Mutational analysis and properties of the msbA gene of Escherichia coli, coding for an essential ABC family transporter

Authors

  • Alessandra Polissi,

    1. Département de Biochimie Médicate, Centre Médical Universitaire, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.
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    • (for correspondence): Glaxo Ricerche, Microbiology Division, Via Fleming 2, 37100 Verona, Italy. E-mail AP44783@ggr.co.uk; Tel. (45) 9218722; Fax (45) 9218196.

  • Costa Georgopoulos

    1. Département de Biochimie Médicate, Centre Médical Universitaire, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.
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Summary

The htrB gene was discovered because its insertional inactivation interfered with Escherichia coli growth and viability at temperatures above 32.5°C, as a result of accumulation of phospholipids. The msbA gene was originally discovered because when cloned on a low-copy-number plasmid vector it was able to suppress the temperature-sensitive growth phenotype of an htrB null mutant as well as the accumulation of phospholipids. The msbA gene product belongs to the superfamily of ABC transporters, a universally conserved family of proteins characterized by a highly conserved ATP-binding domain. The msbA gene is essential for bacterial viability at all temperatures. In order to understand the physiological role of the MsbA protein, we mutated the ATP-binding domain using random PCR mutagenesis. Six independent mutants were isolated and characterized. Four of these mutations resulted in single-amino-acid substitutions in non-conserved residues and were able to support cell growth at 30°C but not at 43°C. The remaining two mutations behaved as recessive lethals, and resulted in single-amino-acid substitutions in Walker motif B, one of the two highly conserved regions of the ATP-binding domain. Despite the fact that neither of these two mutant proteins can support E. coli growth, they both retained the ability to bind ATP in vitro. In addition, we present evidence to show that W-acetyl [3H]-glucosamine, a precursor of lipopolysaccharides, accumulates at the non-permissive temperature in the inner membrane of either htrB null or msbA conditional lethal strains. Translocation of the precursor to the outer membrane is restored by transformation with a plasmid containing the wild-type msbA gene. A possible role for MsbA

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