Structural analysis and knock-out of a Burkholderia pseudomallei homolog of the eukaryotic transcription coactivator PC4

Werten, Sebastiaan; Köhler, Christian; Bayer, N.J.; Steinmetz, Ivo; Hinrichs, W.

doi:10.1016/j.gene.2015.11.037

Cited by 6 publications

(4 citation statements)

References 30 publications

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“…Gene deletions were performed using up-and downstream PCR fragments, which were both cloned into the formally described pBAKA-tetRA plasmid and which were generated with primers listed in S1 Table [100]. The up-and downstream fragments were digested with restriction enzymes as shown in S1 Table . The corresponding sites in the pBAKA-tetRA plasmid were used for insertion of the two fragments.…”

Section: Deletion Of Genes In B Pseudomalleimentioning

confidence: 99%

RegAB Homolog of Burkholderia pseudomallei is the Master Regulator of Redox Control and involved in Virulence

et al. 2021

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Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, often occupies environmental niches and infection sites characterized by limited concentrations of oxygen. Versatile genomic features enable this pathogen to maintain its physiology and virulence under hypoxia, but the crucial regulatory networks employed to switch from oxygen dependent respiration to alternative terminal electron acceptors (TEA) like nitrate, remains poorly understood. Here, we combined a Tn5 transposon mutagenesis screen and an anaerobic growth screen to identify a two-component signal transduction system with homology to RegAB. We show that RegAB is not only essential for anaerobic growth, but also for full virulence in cell lines and a mouse infection model. Further investigations of the RegAB regulon, using a global transcriptomic approach, identified 20 additional regulators under transcriptional control of RegAB, indicating a superordinate role of RegAB in the B. pseudomallei anaerobiosis regulatory network. Of the 20 identified regulators, NarX/L and a FNR homolog were selected for further analyses and a role in adaptation to anaerobic conditions was demonstrated. Growth experiments identified nitrate and intermediates of the denitrification process as the likely signal activateing RegAB, NarX/L, and probably of the downstream regulators Dnr or NsrR homologs. While deletions of individual genes involved in the denitrification process demonstrated their important role in anaerobic fitness, they showed no effect on virulence. This further highlights the central role of RegAB as the master regulator of anaerobic metabolism in B. pseudomallei and that the complete RegAB-mediated response is required to achieve full virulence. In summary, our analysis of the RegAB-dependent modulon and its interconnected regulons revealed a key role for RegAB of B. pseudomallei in the coordination of the response to hypoxic conditions and virulence, in the environment and the host.

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Section: Deletion Of Genes In B Pseudomalleimentioning

confidence: 99%

RegAB Homolog of Burkholderia pseudomallei is the Master Regulator of Redox Control and involved in Virulence

et al. 2021

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“…In humans, the transcriptional coactivator, PC4 is required to stimulate the reconstituted basal transcription by RNA polymerase II in vitro ( Werten et al, 1998 ). PC4 is thought to be evolutionarily conserved in an ssDBD ( Werten et al, 2016 ; Garavis and Calvo, 2017 ). Here, we found that a PRC region (PC4-like Region Conserved) in AtNMDM1 was highly conserved in plants, humans, and yeast ( Figure 6 ).…”

Section: Discussionmentioning

confidence: 99%

Arabidopsis Novel Microgametophyte Defective Mutant 1 Is Required for Pollen Viability via Influencing Intine Development in Arabidopsis

Yang

et al. 2022

Front. Plant Sci.

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The pollen intine layer is necessary for male fertility in flowering plants. However, the mechanisms behind the developmental regulation of intine formation still remain largely unknown. Here, we identified a positive regulator, Arabidopsis novel microgametophyte defective mutant 1 (AtNMDM1), which influences male fertility by regulating intine formation. The AtNMDM1, encoding a pollen nuclei-localized protein, was highly expressed in the pollens at the late anther stages, 10–12. Both the mutations and the knock-down of AtNMDM1 resulted in pollen defects and significantly lowered the seed-setting rates. Genetic transmission analysis indicated that AtNMDM1 is a microgametophyte lethal gene. Calcofluor white staining revealed that abnormal cellulose distribution was present in the aborted pollen. Ultrastructural analyses showed that the abnormal intine rather than the exine led to pollen abortion. We further found, using transcriptome analysis, that cell wall modification was the most highly enriched gene ontology (GO) term used in the category of biological processes. Notably, two categories of genes, Arabinogalactan proteins (AGPs) and pectin methylesterases (PMEs) were greatly reduced, which were associated with pollen intine formation. In addition, we also identified another regulator, AtNMDM2, which interacted with AtNMDM1 in the pollen nuclei. Taken together, we identified a novel regulator, AtNMDM1 that affected cellulose distribution in the intine by regulating intine-related gene expression; furthermore, these results provide insights into the molecular mechanisms of pollen intine development.

