids containing wild-type firA or ssc. Plasmids carrying the omsA allele, or an identical mutant allele prepared by localized mutagenesis, under the control of lac elicited partial complementation. Transcomplementation studies with plasmids carrying various mutant alleles of the S. typhimurium gene indicated that the ability of these plasmids to complement the omsA mutation was similar to their ability to complement the ssc mutation. The antibiotic-supersusceptible phenotype of the omsA mutant closely resembled that of the ssc mutant, i.e., the omsA mutant was supersusceptible to hydrophobic antibiotics and large-peptide antibiotics against which the intact outer membrane is an effective permeability barrier. As previously demonstrated with the omsA mutant, the outer membrane of the ssc mutant became selectively ruptured after incubation for 1 h at the growth-nonpermitting temperature; 82% of the periplasmic beta-lactamase and less than 3% of the cytoplasmic marker enzyme were released into the medium. All of these findings are consistent with our concept that firA is an essential gene involved in generation of the outer membrane.

The Bacterial Actin-Like Cytoskeleton

“…A more sensitive, high-resolution probe for nascent PG insertion in nonfixed cells was recently developed by Daniel and Errington (22). These authors used a fluorescent derivative of the antibiotic vancomycin (a cell wall synthesis inhibitor that binds specifically to PG intermediates) to label nascent PG in gram-positive bacteria by fluorescence microscopy (note that gram-negative bacteria do not stain because their outer membrane presents a permeability barrier to vancomycin) (169). This novel staining method revealed that, at least in B. subtilis, synthesis of the wall occurs in a helical pattern over the cylindrical part of the cell and also specifically at the septum in dividing cells (Fig.…”

The Lipid a Biosynthesis Deficiency of the Escherichia Coli Antibiotic-Supersensitive Mutant LH530 is Suppressed by a Novel Locus, ORF195

“…coli and S . typhimurium strains harbouring mutations in the lipid A biosynthesis genes lpxA and lpxD (Galloway & Raetz, 1990;Hirvas et al, 1991a;Helander et al, 1992Helander et al, , 1993Vuorio & Vaara, 1992b). However, the antibiotic sensitivity of LH530 was not reverted by ZpxA and ZpxD indicating that it did not harbour mutations in these genes.…”

“…Determination of the ratio of LPS to phospholipid biosynthesis. The ratio of LPS and phospholipid biosynthesis in LH530 and its derivatives was assayed (Vuorio & Vaara, 1995) after labelling their fatty acids with radioactive acetate in vivo at 28 "C, 37 "C and 42 "C. In detail, 5 x lo8 bacterial cells (from fresh LB plates grown at 28 "C) were suspended in 5 ml LB broth. After growing (with stirring) for 1 h at the selected temperature (28 "C, 37 "C or 42 "C),lO p1 (074 MBq) [14C]acetate (sodium salt, CFA229, specific activity 1.89 GBq mmol-l ; Amersham) was added and growth was continued for an additional 2 h. The cells were harvested by centrifugation (4300 r.p.m., Heraeus megafuge), washed with 2.5 ml saline, transferred with 1 ml of saline into Sarstedt tubes (1 ml) and collected by centrifugation.…”

Antibiotic-supersusceptible mutants of Escherichia coli and Salmonella typhimurium

“…Sequencing of firA of E. coli revealed that it is 88% identical to ssc of S. typhimunium and that the corresponding proteins are 96% identical (10). Quite recently, Vuorio and Vaara (64) reported that another antibiotic-supersusceptible mutant, the so-called omsA mutant strain CDH23-213 of E. coli (52), resembles SH7622 phenotypically and that omsA is allelic to firA. To date, omsA of CDH23-213 and ssc-1 of SH7622 are the best-characterized firA alleles known.…”