scholarly journals Competence for Genetic Transformation in Streptococcus pneumoniae: Mutations in σABypass the ComW Requirement for Late Gene Expression

2016 ◽  
Vol 198 (17) ◽  
pp. 2370-2378 ◽  
Author(s):  
Yanina Tovpeko ◽  
Junqin Bai ◽  
Donald A. Morrison
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Genetic Transformation ◽  
Sigma Factor ◽  
Specific Protein ◽  
Late Gene ◽  
Late Gene Expression ◽  
Content Type ◽  
Σ Factor

ABSTRACTStreptococcus pneumoniaeis able to integrate exogenous DNA into its genome by natural genetic transformation. Transient accumulation of high levels of the onlyS. pneumoniaealternative σ factor is insufficient for development of full competence without expression of a second competence-specific protein, ComW. The ΔcomWmutant is 104-fold deficient in the yield of recombinants, 10-fold deficient in the amount of σXactivity, and 10-fold deficient in the amount of σXprotein. The critical role of ComW during transformation can be partially obviated by σAmutations clustered on surfaces controlling affinity for core RNA polymerase (RNAP). While strains harboring σAmutations in thecomWmutant background were transforming at higher rates, the mechanism of transformation restoration was not clear. To investigate the mechanism of transformation restoration, we measured late gene expression in σA* suppressor strains. Restoration of late gene expression was observed in ΔcomWσA* mutants, indicating that a consequence of the σA* mutations is, at least, to restore σXactivity. Competence kinetics were normal in ΔcomWσA* strains, indicating that strains with restored competence exhibit the same pattern of transience as wild-type (WT) strains. We also identified a direct interaction between ComW and σXusing the yeast two-hybrid (Y2H) assay. Taken together, these data are consistent with the idea that ComW increases σXaccess to core RNAP, pointing to a direct role of ComW in σ factor exchange during genetic transformation. However, the lack of late gene shutoff in ΔcomWmutants also points to a potential new role for ComW in competence shutoff.IMPORTANCEThe sole alternative sigma factor of the streptococci, SigX, regulates development of competence for genetic transformation, a widespread mechanism of adaptation by horizontal gene transfer in this genus. The transient appearance of this sigma factor is strictly controlled at the levels of transcription and stability. This report shows that it is also controlled at the point of its substitution for SigA by a second transient competence-specific protein, ComW.

scholarly journals A Membrane-Embedded Amino Acid Couples the SpoIIQ Channel Protein to Anti-Sigma Factor Transcriptional Repression during Bacillus subtilis Sporulation

2016 ◽  
Vol 198 (9) ◽  
pp. 1451-1463 ◽  
Author(s):  
Kelly A. Flanagan ◽  
Joseph D. Comber ◽  
Elizabeth Mearls ◽  
Colleen Fenton ◽  
Anna F. Wang Erickson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Sigma Factor ◽  
Late Gene ◽  
Late Gene Expression ◽  
Gene Encoding ◽  
Content Type ◽  
Regulatory Circuit ◽  
Gene Regulatory