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“…Several orthologues of the human positive coactivator PC4 have been discovered in bacteriophages, bacteria, and eukaryotes, showing that it is an evolutionarily conserved factor. All of them are involved in different aspects of DNA metabolism and are characterized by the presence of an ssDBD [ 7 , 8 , 9 , 10 , 11 , 12 ]. The structure of this domain, as a dimer bound to the DNA, has been solved in several of these organisms, disclosing an extraordinary similitude, for instance, between humans [ 13 , 14 , 15 ] and rice [ 7 , 9 , 16 ].…”

Section: Discussionmentioning

confidence: 99%

“…The homology of ScSub1 and PC4 resides in a ssDBD, which has also been described in other organisms [ 7 ], such as in the fission yeast Schizosaccharomyces pombe [ 8 ] and the rice blast fungus Magnaporthe oryzae [ 9 ]. In addition, PC4/Sub1 orthologs have been identified in prokaryotes such as in Lactococcus lactis [ 10 ], Burkholderia pseudomallei [ 11 ], and even in the bacteriophage T5 [ 12 ]. It is also pertinent to note that structures of the DNA binding domains of PC4 [ 13 , 14 , 15 ], MoSub1 [ 7 , 9 , 16 ], L. lactis, and bacteriophage T5 have been elucidated.…”

Section: Introductionmentioning

confidence: 99%

The Role of S. cerevisiae Sub1/PC4 in Transcription Elongation Depends on the C-Terminal Region and Is Independent of the ssDNA Binding Domain

Collin

González-Jiménez

et al. 2022

Cells

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Saccharomyces cerevisiae Sub1 (ScSub1) has been defined as a transcriptional stimulatory protein due to its homology to the ssDNA binding domain (ssDBD) of human PC4 (hPC4). Recently, PC4/Sub1 orthologues have been elucidated in eukaryotes, prokaryotes, and bacteriophages with functions related to DNA metabolism. Additionally, ScSub1 contains a unique carboxyl–terminal region (CT) of unknown function up to date. Specifically, it has been shown that Sub1 is required for transcription activation, as well as other processes, throughout the transcription cycle. Despite the progress that has been made in understanding the mechanism underlying Sub1′s functions, some questions remain unanswered. As a case in point: whether Sub1’s roles in initiation and elongation are differentially predicated on distinct regions of the protein or how Sub1′s functions are regulated. Here, we uncover some residues that are key for DNA–ScSub1 interaction in vivo, localized in the ssDBD, and required for Sub1 recruitment to promoters. Furthermore, using an array of genetic and molecular techniques, we demonstrate that the CT region is required for transcription elongation by RNA polymerase II (RNAPII). Altogether, our data indicate that Sub1 plays a dual role during transcription—in initiation through the ssDBD and in elongation through the CT region.Sub1; PC4; DNA binding proteins; RNA polymerase II; transcription elongation; Saccharomyces cerevisiae

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Structural analysis and knock-out of a Burkholderia pseudomallei homolog of the eukaryotic transcription coactivator PC4

Cited by 6 publications

References 30 publications

RegAB Homolog of Burkholderia pseudomallei is the Master Regulator of Redox Control and involved in Virulence

RegAB Homolog of Burkholderia pseudomallei is the Master Regulator of Redox Control and involved in Virulence

Arabidopsis Novel Microgametophyte Defective Mutant 1 Is Required for Pollen Viability via Influencing Intine Development in Arabidopsis

The Role of S. cerevisiae Sub1/PC4 in Transcription Elongation Depends on the C-Terminal Region and Is Independent of the ssDNA Binding Domain

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