title: 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.
authors.affiliation: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: William A
authors.initials: WA
authors.lastname: Glover
authors.forename: Yanqin
authors.initials: Y
authors.lastname: Yang
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D052160:Benzothiazoles; D003959:Diamines; D004926:Escherichia coli / Q000378:metabolism* / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D016678:Genome; D008954:Models, Biological; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D009930:Organic Chemicals / Q000494:pharmacology; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000900:Anti-Bacterial Agents; D052160:Benzothiazoles; D003959:Diamines; D029968:Escherichia coli Proteins; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I