ABSTRACTSpoIIQ is an essential component of a channel connecting the developing forespore to the adjacent mother cell duringBacillus subtilissporulation. This channel is generally required for late gene expression in the forespore, including that directed by the late-acting sigma factor σG. Here, we present evidence that SpoIIQ also participates in a previously unknown gene regulatory circuit that specifically represses expression of the gene encoding the anti-sigma factor CsfB, a potent inhibitor of σG. ThecsfBgene is ordinarily transcribed in the forespore only by the early-acting sigma factor σF. However, in a mutant lacking the highly conserved SpoIIQ transmembrane amino acid Tyr-28,csfBwas also aberrantly transcribed later by σG, the very target of CsfB inhibition. This regulation ofcsfBby SpoIIQ Tyr-28 is specific, given that the expression of other σF-dependent genes was unaffected. Moreover, we identified a conserved element within thecsfBpromoter region that is both necessary and sufficient for SpoIIQ Tyr-28-mediated inhibition. These results indicate that SpoIIQ is a bifunctional protein that not only generally promotes σGactivity in the forespore as a channel component but also specifically maximizes σGactivity as part of a gene regulatory circuit that represses σG-dependent expression of its own inhibitor, CsfB. Finally, we demonstrate that SpoIIQ Tyr-28 is required for the proper localization and stability of the SpoIIE phosphatase, raising the possibility that these two multifunctional proteins cooperate to fine-tune developmental gene expression in the forespore at late times.IMPORTANCECellular development is orchestrated by gene regulatory networks that activate or repress developmental genes at the right time and place. Late gene expression in the developingBacillus subtilisspore is directed by the alternative sigma factor σG. The activity of σGrequires a channel apparatus through which the adjacent mother cell provides substrates that generally support gene expression. Here we report that the channel protein SpoIIQ also specifically maximizes σGactivity as part of a previously unknown regulatory circuit that prevents σGfrom activating transcription of the gene encoding its own inhibitor, the anti-sigma factor CsfB. The discovery of this regulatory circuit significantly expands our understanding of the gene regulatory network controlling late gene expression in the developingB. subtilisspore.

scholarly journals Inhibition of the Ubiquitin-Proteasome System Prevents Vaccinia Virus DNA Replication and Expression of Intermediate and Late Genes

2009 ◽  
Vol 83 (6) ◽  
pp. 2469-2479 ◽  
Author(s):  
P. S. Satheshkumar ◽  
Luis C. Anton ◽  
Patrick Sanz ◽  
Bernard Moss
Keyword(s):  
Gene Expression ◽  
Vaccinia Virus ◽  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome System ◽  
Late Gene ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Late Genes ◽  
Ubiquitin Proteasome

ABSTRACT The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions. Viruses belonging to several different families utilize or modulate the system for their advantage. Here we showed that the proteasome inhibitors MG132 and epoxomicin blocked a postentry step in vaccinia virus (VACV) replication. When proteasome inhibitors were added after virus attachment, early gene expression was prolonged and the expression of intermediate and late genes was almost undetectable. By varying the time of the removal and addition of MG132, the adverse effect of the proteasome inhibitors was narrowly focused on events occurring 2 to 4 h after infection, the time of the onset of viral DNA synthesis. Further analyses confirmed that genome replication was inhibited by both MG132 and epoxomicin, which would account for the effect on intermediate and late gene expression. The virus-induced replication of a transfected plasmid was also inhibited, indicating that the block was not at the step of viral DNA uncoating. UBEI-41, an inhibitor of the ubiquitin-activating enzyme E1, also prevented late gene expression, supporting the role of the ubiquitin-proteasome system in VACV replication. Neither the overexpression of ubiquitin nor the addition of an autophagy inhibitor was able to counter the inhibitory effects of MG132. Further studies of the role of the ubiquitin-proteasome system for VACV replication may provide new insights into virus-host interactions and suggest potential antipoxviral drugs.

scholarly journals Murine Cytomegalovirus Protein pM49 Interacts with pM95 and Is Critical for Viral Late Gene Expression

2020 ◽  
Vol 94 (6) ◽  
Author(s):  
Tian Han ◽  
Hongyun Hao ◽  
Sirwan S. Sleman ◽  
Baoqin Xuan ◽  
Shubing Tang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virus Infection ◽  
Dna Synthesis ◽  
Gene Transcription ◽  
Murine Cytomegalovirus ◽  
Late Gene ◽  
Single Mutation ◽  
Late Gene Expression ◽  
Viral Dna

