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

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.

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

Journal of Bacteriology ◽

10.1128/jb.00354-16 ◽

2016 ◽

Vol 198 (17) ◽

pp. 2370-2378 ◽

Cited By ~ 9

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.

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Roles of the Escherichia coli heat shock sigma factor 32 in early and late gene expression of bacteriophage T4.

Journal of Bacteriology ◽

10.1128/jb.170.3.1384-1388.1988 ◽

1988 ◽

Vol 170 (3) ◽

pp. 1384-1388 ◽

Cited By ~ 4

Author(s):

M W Frazier ◽

G Mosig

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Heat Shock ◽

Sigma Factor ◽

Bacteriophage T4 ◽

Late Gene ◽

Late Gene Expression

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Roles of the Escherichia coli Heat Shock Sigma Factor 32 in Early and Late Gene Expression of Bacteriophage T4:

Journal of Bacteriology ◽

10.1128/jb.170.9.4428.1988 ◽

1988 ◽

Vol 170 (9) ◽

pp. 4428-4428

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Heat Shock ◽

Sigma Factor ◽

Bacteriophage T4 ◽

Late Gene ◽

Late Gene Expression

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Transcription and translation of the sigG gene is tuned for proper execution of the switch from early to late gene expression in the developing Bacillus subtilis spore

PLoS Genetics ◽

10.1371/journal.pgen.1007350 ◽

2018 ◽

Vol 14 (4) ◽

pp. e1007350 ◽

Cited By ~ 8

Author(s):

Elizabeth B. Mearls ◽

Jacquelin Jackter ◽

Jennifer M. Colquhoun ◽

Veronica Farmer ◽

Allison J. Matthews ◽

...

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Late Gene ◽

Subtilis Spore ◽

Late Gene Expression ◽

Bacillus Subtilis Spore

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Analyses of Single-Amino-Acid Substitution Mutants of Adenovirus Type 5 E1B-55K Protein

Journal of Virology ◽

10.1128/jvi.75.9.4297-4307.2001 ◽

2001 ◽

Vol 75 (9) ◽

pp. 4297-4307 ◽

Cited By ~ 55

Author(s):

Yuqiao Shen ◽

Galila Kitzes ◽

Julie A. Nye ◽

Ali Fattaey ◽

Terry Hermiston

Keyword(s):

Gene Expression ◽

Amino Acid ◽

Human Cancer ◽

Adenovirus Type ◽

Single Amino Acid ◽

Late Gene ◽

Wild Type ◽

Late Gene Expression ◽

Human Cancer Cells ◽

Adenovirus Type 5

ABSTRACT The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus dl309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.

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

Journal of Bacteriology ◽

10.1128/jb.00793-19 ◽

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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