Jean-Claude Lazzaroni

The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein (PAL).

The excC mutants of Escherichia coli are hypersensitive to drugs such as cholic acid and release periplasmic proteins Into the extracellular medium. A 1884 bp fragment carrying the excC gene was isolated and sequenced. It contains the 3′ end of the tolB gene which maps at min 17 on the E. coll map and an open reading frame which encodes the 18748 Da ExcC protein. The protein is composed of a hydrophobic region of 22 residues and displayed an overall hydrophilic configuration. It was shown that the ExcC protein is indeed the PAL (peptidoglycan‐associated lipoprotein) described by Mizuno (1979). The pal gene had not yet been characterized on the E. coli linkage map since no obvious phenotype could be identified for mutations in this gene. A topologic analysis of the PAL protein using PAL–PhoA translational fusions showed that PAL is associated with the outer membrane only by its N‐terminal moiety. The carboxy‐terminal part of the protein is necessary for correct interaction of PAL with the peptidoglycan layer.

Bacterial Peptidoglycan-Associated Lipoprotein: A Naturally Occurring Toll-Like Receptor 2 Agonist That Is Shed into Serum and Has Synergy with Lipopolysaccharide

Sepsis is initiated by interactions between microbial products and host inflammatory cells. Toll-like receptors (TLRs) are central innate immune mediators of sepsis that recognize different components of microorganisms. Peptidoglycan-associated lipoprotein (PAL) is a ubiquitous gram-negative bacterial outer-membrane protein that is shed by bacteria into the circulation of septic animals. We explored the inflammatory effects of purified PAL and of a naturally occurring form of PAL that is shed into serum. PAL is released into human serum by Escherichia coli bacteria in a form that induces cytokine production by macrophages and is tightly associated with lipopolysaccharide (LPS). PAL activates inflammation through TLR2. PAL and LPS synergistically activate macrophages. These data suggest that PAL may play an important role in the pathogenesis of sepsis and imply that physiologically relevant PAL and LPS are shed into serum and act in concert to initiate inflammation in sepsis.

Cloning of the excC and excD genes involved in the release of periplasmic proteins by Escherichia coli K12.

SummaryStrains of Escherichia coli K12 carrying a tolA, tolB, lky or exc mutation located at min 16.5 on the genetic map released periplasmic proteins into the extracellular medium. Wild-type genes defined by these mutations have been cloned from E. coli genomic bank made with plasmid pBR328. Subcloning experiments and complementation studies showed that lky and exc mutations were located either in the previously described tolA and tolB genes or in the newly characterized excC and excD genes. Using minicells, excC and excD gene products were identified as proteins with a molecular mass of 19 and 21 kDa, respectively.

Effects of pho Regulatory Mutations and phoA Gene Amplification on Alkaline Phosphatase Synthesis and Release by lky Mutants of Escherichia coli K12

lky mutants of Escherichia coli K12 spontaneously released alkaline phosphatase (APase) into the extracellular medium to give up to 300 units ml-1. APase is a phosphate repressible periplasmic enzyme encoded by the gene phoA. With a view to establishing a method of easy purification, we have analysed APase synthesis and release patterns of isogenic lky strains containing either a constitutive pho regulatory mutation, or a hybrid plasmid carrying the structural gene phoA+ and pho regulatory genes, or a transducing phi 80 phoA+ phage. In the presence of the phoS2333 mutation, F- lky strains lysogenized with phi 80 phoBin phoA+ phage and grown in high phosphate medium were able to release eight times more APase activity (2300 units ml-1) than haploid strain 2336 (phoS+ lky) grown in low phosphate medium. Neither protein synthesis, the cell export machinery nor leakage mechanisms were limiting for APase release. Sufficient APase was released into the medium to facilitate its purification.