ABSTRACT Late gene expression of betaherpesviruses and gammaherpesviruses is tightly controlled by virus-encoded transactivation factors (vTFs). We recently proved that the 6 vTFs of murine cytomegalovirus (MCMV) form a complex to regulate late gene transcription. pM49, one of the vTFs that has not been studied before, was identified to be a component of the complex that interacts with pM95. In this study, we began to investigate the potential role of pM49 in viral late gene expression. A recombinant MCMV expressing C-terminal FLAG-tagged pM49 was constructed to study the expression kinetics and localization of pM49. pM49 was expressed at the late time of virus infection. Inhibition of viral DNA synthesis by phosphonate sodium phosphonic acid (PAA) abolished pM49 expression, indicating that it is a late protein. pM49 colocalized with pM44 at the viral replication compartment, similarly to other viral vTFs that have been reported. Mutant virus lacking full-length pM49 expression failed to express viral late genes, leading to nonproductive infection. The expression of immediate early and early genes was not affected, and viral DNA synthesis was only minimally affected during pM49-deficient virus infection. All of these data support the role of pM49 in viral late gene expression. After a series of mutagenesis analyses, two key residues, K325 and C326, were identified as required for pM49-pM95 interaction. Cells expressing pM49 with either single mutation of these two residues failed to rescue the late gene expression and support the replication of pM49-deficient virus. Our results indicated that pM49-pM95 interaction is essential for viral late gene expression. IMPORTANCE Cytomegalovirus (CMV) infections result in morbidity and mortality in immunocompromised individuals, and the virus is also a major cause of birth defects in newborns. Currently, because of the unavailability of vaccines against this virus and restricted antiviral therapies with low toxicity, as well as the emergency of resistant strain of this virus, the understanding of viral late gene regulation may provide clues to study new antiviral drugs or vaccines. In this study, we report that MCMV protein pM49 is critical for viral late gene transcription, based on its interaction with pM95. This finding reveals the important role of pM49-pM95 interaction in the regulation of viral late gene expression and that it could be a future potential target for therapeutic intervention in CMV diseases.

scholarly journals The Repressor Function of the Chlamydia Late Regulator EUO Is Enhanced by the Plasmid-Encoded Protein Pgp4

2020 ◽  
Vol 202 (8) ◽  
Author(s):  
Qiang Zhang ◽  
Christopher J. Rosario ◽  
Lauren M. Sheehan ◽  
Syed M. Rizvi ◽  
Julie A. Brothwell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Negative Regulator ◽  
Late Gene ◽  
Late Gene Expression ◽  
New Host ◽  
Content Type ◽  
Late Genes ◽  
Developmental Form

ABSTRACT A critical step in intracellular Chlamydia infection is the production of infectious progeny through the expression of late genes. This differentiation step involves conversion from a reticulate body (RB), which is the replicating form of the bacterium, into an elementary body (EB), which is the developmental form that spreads the infection to a new host cell. EUO is an important chlamydial transcription factor that controls the expression of late genes, but the mechanisms that regulate EUO are not known. We report that a plasmid-encoded protein, Pgp4, enhanced the repressor activity of EUO. Pgp4 did not function as a transcription factor because it did not bind or directly modulate transcription of its target promoters. Instead, Pgp4 increased the ability of EUO to bind and repress EUO-regulated promoters in vitro and physically interacted with EUO in pulldown assays with recombinant proteins. We detected earlier onset of EUO-dependent late gene expression by immunofluorescence microscopy in Pgp4-deficient C. trachomatis and C. muridarum strains. In addition, the absence of Pgp4 led to earlier onset of RB-to-EB conversion in C. muridarum. These data support a role for Pgp4 as a negative regulator of chlamydial transcription that delays late gene expression. Our studies revealed that Pgp4 also has an EUO-independent function as a positive regulator of chlamydial transcription. IMPORTANCE Chlamydia trachomatis is an important human pathogen that causes more than 150 million active cases of genital and eye infection in the world. This obligate intracellular bacterium produces infectious progeny within an infected human cell through the expression of late chlamydial genes. We showed that the ability of a key chlamydial transcription factor, EUO, to repress late genes was enhanced by a plasmid-encoded protein, Pgp4. In addition, studies with Chlamydia Pgp4-deficient strains provide evidence that Pgp4 delays late gene expression in infected cells. Thus, Pgp4 is a novel regulator of late gene expression in Chlamydia through its ability to enhance the repressor function of EUO.

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