Open Access

Regulated ATP-dependent proteolysis is a common feature of developmental processes and plays also a crucial role during environmental perturbations such as stress and starvation. The Bacillus subtilis MgsR regulator controls a subregulon within the stress- and stationary phase σB regulon. After ethanol exposition and a short time-window of activity, MgsR is ClpXP-dependently degraded with a half-life of approximately 6 min. Surprisingly, a protein interaction analysis with MgsR revealed an association with the McsB arginine kinase and an in vivo degradation assay confirmed a strong impact of McsB on MgsR degradation. In vitro phosphorylation experiments with arginine (R) by lysine (K) substitutions in McsB and its activator McsA unraveled all R residues, which are essentially needed for the arginine kinase reaction. Subsequently, site directed mutagenesis of the MgsR substrate was used to substitute all arginine residues with glutamate (R-E) to mimic arginine phosphorylation and to test their influence on MgsR degradation in vivo. It turned out, that especially the R33E and R94/95E residues (RRPI motif), the latter are adjacently located to the two redox-sensitive cysteines in a 3D model, have the potential to accelerate MgsR degradation. These results imply that selective arginine phosphorylation may have favorable effects for Clp dependent degradation of short-living regulatory proteins. We speculate that in addition to its kinase activity and adaptor function for the ClpC ATPase, McsB might also serve as a proteolytic adaptor for the ClpX ATPase in the degradation mechanism of MgsR.

Invasion of the bacterial pathogen Listeria monocytogenes into human host cells requires specialized surface molecules for attachment and induction of phagocytosis. However, efficient invasion is also dependent on factors with house-keeping functions, such as SecA2-dependent secretion of autolysins for post-divisional segregation of daughter cells. Mutations in this pathway prevent degradation of peptidoglycan cross-walls, so that long cell chains are formed that cannot be phagocytosed. The extreme chaining of such mutants manifests as rough colony phenotype. One rough clone was isolated from a transposon library with a transposon insertion in the uncharacterized lmo0720 gene (lftS) together with a spontaneous point mutation in the secA2 gene. We separated both mutations and demonstrated that this point mutation in the intramolecular regulator 2 domain of SecA2 was sufficient to inactivate the protein. In contrast, lftS deletion did not cause a ΔsecA2-like phenotype. lftS is located in an operon with lftR (lmo0719), encoding a PadR-like transcriptional regulator, and lftR deletion affected growth, invasion and day-light dependent coordination of swarming. Inactivation of lftS partially suppressed these phenotypes, suggesting a functional relationship between LftR and LftS. However, the invasion defect of the ΔlftR mutant was only marginally suppressed by lftS removal. LftR regulates expression of the lmo0979–0980 (lieAB) operon, encoding a putative multidrug resistance transporter and lieAB transcription was strongly upregulated in the absence of LftR. Deletion of lieAB in the ΔlftR background restores wild type-like invasion levels. Hence, we conclude that tight transcriptional repression of the lieAB operon is essential for efficient listerial host cell invasion.

Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.