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  • 1
    Online Resource
    Online Resource
    The Origin of Chromosomal Replication Is Asymmetrically Positioned on the Mycobacterial Nucleoid, and the Timing of Its Firing Depends on HupB
    American Society for Microbiology ; 2018
    In:  Journal of Bacteriology Vol. 200, No. 10 ( 2018-05-15)
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 200, No. 10 ( 2018-05-15)
    Abstract: The bacterial chromosome undergoes dynamic changes in response to ongoing cellular processes and adaptation to environmental conditions. Among the many proteins involved in maintaining this dynamism, the most abundant is the nucleoid-associated protein (NAP) HU. In mycobacteria, the HU homolog, HupB, possesses an additional C-terminal domain that resembles that of eukaryotic histones H1/H5. Recently, we demonstrated that the highly abundant HupB protein occupies the entirety of the Mycobacterium smegmatis chromosome and that the HupB-binding sites exhibit a bias from the origin ( oriC ) to the terminus ( ter ). In this study, we used HupB fused with enhanced green fluorescent protein (EGFP) to perform the first analysis of chromosome dynamics and to track the oriC and replication machinery directly on the chromosome during the mycobacterial cell cycle. We show that the chromosome is located in an off-center position that reflects the unequal division and growth of mycobacterial cells. Moreover, unlike the situation in E. coli , the sister oriC regions of M. smegmatis move asymmetrically along the mycobacterial nucleoid. Interestingly, in this slow-growing organism, the initiation of the next round of replication precedes the physical separation of sister chromosomes. Finally, we show that HupB is involved in the precise timing of replication initiation. IMPORTANCE Although our view of mycobacterial nucleoid organization has evolved considerably over time, we still know little about the dynamics of the mycobacterial nucleoid during the cell cycle. HupB is a highly abundant mycobacterial nucleoid-associated protein (NAP) with an indispensable histone-like tail. It was previously suggested as a potential target for antibiotic therapy against tuberculosis. Here, we fused HupB with enhanced green fluorescent protein (EGFP) to study the dynamics of the mycobacterial chromosome in real time and to monitor the replication process directly on the chromosome. Our results reveal that, unlike the situation in Escherichia coli , the nucleoid of an apically growing mycobacterium is positioned asymmetrically within the cell throughout the cell cycle. We show that HupB is involved in controlling the timing of replication initiation. Since tuberculosis remains a serious health problem, studies concerning mycobacterial cell biology are of great importance.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.00044-18
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2018
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 2
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    Online Resource
    Transcriptional Response of Saccharomyces cerevisiae to the Plasma Membrane-Perturbing Compound Chitosan
    American Society for Microbiology ; 2005
    In:  Eukaryotic Cell Vol. 4, No. 4 ( 2005-04), p. 703-715
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    In: Eukaryotic Cell, American Society for Microbiology, Vol. 4, No. 4 ( 2005-04), p. 703-715
    Abstract: Chitosan is a plasma membrane-perturbing compound consisting of linear chains of β-1,4-linked glucosamine residues, which at acidic pHs become positively charged. It is extensively used as an antimicrobial compound, yet its mode of action is still unresolved. Chitosan strongly affected the growth of the yeast Saccharomyces cerevisiae , the food spoilage yeast Zygosaccharomyces bailii , and two human-pathogenic yeasts, Candida albicans and Candida glabrata . Microarray analysis of yeast cells treated with sublethal concentrations of chitosan revealed induction of the environmental stress response and three more major transcriptional responses. The first was a rapid and stable Cin5p-mediated response. Cin5p/Yap4p is a transcription factor involved in various stress responses. Deletion of CIN5 led to increased chitosan sensitivity. The second was a Crz1p-mediated response, which is delayed compared to the Cin5p response. Crz1p is a transcription factor of the calcineurin pathway. Cells deleted for CRZ1 or treated with the calcineurin inhibitor FK506 became hypersensitive to chitosan, supporting the notion that the Crz1p-controlled response offers protection against chitosan. The third was a strong Rlm1p-mediated response which ran parallel in time with the Crz1p-regulated response. Rlm1p is a transcription factor of the cell wall integrity pathway, which is activated by cell wall stress. Importantly, chitosan-treated cells became more resistant to β-1,3-glucanase, which is a well-known response to cell wall stress. We propose that the transcriptional response to chitosan may be representative of other plasma membrane-perturbing compounds.
