Journal of Microbiology

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Unexpected Requirement of Small Amino Acids at Position 183 for DNA Binding in the Escherichia coli cAMP Receptor Protein: Marcus Carranza, Amanda Rea, Daisy Pacheco, Christian Montiel, Jin Park, Hwan Youn; J. Microbiol. 2024;62(10):871-882. Published online September 6, 2024; DOI: https://doi.org/10.1007/s12275-024-00169-2

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The Escherichia coli cAMP receptor protein (CRP) relies on the F-helix, the recognition helix of the helix-turn-helix motif, for DNA binding. The importance of the CRP F-helix in DNA binding is well-established, yet there is little information on the roles of its non-base-contacting residues. Here, we show that a CRP F-helix position occupied by a non-base-contacting residue Val183 bears an unexpected importance in DNA binding. Codon randomization and successive in vivo screening selected six amino acids (alanine, cysteine, glycine, serine, threonine, and valine) at CRP position 183 to be compatible with DNA binding. These amino acids are quite different in their amino acid properties (polar, non-polar, hydrophobicity), but one commonality is that they are all relatively small. Larger amino acid substitutions such as histidine, methionine, and tyrosine were made site-directedly and showed to have no detectable DNA binding, further supporting the requirement of small amino acids at CRP position 183. Bioinformatics analysis revealed that small amino acids (92.15% valine and 7.75% alanine) exclusively occupy the position analogous to CRP Val183 in 1,007 core CRP homologs, consistent with our mutant data. However, in extended CRP homologs comprising 3700 proteins, larger amino acids could also occupy the position analogous to CRP Val183 albeit with low occurrence. Another bioinformatics analysis suggested that large amino acids could be tolerated by compensatory small-sized amino acids at their neighboring positions. A full understanding of the unexpected requirement of small amino acids at CRP position 183 for DNA binding entails the verification of the hypothesized compensatory change(s) in CRP.

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SPD_0410 negatively regulates capsule polysaccharide synthesis and virulence in Streptococcus pneumoniae D39
Ye Tao, Li Lei, Shuhui Wang, Xuemei Zhang, Yibing Yin, Yuqiang Zheng
Frontiers in Microbiology.2025;[Epub] CrossRef

[PROTOCOL] High-throughput cultivation based on dilution-to-extinction with catalase supplementation and a case study of cultivating acI bacteria from Lake Soyang: Suhyun Kim , Miri S. Park , Jaeho Song , Ilnam Kang , Jang-Cheon Cho; J. Microbiol. 2020;58(11):893-905. Published online October 30, 2020; DOI: https://doi.org/10.1007/s12275-020-0452-2

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Abstract

Multi-omics approaches, including metagenomics and single- cell amplified genomics, have revolutionized our understanding of the hidden diversity and function of microbes in nature. Even in the omics age, cultivation is an essential discipline in microbial ecology since microbial cultures are necessary to assess the validity of an in silico prediction about the microbial metabolism and to isolate viruses infecting bacteria and archaea. However, the ecophysiological characteristics of predominant freshwater bacterial lineages remain largely unknown due to the scarcity of cultured representatives. In an ongoing effort to cultivate the uncultured majority of freshwater bacteria, the most abundant freshwater Actinobacteria acI clade has recently been cultivated from Lake Soyang through catalase-supplemented high-throughput cultivation based on dilution-to-extinction. This method involves physical isolation of target microbes from mixed populations, culture media simulating natural habitats, and removal of toxic compounds. In this protocol, we describe detailed procedures for isolating freshwater oligotrophic microbes, as well as the essence of the dilution-to-extinction culturing. As a case study employing the catalase-supplemented dilution-to-extinction protocol, we also report a cultivation trial using a water sample collected from Lake Soyang. Of the 480 cultivation wells inoculated with a single lake-water sample, 75 new acI strains belonging to 8 acI tribes (acI-A1, A2, A4, A5, A6, A7, B1, B4, C1, and C2) were cultivated, and each representative strain per subclade could be revived from glycerol stocks. These cultivation results demonstrate that the protocol described in this study is efficient in isolating freshwater bacterioplankton harboring streamlined genomes.

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Environmental Microbiome.2022;[Epub] CrossRef
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Journal of Microbiology.2021; 59(3): 311. CrossRef
Heme auxotrophy in abundant aquatic microbial lineages
Suhyun Kim, Ilnam Kang, Jin-Won Lee, Che Ok Jeon, Stephen J. Giovannoni, Jang-Cheon Cho
Proceedings of the National Academy of Sciences.2021;[Epub] CrossRef
Recent trend, biases and limitations of cultivation-based diversity studies of microbes
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FEMS Microbiology Letters.2021;[Epub] CrossRef
Cultivation of Dominant Freshwater Bacterioplankton Lineages Using a High-Throughput Dilution-to-Extinction Culturing Approach Over a 1-Year Period
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Frontiers in Microbiology.2021;[Epub] CrossRef

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