Cyclic di-GMP Regulation of Gene Expression

Hsieh, Meng-Lun; Hinton, Deborah M.; Waters, Christopher M.

doi:10.1007/978-3-030-33308-9_23

Microbial Cyclic Di-Nucleotide Signaling 2020

DOI: 10.1007/978-3-030-33308-9_23

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Cyclic di-GMP Regulation of Gene Expression

Meng-Lun Hsieh

Deborah M. Hinton

Christopher M. Waters

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Cited by 4 publications

(2 citation statements)

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“…A major mechanism by which c-di-GMP controls biofilm formation and motility is through regulation of transcription (21). Bacterial RNA polymerase (RNAP), which is responsible for transcription, is comprised of five subunits (␤, ␤=, two ␣s, and ) and a specificity factor, (22).…”

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“…In V. cholerae, the transcriptional activators VpsR and VpsT are required to activate transcription from the biofilm formation operons (21,(29)(30)(31)(32)(33)(34)(35)(36). Though both regulators bind c-di-GMP with a dissociation constant (K d ) of 1.6 M and 3.2 M for VpsR and VpsT, respectively (32,35), the binding of c-di-GMP to VpsT increases its binding to DNA (30,34,35,37), while the binding of c-di-GMP to VpsR has no effect on its affinity for the vpsL promoter (P vpsL ) (18,31).…”

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VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae

2020

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Vibrio cholerae biofilm biogenesis, which is important for survival, dissemination, and persistence, requires multiple genes in the Vibrio polysaccharides (vps) operons I and II as well as the cluster of ribomatrix (rbm) genes. Transcriptional control of these genes is a complex process that requires several activators/repressors and the ubiquitous signaling molecule, cyclic di-GMP (c-di-GMP). Previously, we demonstrated that VpsR directly activates RNA polymerase containing σ70 (σ70-RNAP) at the vpsL promoter (PvpsL), which precedes the vps-II operon, in a c-di-GMP-dependent manner by stimulating formation of the transcriptionally active, open complex. Using in vitro transcription, electrophoretic mobility shift assays, and DNase I footprinting, we show here that VpsR also directly activates σ70-RNAP transcription from other promoters within the biofilm formation cluster: PvpsU, at the beginning of the vps-I operon; PrbmA, at the start of the rbm cluster; and PrbmF, which lies upstream of the divergent rbmF/E genes. In this capacity, we find that VpsR is able to behave both as a Class II activator, which functions immediately adjacent/overlapping the core promoter sequence (PvpsL and PvpsU), and as a Class I activator, which functions farther upstream (PrbmA and PrbmF). Because these promoters vary in VpsR-DNA binding affinity in the absence and presence of c-di-GMP, we speculate that VpsR's mechanism of activation is dependent on both the concentration of VpsR and the level of c-di-GMP to increase transcription, resulting in finely tuned regulation. Importance Vibrio cholerae, the bacterial pathogen that is responsible for the disease cholera, uses biofilms to aid in survival, dissemination, and persistence. VpsR, which directly senses the second messenger c-di-GMP, is a major regulator of this process. Together with c-di-GMP, VpsR directly activates transcription by RNA polymerase containing σ70 from the vpsL biofilm biogenesis promoter. Using biochemical methods, we demonstrate for the first time that VpsR/c-di-GMP directly activates σ70-RNA polymerase at the first genes of the vps and ribomatrix operons. In this regard, it functions as either a Class I or Class II activator. Our results broaden the mechanism of c-di-GMP-dependent transcription activation and the specific role of VpsR in biofilm formation.

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VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae

2020

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The Vibrio cholerae master regulator for the activation of biofilm biogenesis genes, VpsR, senses both cyclic di-GMP and phosphate

Hsieh

Kiel

Jenkins

et al. 2022

Nucleic Acids Research

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Vibrio cholerae biofilm formation/maintenance is controlled by myriad factors; chief among these are the regulator VpsR and cyclic di-guanosine monophosphate (c-di-GMP). VpsR has strong sequence similarity to enhancer binding proteins (EBPs) that activate RNA polymerase containing sigma factor σ54. However, we have previously shown that transcription from promoters within the biofilm biogenesis/maintenance pathways uses VpsR, c-di-GMP and RNA polymerase containing the primary sigma factor (σ70). Previous work suggested that phosphorylation of VpsR at a highly conserved aspartate, which is phosphorylated in other EBPs, might also contribute to activation. Using the biofilm biogenesis promoter PvpsL, we show that in the presence of c-di-GMP, either wild type or the phospho-mimic VpsR D59E activates PvpsL transcription, while the phospho-defective D59A variant does not. Furthermore, when c-di-GMP levels are low, acetyl phosphate (Ac∼P) is required for significant VpsR activity in vivo and in vitro. Although these findings argue that VpsR phosphorylation is needed for activation, we show that VpsR is not phosphorylated or acetylated by Ac∼P and either sodium phosphate or potassium phosphate, which are not phosphate donors, fully substitutes for Ac∼P. We conclude that VpsR is an unusual regulator that senses phosphate directly, rather than through phosphorylation, to aid in the decision to form/maintain biofilm.

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Shigella flexneri Diguanylate Cyclases Regulate Virulence

et al. 2021

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Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli , and genome comparisons reveal that S. flexneri has lost approximately 20% of its genes through the process of pathoadaptation, including a disproportionate number of genes associated with the turnover of the nucleotide-based second messenger cyclic di-guanosine monophosphate (c-di-GMP); however, the remaining c-di-GMP turnover enzymes are highly conserved. C-di-GMP regulates many behavioral changes in other bacteria in response to changing environmental conditions, including biofilm formation, but this signaling system has not been examined in S. flexneri . In this study, we expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae , in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation, and reduced acid resistance, host cell invasion, and plaque size. We examined the impact of VCA0956 expression on the S. flexneri transcriptome, and found that genes related to acid resistance were repressed, and this corresponded with decreased survival to acid shock. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation, reduced host cell invasion and plaque size, as well as increased resistance to acid shock. This study highlights the importance of c-di-GMP signaling in regulating S. flexneri virulence phenotypes Importance The intracellular human pathogen Shigella causes dysentery, resulting in as many as one million deaths per year. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance amongst Shigella species is on the rise. Here, we explore how the widely conserved c-di-GMP bacterial signaling system alters Shigella behaviors associated with pathogenesis. We find that expressing or removing enzymes associated with c-di-GMP synthesis results in changes in Shigella ’s ability to form biofilms, invade host cells, form lesions in host cell monolayers, and resist acid stress.

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Cyclic di-GMP Regulation of Gene Expression

Cited by 4 publications

References 76 publications

VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae

VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae

The Vibrio cholerae master regulator for the activation of biofilm biogenesis genes, VpsR, senses both cyclic di-GMP and phosphate

Shigella flexneri Diguanylate Cyclases Regulate Virulence

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