Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlAm) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlAm), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlAm significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa.

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During the late summer to early fall, Horsetooth Reservoir in Fort Collins, CO, USA is fully stratified and exhibits seasonally high fluxes of iron, manganese, and metal-rich particles into the water column. Particles were collected from the mid-region of the hypolimnion and examined for metal content. Nucleic acids extracted from the particles were used to construct bacterial and archaeal 16S rDNA clone libraries. Surprisingly, 50% of cloned bacterial genes were closely related to a coherent cluster within Candidate Division OP10. To our knowledge, this is the first report of an environmental gene clone library that exhibits a dominance of OP10-related clones. Several other sequences, many with long branch lengths, clustered within eight separate bacterial divisions and the diatom chloroplasts. Most of these divisions are commonly found in freshwater environments. However, gene sequences from characterized metal-oxidizing or metal-reducing bacteria were not identified. The archaeal gene clone libraries contained diverse sequences, most with close homology to previously characterized gene clones of methanogens or uncultivated Crenarchaeota from soil and lacustrine environments. This study identified a unique environment where OP10 bacteria are potentially abundant. Furthermore, we demonstrated that the metal-rich particles from this reservoir support a diverse and interesting community of microorganisms.

Bacteria require explicit control over their proteomes in order to compete and survive in dynamic environments. The Lyme disease spirochete Borrelia burgdorferi undergoes substantial protein profile changes during its cycling between vector ticks and vertebrate hosts. In an effort to understand regulation of these transitions, we recently isolated and functionally characterized the borrelial nucleic acid-binding protein BpuR, a PUR domain-containing protein. We now report that this regulatory protein governs its own synthesis through direct interactions with bpuR mRNA. In vitro and in vivo techniques indicate that BpuR binds with high affinity and specificity to the 5= region of its message, thereby inhibiting translation. This negative feedback could permit the bacteria to fine-tune cellular BpuR concentrations. These data add to the understanding of this newly described class of prokaryotic DNA-and RNA-binding regulatory proteins.

This unit describes general procedures for the lab cultivation and storage of the Grampositive facultative intracellular bacterium Listeria monocytogenes. The basic protocols are relevant for a wide scope of applications including microbial genetics and both in vitro and in vivo infection studies. Commonly used L. monocytogenes strains, serotypes, and growth parameters are also discussed.