Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure1. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia encodes genes for PG biosynthesis2–7 and exhibits susceptibility to "anti-PG" antibiotics8,9, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’10). We employed a novel approach to metabolically label chlamydial PG using D-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis was labeled with the probes throughout its biphasic, developmental life cycle, and differential probe incorporation experiments conducted in the presence of ampicillin is consistent with the presence of chlamydial PG modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence to date that chlamydial species possess functional PG.

Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylase (LDC) activity is present in Ϸ90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these ''black holes,'' deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.

Virulent strains of Shigellaflexneri invade HeLa cells with high efficiency. This crucial step in the pathogenic process is encoded by a 140-megadalton plasmid which induces phagocytosis of the bacteria by host cells. In this report we used pWR100, the virulence plasmid of S. flexneri serotype 5, and pHS4108, a 32-megadalton subclone of pWR100, to demonstrate that the plasmid is also responsible for rapid intracellular growth of the bacteria. The ability to replicate intracellularly was not correlated with induction of Shiga toxin. However, plasmid-mediated intracellular multiplication was strongly correlated with the ability of the bacteria to rapidly and efficiently lyse the phagocytic vacuole and replicate freely in the cytoplasm. Temperature-regulated plasmid-mediated contact hemolytic activity strongly correlated with both phagosomal membrane lysis and efficient intracellular multiplication. We propose this virulence plasmid-associated hemolysin to be an important factor in the invasion and proliferation of Shigella spp. in mammalian cells.

Genes required for the full expression of Shigella virulence are on both the chromosome and a large virulence-associated plasmid. Expression of one or more virulence (vir) genes is temperature-regulated, wild-type strains being virulent (invasive) when grown at 370C but phenotypically avirulent (noninvasive) at 30TC. A vir.:lac operon fusion located on the virulence plasmid, which brings the lac genes under control of a temperature-regulated vir gene promoter, was used to select regulatory mutants constitutive for the Lac + phenotype at the nonpermissive temperature. A transposon TnlO-induced mutant that was Lac' at 300C and 37C was isolated, and the TnlO insertion was transduced into a wildtype strain. The transductants all simultaneously became deregulated for virulence and invaded HeLa cells equally well at 300C and 37TC. Other virulence-associated phenotypes were also deregulated and expressed at 30TC. Southern hybridization with a probe for TnWO determined the insertion to be on the chromosome. Fine mapping by transduction with phage P1L4 positioned the mutation between the galU and tip genes. A cosmid cloned fragment of Shigella chromosomal DNA containing the region around galU was used in complementation studies and showed that the closely linked regulatory gene was able to complement, in trans, the TnlO-induced mutation. We propose that this mutation defines a regulatory gene, virR, and that insertion of TnlO into this gene inactivates a repressor that normally blocks expression of vir genes at 300C.The primary characteristic of Shigella pathogenicity is the ability of the bacteria to penetrate into and replicate within human colonic epithelial cells (1). Infection results in destruction of colonic epithelium and ulcerative lesions, producing the bloody diarrhea characteristic of bacillary dysentery. Laboratory methods for studying Shigella virulence use both animal and cell culture models for measuring penetration capacity and the ability to sustain growth inside mammalian cells. Use of these assays has permitted the identification of chromosomal virulence (vir) genes (2, 3) as well as the demonstration of the involvement of a 220-kilobase (kb) plasmid in expression of the virulence phenotype of Shigella flexneri (4). Large virulence-associated plasmids homologous to that found in S. flexneri are present in Shigella sonnei, Shigella dysenteriae, and enteroinvasive strains of Escherichia coli (5, 6). It also has been shown that expression of the invasive phenotype by Shigella spp. is regulated by growth temperature (7). Bacteria grown at 370C are virulent and invade epithelial cells, whereas the same virulent strains are noninvasive when grown at 30TC. Loss of invasive ability at 30'C reflects a reversible phenotypic change and not a genotypic change, since virulence is completely restored after shifting the growth temperature back to 37TC.We have taken advantage of gene fusion technology to study the mechanism of vir gene regulation. In a previous study, an operon fusion was constructed such tha...