Insights into the Molecular Basis of L-Form Formation and Survival in Escherichia coli

Abstract

L-forms have been shown to occur among many species of bacteria and are suspected to be involved in persistent infections. Since their discovery in 1935, numerous studies characterizing L-form morphology, growth, and pathogenic potential have been conducted. However, the molecular mechanisms underlying the formation and survival of L-forms remain unknown. Using unstable L-form colonies of Escherichia coli as a model, we performed genome-wide transcriptome analysis and screened a deletion mutant library to study the molecular mechanisms involved in formation and survival of L-forms. Microarray analysis of L-form versus classical colonies revealed many up-regulated genes of unknown function as well as multiple over-expressed stress pathways shared in common with persister cells and biofilms. Mutant screens identified three groups of mutants which displayed varying degrees of defects in L-form colony formation. Group 1 mutants, which showed the strongest defect in L-form colony formation, belonged to pathways involved in cell envelope stress, DNA repair, iron homeostasis, outer membrane biogenesis, and drug efflux/ABC transporters. Four (Group 1) mutants, rcsB, a positive response regulator of colanic acid capsule synthesis, ruvA, a recombinational junction binding protein, fur, a ferric uptake regulator and smpA a small membrane lipoprotein were selected for complementation. Complementation of the mutants using a high-copy overexpression vector failed, while utilization of a low-copy inducible vector successfully restored L-form formation. This work represents the first systematic genetic evaluation of genes and pathways involved in the formation and survival of unstable L-form bacteria. Our findings provide new insights into the molecular mechanisms underlying L-form formation and survival and have implications for understanding the emergence of antibiotic resistance, bacterial persistence and latent infections and designing novel drugs and vaccines.

Colanic Acid Intermediates Prevent De Novo Shape Recovery of Escherichia coli Spheroplasts, Calling into Question Biological Roles Previously Attributed to Colanic Acid

“…CA has been deemed to be essential for the continued growth and longterm survival of wall-less E. coli cells (variously described as Lforms or L-form-like cells) (15,16,20). However, we find that, in the complete absence of the entire CA biosynthetic pathway, LI spheroplasts survive, grow, and revert to a wild-type rod shape.…”

“…Triggering the Rcs system induces expression of this operon, leading to the production of copious amounts of this extracellular capsular carbohydrate (21,32). Because CA was reported to be essential for creating L-forms (15,16,20), it seemed possible that Rcs-deficient spheroplasts would not recover, because they could not synthesize this compound. Consistent with this expectation, spheroplasts lacking wza did not revert to a normal rod shape but instead expanded to become giant spheroids that lysed (Fig.…”

“…Though both spheroplasts and L-forms may sometimes regenerate a properly formed cell wall (13), L-forms must be able to divide and survive in the absence of a cell wall, and several genes and proteins are important for this transformation (15)(16)(17). One component reported to be required for L-form survival is the production of colanic acid (CA), a product of the Rcs stress response that regulates about 150 genes (15,16,(18)(19)(20). CA is an extracellular capsular polysaccharide common to members of Enterobacteriaceae but absent from other bacteria (21), and its putative role in L-form survival is undefined.…”

Structural Basis of Outer Membrane Protein Biogenesis in Bacteria

“…Proteins inhibiting ␤-lactamases typically comprise a tandem fold of (ϳ70 -100) 2 residues (59), which is the approximate size of dimeric BamE. The function of natural ␤-lactamase inhibitors on the basis of proteins is still under debate, but they may be involved in the regulation of cell wall growth (60), a function which could, e.g., reflect a reduced stalk biosynthesis in Caulobacter crescentus (61) and a function in L-form formation of E. coli has been reported recently (62). Together these observations support our view of a BamE function in cell wall maintenance, possibly by a direct or indirect effect involving the ␤-lactamase AmpC from E. coli.…”

Fluorescence Reporter in Staphylococcus aureus as a Useful Tool for Studying L-forms and Virulence

“…This indicates that the promoter of sarA which drives GFP expression in Newman-pCM29 is also expressed in bacterial L-form in addition to its expression in typically cell wall form. In fact, the close similarity of gene expression profiling of bacterial L-form was observed in Escherichia coli (24).…”