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An Optimized Method for Reconstruction of Transcriptional Regulatory Networks in Bacteria Using ChIP-exo and RNA-seq Datasets
Minchang Jang, Joon Young Park, Gayeon Lee, Donghyuk Kim
J. Microbiol. 2024;62(12):1075-1088.   Published online November 11, 2024
DOI: https://doi.org/10.1007/s12275-024-00181-6
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AbstractAbstract
Transcriptional regulatory networks (TRNs) in bacteria are crucial for elucidating the mechanisms that regulate gene expression and cellular responses to environmental stimuli. These networks delineate the interactions between transcription factors (TFs) and their target genes, thereby uncovering the regulatory processes that modulate gene expression under varying environmental conditions. Analyzing TRNs offers valuable insights into bacterial adaptation, stress responses, and metabolic optimization from an evolutionary standpoint. Additionally, understanding TRNs can drive the development of novel antimicrobial therapies and the engineering of microbial strains for biofuel and bioproduct production. This protocol integrates advanced data analysis pipelines, including ChEAP, DEOCSU, and DESeq2, to analyze omics datasets that encompass genome-wide TF binding sites and transcriptome profiles derived from ChIP-exo and RNA-seq experiments. This approach minimizes both the time required and the risk of bias, making it accessible to non-expert users. Key steps in the protocol include preprocessing and peak calling from ChIP-exo data, differential expression analysis of RNA-seq data, and motif and regulon analysis. This method offers a comprehensive and efficient framework for TRN reconstruction across various bacterial strains, enhancing both the accuracy and reliability of the analysis while providing valuable insights for basic and applied research.

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  • ChIP-mini: a low-input ChIP-exo protocol for elucidating DNA-binding protein dynamics in intracellular pathogens
    Joon Young Park, Minchang Jang, Eunna Choi, Sang-Mok Lee, Ina Bang, Jihoon Woo, Seonggyu Kim, Eun-Jin Lee, Donghyuk Kim
    Nucleic Acids Research.2025;[Epub]     CrossRef
Characterization of MocR, a GntR-like transcriptional regulator, in Bradyrhizobium japonicum: its impact on motility, biofilm formation, and soybean nodulation
May Nyan Taw , Hae-In Lee , Sang-Ho Lee , Woo-Suk Chang
J. Microbiol. 2015;53(8):518-525.   Published online July 31, 2015
DOI: https://doi.org/10.1007/s12275-015-5313-z
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AbstractAbstract
Bradyrhizobium japonicum is a Gram-negative soil bacterium that can fix nitrogen into ammonia by developing a symbiotic relationship with the soybean plant. MocR proteins make up a subfamily of GntR superfamily, one of the most widely distributed and prolific groups of the helix-turn-helix transcription factors. In this study, we constructed a mutant strain for mocR (blr6977) to investigate its role in cellular processes and symbiosis in B. japonicum. Although growth rate and morphology of the mutant were indistinguishable from those of the wild type, the mutant showed significant differences in motility and attachment (i.e., biofilm formation) from the wild type. The mutant displayed a decrease in biofilm formation, but was more motile than the wild type. The inactivation of mocR did not affect the number of nodules on soybean roots, but caused delayed nodulation. Delayed nodulation intrigued us to study competitiveness of the mutant infecting soybeans. The mutant was less competitive than the wild type, indicating that delayed nodulation might be due to competitiveness. Gene expressions of other MocR subfamily members were also compared between the wild type and mutant strains. None of the mocR-like genes examined in this study were differentially expressed between both strains.

