In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins, which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ϳ50% identical to MxaF and ϳ90% identical to each other. It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM, which are required for the expression of the methanol dehydrogenase genes.Methylobacterium extorquens AM1 is a methylotrophic bacterium ubiquitous in the environment and in particular on the undersides of leaves. This organism is able to metabolize both single-carbon compounds like methanol and multicarbon compounds like succinate and pyruvate (1,11,19). M. extorquens AM1 is a model organism for understanding the process of methylotrophic growth and has been studied biochemically and genetically for over 50 years (reviewed in reference 7). To utilize methanol as a sole source of carbon and energy, methanol is first oxidized to formaldehyde in the periplasm via methanol dehydrogenase, a soluble quinoprotein (2-4). This enzyme uses pyrroloquinoline quinone (PQQ) to sequentially transfer two electrons to cytochrome c L , which enters the electron transport chain, resulting in ϳ1 molecule of ATP per molecule of methanol oxidized (9). Methanol dehydrogenase is a heterotetrameric protein consisting of two 66-kDa large subunits (MxaF) and two small 8.5-kDa subunits (MxaI) (28, 37). The large subunit contains the active-site residues and the PQQ prosthetic group, which is coordinated to a calcium ion in the active site (37). The loss of this enzyme in M. extorquens

Salmonella enterica serovar Typhimurium induces inflammatory diarrhea and bacterial uptake into intestinal epithelial cells using the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS). HilA activates transcription of the SPI1 structural components and effector proteins. Expression of hilA is activated by HilD, HilC, and RtsA, which act in a complex feed-forward regulatory loop. Many environmental signals and other regulators are integrated into this regulatory loop, primarily via HilD. After the invasion of Salmonella into host intestinal epithelial cells or during systemic replication in macrophages, the SPI T3SS is no longer required or expressed. We have shown that the two-component regulatory system PhoPQ, required for intracellular survival, represses the SPI1 T3SS mostly by controlling the transcription of hilA and hilD. Here we show that PinT, one of the PhoPQ-regulated small RNAs (sRNAs), contributes to this regulation by repressing hilA and rtsA translation. PinT base pairs with both the hilA and rtsA mRNAs, resulting in translational inhibition of hilA, but also induces degradation of the rts transcript. PinT also indirectly represses expression of FliZ, a posttranslational regulator of HilD, and directly represses translation of ssrB, encoding the primary regulator of the SPI2 T3SS. Our in vivo mouse competition assays support the concept that PinT controls a series of virulence genes at the posttranscriptional level in order to adapt Salmonella from the invasion stage to intracellular survival. IMPORTANCE Salmonella is one of the most important food-borne pathogens, infecting over one million people in the United States every year. These bacteria use a needle-like device to interact with intestinal epithelial cells, leading to invasion of the cells and induction of inflammatory diarrhea. A complex regulatory network controls expression of the invasion system in response to numerous environmental signals. Here we explore the molecular mechanisms by which the small RNA PinT contributes to this regulation, facilitating inactivation of the system after invasion. PinT controls several important virulence systems in Salmonella, tuning the transition between different stages of infection.

Animal models play an important role in understanding the mechanisms of bacterial pathogenesis. Here we review recent studies of Salmonella infection in various animal models. Although mice are a classic animal model for Salmonella, mice do not normally get diarrhea, raising the question of how well the model represents normal human infection. However, pretreatment of mice with oral streptomycin, which apparently reduces the normal microbiota, leads to an inflammatory diarrheal response upon oral infection with Salmonella. This has led to a re-evaluation of the role of various Salmonella virulence factors in colonization of the intestine and induction of diarrhea. Indeed, it is now clear that Salmonella purposefully induces inflammation, which leads to the production of both carbon sources and terminal electron acceptors by the host that allow Salmonella to outgrow the normal intestinal microbiota. Overall use of this modified mouse model provides a more nuanced understanding of Salmonella intestinal infection in the context of the microbiota with implications for the ability to predict human risk.

Salmonella must rapidly adapt to various niches in the host during infection. Relevant virulence factors must be appropriately induced, and systems that are detrimental in a particular environment must be turned off. Salmonella infects intestinal epithelial cells using a type 3 secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). The system is controlled by three AraC-like regulators, HilD, HilC, and RtsA, which form a complex feed-forward loop to activate expression of hilA, encoding the main transcriptional regulator of T3SS structural genes. This system is tightly regulated, with many of the activating signals acting at the level of hilD translation or HilD protein activity. Once inside the phagosomes of epithelial cells, or in macrophages during systemic stages of disease, the SPI1 T3SS is no longer required or expressed. Here, we show that the PhoPQ two-component system, critical for intracellular survival, appears to be the primary mechanism by which Salmonella shuts down the SPI1 T3SS. PhoP negatively regulates hilA through multiple distinct mechanisms: direct transcriptional repression of the hilA promoter, indirect transcriptional repression of both the hilD and rtsA promoters, and activation of the small RNA (sRNA) PinT. Genetic analyses and electrophoretic mobility shift assays suggest that PhoP specifically binds the hilA promoter to block binding of activators HilD, HilC, and RtsA as a mechanism of repression. IMPORTANCE Salmonella is one of the most common foodborne pathogens, causing an estimated 1.2 million illnesses per year in the United States. A key step in infection is the activation of the bacterial invasion machinery, which induces uptake of the bacterium into epithelial cells and leads to induction of inflammatory diarrhea. Upon entering the vacuolar compartments of host cells, Salmonella senses an environmental transition and represses the invasion machinery with a two-component system relevant for survival within the vacuole. This adaptation to specific host niches is an important example of how signals are integrated for survival of the pathogen.

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