SummaryDrug efflux systems play a major role in resistance to a wide range of noxious compounds in several Gram negative species. Here, we report the drug resistance and virulence phenotypes of Salmonella mutants defective in either resistance-nodulation-division (RND)-type systems and/or in drug efflux systems belonging to the major facilitator (MFS), multidrug and toxic compound extrusion (MATE), and ATPbinding cassette (ABC) superfamilies. We determined that nine potential drug transporters contribute to drug resistance of Salmonella and found that the Salmonella -specific MdsABC system conferred resistance to a variety of toxic compounds. The RND-type MdsAB system could function with either MdsC, which is encoded in the same operon, or TolC as the outer membrane component. Although the Salmonella EmrAB, MdfA and MdtK are 90% identical in their amino acid sequences to their Escherichia coli homologues, the drug specificity of Salmonella transporters was different from that reported for equivalent E. coli transporters. Deletion of the macAB genes attenuated Salmonella virulence and a strain lacking all drug efflux systems was avirulent when mice were inoculated by the oral route. The promoter region of the macAB drug efflux system genes harbours a binding site for the response regulator PhoP, which functions to repress macAB transcription. The PhoP/PhoQ two-component system is a major regulator of Salmonella virulence, which underscores the connection between drug efflux systems and virulence.
Most RNA molecules require Mg(2+) for their structure and enzymatic properties. Here we report the first example of an RNA serving as sensor for cytoplasmic Mg(2+). We establish that expression of the Mg(2+) transporter MgtA of Salmonella enterica serovar Typhimurium is controlled by its 5' untranslated region (5'UTR). We show that the 5'UTR of the mgtA gene can adopt different stem-loop structures depending on the Mg(2+) levels, which determine whether transcription reads through into the mgtA coding region or stops within the 5'UTR. We could recapitulate the Mg(2+)-regulated transcription using a defined in vitro transcription system with RNA polymerase as the only protein component. The initiation of mgtA transcription responds to extracytoplasmic Mg(2+) and its elongation into the coding region to cytoplasmic Mg(2+), providing a singular example in which the same ligand is sensed in different cellular compartments to regulate disparate steps in gene transcription.
Genetic and genomic approaches have been successfully used to assign genes to distinct regulatory networks. However, the present challenge of distinguishing differentially regulated genes within a network is particularly hard because members of a given network tend to have similar regulatory features. We have addressed this challenge by developing a method, termed Gene Promoter Scan, that discriminates coregulated promoters by simultaneously considering both multiple cis promoter features and gene expression. Here, we apply this method to probe the regulatory networks governed by the PhoP͞PhoQ two-component system in the enteric bacteria Escherichia coli and Salmonella enterica. Our analysis uncovered members of the PhoP regulon and interactions with other regulatory systems that were not discovered in previous approaches. The predictions made by Gene Promoter Scan were experimentally validated to establish that the PhoP protein uses multiple mechanisms to control gene transcription, regulates acid resistance determinants, and is a central element in a highly connected network.promoter ͉ machine learning ͉ gene transcription ͉ acid pH
Vascular calcification is common in people with diabetes and its presence predicts premature mortality. To clarify the underlying mechanisms, we used low density lipoprotein receptor-deficient (LDLR ؊/؊) mice to study vascular calcification in the ascending aorta. LDLR ؊/؊ mice on a chow diet did not develop obesity, diabetes, atheroma, or vascular calcification. In contrast, LDLR ؊/؊ mice on high fat diets containing cholesterol developed obesity, severe hyperlipidemia, hyperinsulinemic diabetes, and aortic atheroma. A high fat diet without cholesterol also induced obesity and diabetes, but caused only moderate hyperlipidemia and did not result in significant aortic atheroma formation. Regardless of cholesterol content, high fat diets induced mineralization of the proximal aorta (assessed by von Kossa staining) and promoted aortic expression of Msx2 and Msx1, genes encoding homeodomain transcription factors that regulate mineralization and osseous differentiation programs in the developing skull. Osteopontin (Opn), an osteoblast matrix protein gene also expressed by activated macrophages, was up-regulated in the aorta by these high fat diets. In situ hybridization showed that peri-aortic adventitial cells in high fat-fed mice express Msx2. Opn was also detected in this adventitial cell population, but in addition was expressed by aortic vascular smooth muscle cells and macrophages of the intimal atheroma.High fat diets associated with hyperinsulinemic diabetes activate an aortic osteoblast transcriptional regulatory program that is independent of intimal atheroma formation. The spatial pattern of Msx2 and Opn gene expression strongly suggests that vascular calcification, thought to be limited to the media, is an active process that can originate from an osteoprogenitor cell population in the adventitia.More than a century ago, Virchow (1) recognized two components of vascular disease, atheroma formation and calcification. Cholesterol-containing lipoproteins are probably necessary but not sufficient for atheroma formation. Their presence sets the stage for macrophage activation, foam cell formation, recruitment of smooth muscle cells, and fibrosis (2). In contrast, vascular calcification is largely uncharacterized.Vascular calcification is particularly relevant to people with diabetes. Atherosclerosis accounts for 80% of diabetes-related deaths, and vascular disease in diabetics carries a far worse prognosis than in nondiabetics (3-5). Patients with type II diabetes are more likely than nondiabetics to develop macrovascular calcification (6), which is associated with accelerated cardiovascular event rates and death (7). The underlying mechanisms are unknown.Sometimes considered a passive process of calcium deposition in necrotic tissue, vascular calcification can also be active, associated with expression of osteoblast bone matrix proteins that regulate mineralization. A transcriptional hierarchy controlling osteoblast gene expression and mineralization has emerged (8). The runt domain transcription factor Cbfa1...
Related organisms typically rely on orthologous regulatory proteins to respond to a given signal. However, the extent to which (or even if) the targets of shared regulatory proteins are maintained across species has remained largely unknown. This question is of particular significance in bacteria due to the widespread effects of horizontal gene transfer. Here, we address this question by investigating the regulons controlled by the DNA-binding PhoP protein, which governs virulence and Mg2+ homeostasis in several bacterial species. We establish that the ancestral PhoP protein directs largely different gene sets in ten analyzed species of the family Enterobacteriaceae, reflecting both regulation of species-specific targets and transcriptional rewiring of shared genes. The two targets directly activated by PhoP in all ten species (the most distant of which diverged >200 million years ago), and coding for the most conserved proteins are the phoPQ operon itself and the lipoprotein-encoding slyB gene, which decreases PhoP protein activity. The Mg2+-responsive PhoP protein dictates expression of Mg2+ transporters and of enzymes that modify Mg2+-binding sites in the cell envelope in most analyzed species. In contrast to the core PhoP regulon, which determines the amount of active PhoP and copes with the low Mg2+ stress, the variable members of the regulon contribute species-specific traits, a property shared with regulons controlled by dissimilar regulatory proteins and responding to different signals.
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