No abstract

A polymerase chain reaction (PCR)-based procedure without any cloning step was developed for a rapid mutagenesis/deletion of chromosomal target genes in Yersinia. For this purpose, a PCR fragment carrying an antibiotic resistance gene flanked by regions homologous to the target locus is electroporated into a recipient strain expressing the highly proficient homologous recombination system encoded by plasmid pKOBEG-sacB. Two PCR procedures were tested to generate an amplification product formed of an antibiotic resistance gene flanked by short (55 bp) or long (500 bp) homology extensions. Using this method, three chromosomal loci were successfully disrupted in Yersinia pseudotuberculosis. The use of this technique allows rapid and efficient large-scale mutagenesis of Yersinia target chromosomal genes.

The bacA gene product of Escherichia coli was recently purified to near homogeneity and identified as an undecaprenyl pyrophosphate phosphatase activity (El Ghachi, M., Bouhss, A., Blanot, D., and Mengin-Lecreulx, D. (2004) J. Biol. Chem. 279, 30106 -30113). The enzyme function is to synthesize the carrier lipid undecaprenyl phosphate that is essential for the biosynthesis of peptidoglycan and other cell wall components. The inactivation of the chromosomal bacA gene was not lethal but led to a significant, but not total, depletion of undecaprenyl pyrophosphate phosphatase activity in E. coli membranes, suggesting that other(s) protein(s) should exist and account for the residual activity and viability of the mutant strain. Here we report that inactivation of two additional genes, ybjG and pgpB, is required to abolish growth of the bacA mutant strain. Overexpression of either of these genes, or of a fourth identified one, yeiU, is shown to result in bacitracin resistance and increased levels of undecaprenyl pyrophosphate phosphatase activity, as previously observed for bacA. A thermosensitive conditional triple mutant ⌬bacA,⌬ybjG,⌬pgpB in which the expression of bacA is impaired at 42°C was constructed. This strain was shown to accumulate soluble peptidoglycan nucleotide precursors and to lyse when grown at the restrictive temperature, due to the depletion of the pool of undecaprenyl phosphate and consequent arrest of cell wall synthesis. This work provides evidence that two different classes of proteins exhibit undecaprenyl pyrophosphate phosphatase activity in E. coli and probably other bacterial species; they are the BacA enzyme and several members from a superfamily of phosphatases that, different from BacA, share in common a characteristic phosphatase sequence motif.An essential carrier lipid, undecaprenyl phosphate (C 55 -P), 1 is required for the synthesis of various bacterial cell wall polymers such as peptidoglycan, lipopolysaccharides, and teichoic acids (1-5) (Scheme 1). It is synthesized as a pyrophosphate precursor (C 55 -PP) by the addition of eight isoprene units to farnesyl pyrophosphate, a reaction catalyzed by the well characterized cis-prenyl-pyrophosphate synthase UppS (6 -10). However, genes and enzymes involved in subsequent steps of C 55 -P synthesis and recycling still remained to be identified. Bacitracin is a dodecapeptide antibiotic known to specifically block this metabolism by forming a specific complex with C 55 -PP. As a result, cell wall biosynthesis is inhibited and cell lysis finally occurs (11)(12)(13)(14). Bacillus licheniformis strains that produce bacitracin are resistant to this antibiotic due to the presence of an appropriate ABC transporter efflux system (15, 16). Several mutations leading to bacitracin resistance were identified in Escherichia coli and other Gram-negative bacteria. Interestingly, all these mutations were shown to block the synthesis of non-essential cell envelope polymers such as osmoregulated periplasmic glycans and capsule polysaccharides that also requ...