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Research Support, Non-U.S. Gov't
Functional Analysis of SGR4635-Induced Enhancement of Pigmented Antibiotic Production in Streptomyces lividans
Won-Jae Chi , Soon-Youl Lee , JaeHag Lee
J. Microbiol. 2011;49(5):828-833.   Published online November 9, 2011
DOI: https://doi.org/10.1007/s12275-011-1100-7
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AbstractAbstract
The Gram-positive mycelium-producing bacterium Streptomyces undergoes complex morphological differentiation after autolytic degradation of the vegetative mycelium. Cell-wall breakdown during growth stimulates cell development and secondary metabolite production by Streptomyces. N-acetylglucosamine (GlcNAc) produced by cell-wall lysis acts as a signal molecule, triggering the production of secondary metabolites in S. coelicolor A3(2). Here, we report that introduction of multiple copies of the GlcNAc-internalizing gene (sgr4635, encoding nagE2) of S. griseus activates actinorhodin and undecylprodigiosin production during the late growth of S. lividans in the absence of GlcNAc. Furthermore, the repressor-type transcriptional regulator DasR binds to two operator sites upstream of sgr4635. Our findings indicate that sgr4635 induces DasR-mediated antibiotic production by internalizing the GlcNAc accumulated from cell-wall lysis.
Review
REVIEW] Transcriptional Regulatory Elements in Fungal Secondary Metabolism
Wenbing Yin , Nancy P. Keller
J. Microbiol. 2011;49(3):329-339.   Published online June 30, 2011
DOI: https://doi.org/10.1007/s12275-011-1009-1
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AbstractAbstract
Filamentous fungi produce a variety of secondary metabolites of diverse beneficial and detrimental activities to humankind. The genes required for a given secondary metabolite are typically arranged in a gene cluster. There is considerable evidence that secondary metabolite gene regulation is, in part, by transcriptional control through hierarchical levels of transcriptional regulatory elements involved in secondary metabolite cluster regulation. Identification of elements regulating secondary metabolism could potentially provide a means of increasing production of beneficial metabolites, decreasing production of detrimental metabolites, aid in the identification of ‘silent’ natural products and also contribute to a broader understanding of molecular mechanisms by which secondary metabolites are produced. This review summarizes regulation of secondary metabolism associated with transcriptional regulatory elements from a broad view as well as the tremendous advances in discovery of cryptic or novel secondary metabolites by genomic mining.

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Research Support, Non-U.S. Gov'ts
Cys-92, Cys-95, and the C-Terminal 12 Residues of the Vibrio harveyi Ferric Uptake Regulator (Fur) are Functionally Inessential
Kun Sun , Shuang Cheng , Min Zhang , Fang Wang , Li Sun
J. Microbiol. 2008;46(6):670-680.   Published online December 24, 2008
DOI: https://doi.org/10.1007/s12275-008-0113-3
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AbstractAbstract
Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (FurVh) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. FurVh shares 77% overall sequence identity with the Escherichia coli Fur (FurEc) and could complement a mutant of FurEc. Like FurEc, FurVh possesses two cysteine residues at positions 92 and 95, yet unlike FurEc, in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of FurVh proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of FurVh are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of FurVh are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of FurVh is possibly different from that of FurEc; and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of FurVh; and (iii) provided insights into the potential function of the local structure involving C137 and K138.
The GntR-Type Regulators GtrA and GtrB Affect Cell Growth and Nodulation of Sinorhizobium meliloti
Yi Wang , Ai-Min Chen , Ai-Yuan Yu , Li Luo , Guan-Qiao Yu , Jia-Bi Zhu , Yan-Zhang Wang
J. Microbiol. 2008;46(2):137-145.   Published online June 11, 2008
DOI: https://doi.org/10.1007/s12275-007-0145-0
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AbstractAbstract
GntR-type transcriptional regulators are involved in the regulation of various biological processes in bacteria, but little is known about their functions in Sinorhizobium meliloti. Here, we identified two GntR-type transcriptional regulator genes, gtrA and gtrB, from S. meliloti strain 1021. Both the gtrA1 mutant and the gtrB1 mutant had lower growth rates and maximal cell yields on rich and minimal media, as well as lower cell motility on swimming plates, than did the wild-type strain. Both mutants were also symbiotically deficient. Alfalfa plants inoculated with wild-type strain 1021 formed pink elongated nodules on primary roots. In contrast, the plants inoculated with the gtrA1 and gtrB1 mutants formed relatively smaller, round, light pink nodules mainly on lateral roots. During the first 3~4 weeks post-inoculation, the plants inoculated with the gtrA1 and gtrB1 mutants were apparently stunted, with lower levels of nitrogenase activity, but there was a remarkable increase in the number of nodules compared to those inoculated with the wild-type strain. Moreover, the gtrA1 and gtrB1 mutants not only showed delayed nodulation, but also showed markedly reduced nodulation competition. These results demonstrated that both GtrA and GtrB affect cell growth and effective symbiosis of S. meliloti. Our work provides new insight into the functions of GntR-like transcriptional regulators.

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