Morphotypic Conversion inListeria monocytogenesBiofilm Formation: Biological Significance of Rough Colony Isolates

Abstract

Adherence to a stainless steel surface selected isolates of Listeria monocytogenes with enhanced surface colonization abilities and a change in phenotype from the common smooth colony morphology to a succession of rough colony morphotypes. Growth in broth culture of the best-adapted, surface-colonizing rough colony morphotype gave a smooth colony revertant. Comparative analysis revealed that the smooth and rough variants had similar phenotypic and biochemical characteristics (e.g., identical growth rates and tolerances to antibiotics and environmental stressors). Rough colony isolates, however, failed to coordinate motility or induce autolysis. The defect in autolysis of rough colony isolates, which involved impaired cellular localization of several peptidoglycan-degrading enzymes, including cell wall hydrolase A (CwhA), suggested a link to a secretory pathway defect. The genetic basis for the impairment was studied at the level of the accessory secretory pathway component SecA2. DNA sequencing of the secA2 gene in smooth and rough colony isolates found no mutations in the coding or promoter regions. Analysis of SecA2 expression with an integrated secA2-FLAG tag construct found the protein to be upregulated in the rough and revertant backgrounds compared to the parental smooth colony isolate. A compensatory mechanism involving the SecA2 secretion pathway components is postulated to control smooth to rough interconversion of L. monocytogenes. Such phenotypic variation may enhance the ability of this opportunistic pathogen to colonize environments as diverse as processing surfaces, food products, and animal hosts.Changes in bacterial colony morphology often accompany microbial adaptation to new environments and ecological niches (12,19,32,34). Understanding the molecular basis of morphotypic change during biofilm formation on abiotic surfaces and the subsequent seeding and adaptation to planktonic growth have important implications for the food industry and medicine. For example, the fouling of conveyor belts and prosthetic devices can lead to contamination of food products and the spread of infection, respectively (16,36). Experimental models that are amenable to molecular methods are needed so that these problems can be circumvented. The readily cultured, intracellular, food-borne pathogen Listeria monocytogenes provides a practical model to explore morphotypic variation and its role in microbial adaptation.Variant rough colony morphotypes were first described within a decade of the discovery of smooth-colony-forming L. monocytogenes (17). The rough colony morphotype was thought to occur spontaneously and irreversibly at low frequency during prolonged culture in the laboratory. Apart from obvious physical differences, such as the absence of a bluegreen sheen upon Henry illumination and impaired cell separation that gave chaining cells without coordinated motility, the fermentative and biochemical profiles of rough and smooth colonies were considered identical (16,17,43).Characterization of the L. monocytogenes s...

A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

“…One possibility is that secA2 mutants have an advantage under certain conditions. Indeed, secA2 mutants arise spontaneously in the lab as rough colonies that emanate from a smooth colony and spread on solid media (25)(26)(27). At ambient temperatures, these mutants readily form distinct filamentous biofilms, which are thicker than those formed by the WT strain (26,72).…”

“…Indeed, secA2 mutants arise spontaneously in the lab as rough colonies that emanate from a smooth colony and spread on solid media (25)(26)(27). At ambient temperatures, these mutants readily form distinct filamentous biofilms, which are thicker than those formed by the WT strain (26,72). The spontaneous and reversible (25,73) morphological conversion to a rough phenotype may provide a potential advantage to L. monocytogenes in its saprophytic phase by enhancing the ability to colonize abiotic surfaces.…”

“…Spontaneous rough mutants of L. monocytogenes commonly arise on solid media, and many of these contain mutations in secA2 (25)(26)(27). Mutants in secA2 exhibit a chaining phenotype and are approximately 1,000-fold less virulent in animal models of infection (14,25,28).…”

Simultaneous Deficiency of both MurA and p60 Proteins Generates a Rough Phenotype inListeria monocytogenes

“…The subcellular fractionation procedure was as described by Monk and colleagues (13,23) with slight modifications. Briefly, 50-ml cultures of L. monocytogenes were grown in BHI at 37°C to log phase (optical density at 600 nm [OD 600 ], Ϸ1.0), and the cells were subsequently harvested by centrifugation at 6,200 ϫ g for 15 min at 4°C.…”

Phenotypic Diversification and Adaptation ofSerratia marcescensMG1 Biofilm-Derived Morphotypes

“…Significant phenotypic variation has been found to occur within many different biofilm communities (25,32,41,45). Although the biofilm is proposed to provide protection from external stresses, high cell densities inside a biofilm may result in stressful environments characterized by a scarcity of nutrients and oxygen, nonoptimal pH, and accumulation of metabolic by-products (8,16,51).…”