Identification of Multiple Genes Encoding Membrane Proteins with Undecaprenyl Pyrophosphate Phosphatase (UppP) Activity in Escherichia coli

Abstract

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...

An Escherichia coli undecaprenyl-pyrophosphate phosphatase implicated in undecaprenyl phosphate recycling

“…The E. coli K-12 proteins YbjG, YeiU and PgpB have a similar motif to that found in Cwh8 ( Fig. 1) (El Ghachi et al, 2005). However, their function in the recycling of Und-PP remains to be determined.…”

“…The de novo synthesis and regeneration of Und-PP at opposite sides of the membrane suggests the requirement for pyrophosphatases whose active sites are in cytosolic and periplasmic environments, respectively. Recently, El Ghachi et al (2004Ghachi et al ( , 2005 have identified four genes in Escherichia coli K-12 encoding membrane proteins with Und-PP pyrophosphatase activities. Among these, UppP (formerly BacA) accounts for 75 % of the cellular Und-PP pyrophosphatase activity (El Ghachi et al, 2004), while the proteins YbjG, YeiU and PgpB account for the remaining activity (El Ghachi et al, 2005).…”

“…Recently, El Ghachi et al (2004Ghachi et al ( , 2005 have identified four genes in Escherichia coli K-12 encoding membrane proteins with Und-PP pyrophosphatase activities. Among these, UppP (formerly BacA) accounts for 75 % of the cellular Und-PP pyrophosphatase activity (El Ghachi et al, 2004), while the proteins YbjG, YeiU and PgpB account for the remaining activity (El Ghachi et al, 2005). However, it is not clear which of these proteins function in Und-PP recycling.…”

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP

“…They also showed that disruption of the bacA gene alone in E. coli was not lethal despite accounting for 75 % of the undecaprenyl pyrophosphate phosphatase activity. These investigators hypothesized that additional enzymes were necessary to account for the remaining~25 % of the undecaprenyl pyrophosphate phosphatase activity and demonstrated that disruption of two additional genes, ybjG and pgpB, along with bacA/uppP caused bacterial cell lysis (El Ghachi et al, 2005). We subsequently use the combined name BacA/UppP to both address activity and distinguish from other enzymes containing undecaprenyl pyrophosphate phosphatase activity.…”

Colonize, evade, flourish

“…3 Subsequently, UndPP is mono-dephosphorylated by an undecaprenyl pyrophosphate phosphatase and recycled back to the cytoplasmic face of the inner membrane to repeat the cycle. 10 Mature LPS must then be transported across the periplasm and shuttled across the outer membrane for final surface presentation; these steps are performed by the lipopolysaccharide transport (Lpt) machinery. 11 H. pylori possesses homologs to several the environment, the O-antigen chain shields the bacterium from host complement deposition and may contain sugar subunits that mimic human sugars, as discussed in detail later in this review.…”