    Type of Medium: Online Resource
    ISSN: 1535-9778 , 1535-9786
    URL: Article
    DOI: 10.1128/EC.4.4.703-715.2005
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2005
    detail.hit.zdb_id: 2071564-X
    SSG: 12
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  • 3
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    Online Resource
    AfsK-Mediated Site-Specific Phosphorylation Regulates DnaA Initiator Protein Activity in Streptomyces coelicolor
    American Society for Microbiology ; 2020
    In:  Journal of Bacteriology Vol. 202, No. 3 ( 2020-01-15)
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 202, No. 3 ( 2020-01-15)
    Abstract: In all organisms, chromosome replication is regulated mainly at the initiation step. Most of the knowledge about the mechanisms that regulate replication initiation in bacteria has come from studies on rod-shaped bacteria, such as Escherichia coli and Bacillus subtilis . Streptomyces is a bacterial genus that is characterized by distinctive features and a complex life cycle that shares some properties with the developmental cycle of filamentous fungi. The unusual lifestyle of streptomycetes suggests that these bacteria use various mechanisms to control key cellular processes. Here, we provide the first insights into the phosphorylation of the bacterial replication initiator protein, DnaA, from Streptomyces coelicolor . We suggest that phosphorylation of DnaA triggers a conformational change that increases its ATPase activity and decreases its affinity for the replication origin, thereby blocking the formation of a functional orisome. We suggest that the phosphorylation of DnaA is catalyzed by Ser/Thr kinase AfsK, which was shown to regulate the polar growth of S. coelicolor . Together, our results reveal that phosphorylation of the DnaA initiator protein functions as a negative regulatory mechanism to control the initiation of chromosome replication in a manner that presumably depends on the cellular localization of the protein. IMPORTANCE This work provides insights into the phosphorylation of the DnaA initiator protein in Streptomyces coelicolor and suggests a novel bacterial regulatory mechanism for initiation of chromosome replication. Although phosphorylation of DnaA has been reported earlier, its biological role was unknown. This work shows that upon phosphorylation, the cooperative binding of the replication origin by DnaA may be disturbed. We found that AfsK kinase is responsible for phosphorylation of DnaA. Upon upregulation of AfsK, chromosome replication occurred further from the hyphal tip. Orthologs of AfsK are exclusively found in mycelial actinomycetes that are related to Streptomyces and exhibit a complex life cycle. We propose that the AfsK-mediated regulatory pathway serves as a nonessential, energy-saving mechanism in S. coelicolor .
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.00597-19
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2020
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 4
    Online Resource
    Online Resource
    Cellular Processes and Pathways That Protect Saccharomyces cerevisiae Cells against the Plasma Membrane-Perturbing Compound Chitosan
    American Society for Microbiology ; 2007
    In:  Eukaryotic Cell Vol. 6, No. 4 ( 2007-04), p. 600-608
    Details
    In: Eukaryotic Cell, American Society for Microbiology, Vol. 6, No. 4 ( 2007-04), p. 600-608
    Abstract: Global fitness analysis makes use of a genomic library of tagged deletion strains. We used this approach to study the effect of chitosan, which causes plasma membrane stress. The data were analyzed using T-profiler, which was based on determining the sensitivities of groups of deletion strains to chitosan, as defined by Gene Ontology (GO) and by genomic synthetic lethality screens, in combination with t statistics. The chitosan-hypersensitive groups included a group of deletion strains characterized by a defective HOG (high-osmolarity glycerol) signaling pathway, indicating that the HOG pathway is required for counteracting chitosan-induced stress. Consistent with this, activation of this pathway in wild-type cells by hypertonic conditions offered partial protection against chitosan, whereas hypotonic conditions sensitized the cells to chitosan. Other chitosan-hypersensitive groups were defective in RNA synthesis and processing, actin cytoskeleton organization, protein N-glycosylation, ergosterol synthesis, endocytosis, or cell wall formation, predicting that these cellular functions buffer the cell against the deleterious effect of chitosan. These predictions were supported by showing that tunicamycin, miconazole, and staurosporine (which target protein N-glycosylation, ergosterol synthesis, and the cell wall integrity pathway, respectively) sensitized Saccharomyces cerevisiae cells to chitosan. Intriguingly, the GO-defined group of deletion strains belonging to the “cytosolic large ribosomal subunit” was more resistant to chitosan. We propose that global fitness analysis of yeast in combination with T-profiler is a powerful tool to identify specific cellular processes and pathways that are required for survival under stress conditions.
    Type of Medium: Online Resource
    ISSN: 1535-9778 , 1535-9786
    URL: Article
    DOI: 10.1128/EC.00355-06
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2007
    detail.hit.zdb_id: 2071564-X
    SSG: 12
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