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Background: Many bacteria transport cellobiose via a phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). In Listeria monocytogenes, two pairs of soluble PTS components (EIIACel1/EIIBCel1 and EIIACel2/EIIBCel2) and the permease EIICCel1 were suggested to contribute to cellobiose uptake. Interestingly, utilization of several carbohydrates, including cellobiose, strongly represses virulence gene expression by inhibiting PrfA, the virulence gene activator., Results: The LevR-like transcription regulator CelR activates expression of the cellobiose-induced PTS operons celB1-celC1-celA1, celB2-celA2, and the EIIC-encoding monocistronic celC2. Phosphorylation by P∼His-HPr at His550 activates CelR, whereas phosphorylation by P∼EIIBCel1 or P∼EIIBCel2 at His823 inhibits it. Replacement of His823 with Ala or deletion of both celA or celB genes caused constitutive CelR regulon expression. Mutants lacking EIICCel1, CelR or both EIIACel exhibitedslow cellobiose consumption. Deletion of celC1 or celR prevented virulence gene repression by the disaccharide, but not by glucose and fructose. Surprisingly, deletion of both celA genes caused virulence gene repression even during growth on non-repressing carbohydrates. No cellobiose-related phenotype was found for the celC2 mutant., Conclusion: The two EIIA/BCel pairs are similarly efficient as phosphoryl donors in EIICCel1-catalyzed cellobiose transport and CelR regulation. The permanent virulence gene repression in the celA double mutant further supports a role of PTSCel components in PrfA regulation., (© 2019 S. Karger AG, Basel.)

Protein phosphorylation is a major mechanism of signal transduction in bacteria. Here, we analyzed the proteome and phosphoproteome of a wild-type strain of the food-borne pathogen Listeria monocytogenes that was grown in either chemically defined medium or rich medium containing glucose. We then compared these results with those obtained from an isogenic prfA* mutant that produced a constitutively active form of PrfA, the main transcriptional activator of virulence genes. In the prfA* mutant grown in rich medium, we identified 256 peptides that were phosphorylated on serine (S), threonine (T), or tyrosine (Y) residues, with a S/T/Y ratio of 155:75:12. Strikingly, we detected five novel phosphosites on the virulence protein ActA. This protein was known to be phosphorylated by a cellular kinase in the infected host, but phosphorylation by a listerial kinase had not previously been reported. Unexpectedly, SILAC experiments with the prfA* mutant grown in chemically defined medium revealed that, in addition to previously described PrfA-regulated proteins, several other proteins were significantly overproduced, among them were several proteins involved in purine biosynthesis. This work provides new information for our understanding of the correlation among protein phosphorylation, virulence mechanisms, and carbon metabolism.

Despite the public health challenges associated with the emergence of new pathogenic bacterial strains and/or serotypes, there is a dearth of information regarding the molecular mechanisms that drive this variation. Here, we began to address the mechanisms behind serotype-specific variation between serotype M1 and M3 strains of the human pathogen Streptococcus pyogenes (the group A Streptococcus [GAS]). Spatially diverse contemporary clinical serotype M3 isolates were discovered to contain identical inactivating mutations within genes encoding two regulatory systems that control the expression of important virulence factors, including the thrombolytic agent streptokinase, the protease inhibitor-binding protein-G-related α2-macroglobulin-binding (GRAB) protein, and the antiphagocytic hyaluronic acid capsule. Subsequent analysis of a larger collection of isolates determined that M3 GAS, since at least the 1920s, has harbored a 4-bp deletion in the fasC gene of the fasBCAX regulatory system and an inactivating polymorphism in the rivR regulator-encoding gene. The fasC and rivR mutations in M3 isolates directly affect the virulence factor profile of M3 GAS, as evident by a reduction in streptokinase expression and an enhancement of GRAB expression. Complementation of the fasC mutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of the rivR mutation in M3 GAS restored the regulation of grab mRNA abundance but did not alter capsule mRNA levels. While important, the fasC and rivR mutations do not provide a full explanation for why serotype M3 strains are associated with unusually severe invasive infections; thus, further investigation is warranted.

Numerous bacteria possess transcription activators and antiterminators composed of regulatory domains phosphorylated by components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). These domains, called PTS regulation domains (PRDs), usually contain two conserved histidines as potential phosphorylation sites. While antiterminators possess two PRDs with four phosphorylation sites, transcription activators contain two PRDs plus two regulatory domains resembling PTS components (EIIA and EIIB). The activity of these transcription regulators is controlled by up to five phosphorylations catalyzed by PTS proteins. Phosphorylation by the general PTS components EI and HPr is usually essential for the activity of PRD-containing transcription regulators, whereas phosphorylation by the sugar-specific components EIIA or EIIB lowers their activity. For a specific regulator, for example the Bacillus subtilis mtl operon activator MtlR, the functional phosphorylation sites can be different in other bacteria and consequently the detailed mode of regulation varies. Some of these transcription regulators are also controlled by an interaction with a sugar-specific EIIB PTS component. The EIIBs are frequently fused to the membrane-spanning EIIC and EIIB-mediated membrane sequestration is sometimes crucial for the control of a transcription regulator. This is also true for the Escherichia coli repressor Mlc, which does not contain a PRD but nevertheless interacts with the EIIB domain of the glucose-specific PTS. In addition, some PRD-containing transcription activators interact with a distinct EIIB protein located in the cytoplasm. The phosphorylation state of the EIIB components, which changes in response to the presence or absence of the corresponding carbon source, affects their interaction with transcription regulators. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The bacterial pathogen group A Streptococcus (GAS) causes human diseases ranging from self-limiting pharyngitis (also known as strep throat) to severely invasive necrotizing fasciitis (also known as the flesh-eating syndrome). To control virulence factor expression, GAS utilizes both protein- and RNA-based mechanisms of regulation. Here we report that the transcription factor RivR (RofA-like protein IV) negatively regulates the abundance of mRNAs encoding the hyaluronic acid capsule biosynthesis proteins (hasABC; ∼7-fold) and the protein G-related α(2)-macroglobulin-binding protein (grab; ∼29-fold). Our data differ significantly from those of a previous study of the RivR regulon. Given that grab and hasABC are also negatively regulated by the two-component system CovR/S (control of virulence), we tested whether RivR functions through CovR/S. A comparison of riv and cov single and double mutant strains showed that RivR requires CovR activity for grab and hasABC regulation. Analysis of the upstream region of rivR identified a novel promoter the deletion of which reduced rivR mRNA abundance by 70%. A rivR mutant strain had a reduced ability to adhere to human keratinocytes relative to that of the parental and complemented strains, a phenotype that was abolished upon GAS pretreatment with hyaluronidase, highlighting the importance of capsule regulation by RivR during colonization. The rivR mutant strain was also attenuated for virulence in a murine model of bacteremia infection. Thus, we identify RivR as an important regulator of GAS virulence and provide new insight into the regulatory networks controlling virulence factor production in this pathogen.