SUMMARY Several intracellular pathogens including Salmonella enterica and Mycobacterium tuberculosis require the virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that, unlike secreted virulence factors that target the host vacuolar ATPase to withstand phagosomal acidity, the MgtC protein acts on Salmonella's own F1Fo ATP synthase. This complex couples proton translocation to ATP synthesis/ hydrolysis and is required for virulence. We establish that MgtC interacts with the a subunit of the F1Fo ATP synthase, hindering ATP-driven proton translocation and NADH-driven ATP synthesis in inverted vesicles. An mgtC null mutant displays heightened ATP levels and an acidic cytoplasm whereas mgtC overexpression decreases ATP levels. A single amino acid substitution in MgtC that prevents binding to the F1Fo ATP synthase abolishes control of ATP levels and attenuates pathogenicity. MgtC provides a singular example of a virulence protein that promotes pathogenicity by interfering with another virulence protein.

Organisms must maintain physiological levels of Mg2+ because this divalent cation is critical for the stabilization of membranes and ribosomes, the neutralization of nucleic acids, and as a cofactor in a variety of enzymatic reactions. In this review, we describe the mechanisms that bacteria utilize to sense the levels of Mg2+ both outside and inside the cytoplasm. We examine how bacteria achieve Mg2+ homeostasis by adjusting the expression and activity of Mg2+ transporters, and by changing the composition of their cell envelope. We discuss the connections that exist between Mg2+ sensing, Mg2+ transport and bacterial virulence. Additionally, we explore the logic behind the fact that bacterial genomes encode multiple Mg2+ transporters and distinct sensing systems for cytoplasmic and extracytoplasmic Mg2+. These analyses may be applicable to the homeostatic control of other cations.

Bacteria can withstand killing by bactericidal antibiotics through phenotypic changes mediated by their preexisting genetic repertoire. These changes can be exhibited transiently by a large fraction of the bacterial population, giving rise to tolerance, or displayed by a small subpopulation, giving rise to persistence. Apart from undermining the use of antibiotics, tolerant and persistent bacteria foster the emergence of antibiotic-resistant mutants. Persister formation has been attributed to alterations in the abundance of particular proteins, metabolites, and signaling molecules, including toxin-antitoxin modules, adenosine triphosphate, and guanosine (penta) tetraphosphate, respectively. Here, we report that persistent bacteria form as a result of slow growth alone, despite opposite changes in the abundance of such proteins, metabolites, and signaling molecules. Our findings argue that transitory disturbances to core activities, which are often linked to cell growth, promote a persister state regardless of the underlying physiological process responsible for the change in growth.

Summary The synthesis of ribosomes is regulated by both amino acid abundance and the availability of adenosine triphosphate (ATP), which regenerates guanosine triphosphate (GTP), powers ribosomes, and promotes transcription of ribosomal RNA genes. We now report that bacteria supersede both of these controls when experiencing low cytosolic magnesium (Mg2+), a divalent cation essential for ribosome stabilization and for neutralization of ATP’s negative charge. We uncover a regulatory circuit that responds to low cytosolic Mg2+ by promoting expression of proteins that import Mg2+ and that lower ATP amounts. This response reduces the levels of ATP and ribosomes, making Mg2+ ions available for translation. Mutants defective in Mg2+ uptake and unable to reduce ATP levels accumulate non-functional ribosomal components and undergo translational arrest. Our findings establish a new paradigm whereby cells reduce the amounts of translating ribosomes to carry out protein synthesis.

Cellulose is the most abundant organic polymer on Earth. In bacteria, cellulose confers protection against environmental insults and is a constituent of biofilms typically formed on abiotic surfaces. We report that, surprisingly, Salmonella enterica serovar Typhimurium makes cellulose when inside macrophages. We determine that preventing cellulose synthesis increases virulence, whereas stimulation of cellulose synthesis inside macrophages decreases virulence. An attenuated mutant lacking the mgtC gene exhibited increased cellulose levels due to increased expression of the cellulose synthase gene bcsA and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of bcsA restored wild-type virulence to the Salmonella mgtC mutant, but not to other attenuated mutants displaying a wild-type phenotype regarding cellulose. Our findings indicate that a virulence determinant can promote pathogenicity by repressing a pathogen's antivirulence trait. Moreover, they suggest that controlling antivirulence traits increases long-term pathogen fitness by mediating a tradeoff between acute virulence and transmission.biofilm | magnesium | ATP

Members of the genus Arsenophonus comprise a large group of bacterial endosymbionts that are widely distributed in arthropods of medical, veterinary, and agricultural importance. At present, little is known about the role of these bacteria in arthropods, because few representatives have been isolated and cultured in the laboratory. In the current study, we describe the isolation and pure culture of an Arsenophonus endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis. We propose provisional nomenclature for this bacterium in the genus Arsenophonus as "Candidatus Arsenophonus arthropodicus." Phylogenetic analyses indicate that "Candidatus Arsenophonus arthropodicus" is closely related to the Arsenophonus endosymbionts found in psyllids, whiteflies, aphids, and mealybugs. The pure culture of this endosymbiont offers new opportunities to examine the role of Arsenophonus in insects. To this end, we describe methods for the culture of "Candidatus Arsenophonus arthropodicus" in an insect cell line and the transformation of this bacterium with a broad-host-range plasmid.Many members of the class Insecta maintain mutualistic associations with one or more specialized symbiotic bacteria (2). Bacteria that participate in these associations are classified either as primary (P) or secondary (S) endosymbionts, because they often coexist in a single insect host. The P-endosymbionts are predicted to be ancient in origin because their phylogenies are concordant with those of their host insects over a substantial period of evolutionary time, indicating long-term coevolution. On the other hand, the S-endosymbionts are predicted to be recent in origin because their phylogenies show little or no concordance with their insect hosts, indicating recent acquisition.While the ancient P-endosymbionts are known to have defined mutualistic functions in their insect hosts, the role of the S-endosymbionts is not yet well understood. From an evolutionary standpoint it seems likely that S-endosymbionts have beneficial (mutualistic) roles in their insect hosts because they are maintained predominantly through a maternal (vertical) transmission strategy. Several recent studies have provided experimental evidence for a number of beneficial effects conferred by the S-endosymbionts of aphids, which recently received new nomenclature (20). These benefits include host plant specialization (17, 27), increased resistance to hymenopteran parasitoids (21, 22), and increased tolerance to heat stress (5, 19). In addition, there is evidence indicating that S-endosymbionts can provide some level of functional compensation for the loss of P-endosymbionts in a laboratory population of aphids (16). While these studies are both exciting and encouraging, the ability to perform experimentation in these systems would be greatly enhanced with the opportunity to genetically manipulate S-endosymbionts. The application of recombinant DNA technology would permit functional analysis of individual genes in the endosymbiont genomes, providing a platform to e...

Adenosine triphosphate (ATP) is the energy currency of living cells. Even though ATP powers virtually all energy-dependent activity, most cellular ATP is utilized in protein synthesis via tRNA aminoacylation and GTP regeneration. Magnesium (Mg2+), the most common divalent cation in living cells, plays crucial roles in protein synthesis by maintaining the structure of ribosomes, participating in the biochemistry of translation initiation and functioning as a counter-ion for ATP. A non-physiological increase in ATP levels hinders growth in cells experiencing Mg2+ limitation because ATP is the most abundant nucleotide triphosphate in the cell and Mg2+ is also required for the stabilization of the cytoplasmic membrane and as a cofactor for essential enzymes. We propose that organisms cope with Mg2+ limitation by decreasing ATP levels and ribosome production, thereby reallocating Mg2+ to indispensable cellular processes.

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