Journal of Microbiology

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[PROTOCOL] Determination of protein phosphorylation by polyacrylamide gel electrophoresis: Chang-Ro Lee , Young-Ha Park , Huitae Min , Yeon-Ran Kim , Yeong-Jae Seok; J. Microbiol. 2019;57(2):93-100. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-9021-y

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Phosphorylation is the most important modification for protein regulation; it controls many signal transduction pathways in all organisms. While several tools to detect phosphorylated proteins have been developed to study a variety of basic cellular processes involving protein phosphorylation, these methods have several limitations. Many proteins exhibit a phosphorylation-dependent electrophoretic mobility shift (PDEMS) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mechanism responsible for this phenomenon has been elucidated recently. The method for detecting phosphorylated proteins can be simplified by the application of the PDEMS. Herein, we present a novel simple method to detect protein phosphorylation, which is based on the construction of a variant protein displaying a PDEMS. The PDEMS of proteins is caused by the distribution of negatively charged amino acids around the phosphorylation site, i.e. an electrophoretic mobility shift (EMS)-related motif (ΘX1-3ΘX1-3Θ, where Θ corresponds to an acidic or phosphorylated amino acid and X represents any amino acid). The EMS-related motif can be constructed by the introduction of a negative charge by phosphorylation; it results in the decreased binding of SDS to the proteins, consequently inducing the retardation of the mobility of the protein during SDS-PAGE. Based on these molecular analyses of the PDEMS, a protein with the EMSrelated motif is designed and used to determine the in vivo phosphorylation state of the protein. This method may be used as a general strategy to easily measure the ratio of protein phosphorylation in cells.

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Research Support, Non-U.S. Gov'ts

NOTE] Probing the ArcA Regulon in the Rumen Bacterium Mannheimia succiniciproducens by Genome-Wide Expression Profiling: Seulgi Yun , Jong Moon Shin , Oh-Cheol Kim , Young Ryul Jung , Doo-Byoung Oh , Sang Yup Lee , Ohsuk Kwon; J. Microbiol. 2012;50(4):665-672. Published online July 21, 2012; DOI: https://doi.org/10.1007/s12275-012-2007-7

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Abstract: In this study, the putative target genes of the Arc two-component system of the rumen bacterium Mannheimia succiniciproducens were determined by analyzing the transcriptome of the ArcA overexpression strain and by the in silico scanning of the entire genome sequence with the position weight matrix of the ArcA binding sequence developed for Escherichia coli. The majority of 79 repressed genes were involved in energy metabolism and carbohydrate transport and metabolism, while the majority of 82 induced genes were involved in hypothetical or unknown functions. Our results suggest that the Arc system in M. succiniciproducens has a specific function that differs from that in E. coli.

NOTE] Evidence Against the Physiological Role of Acetyl Phosphate in the Phosphorylation of the ArcA Response Regulator in Escherichia coli: Xueqiao Liu , Gabriela R. Peña Sandoval , Barry L. Wanner , Won Seok Jung , Dimitris Georgellis , Ohsuk Kwon; J. Microbiol. 2009;47(5):657-662. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0087-9

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Abstract: The Arc two-component signal transduction system of Escherichia coli comprises the ArcB sensor kinase and the ArcA response regulator. Under anoxic growth conditions, ArcB autophosphorylates and transphosphorylates ArcA, which, in turn, represses or activates its target operons. ArcA has been shown to be able to autophosphorylate in vitro at the expense of acetyl-P. Here, the in vivo effect of acetyl phosphate on the redox signal transduction by the Arc system was assessed. Our results indicate that acetyl phosphate can modulate the expression of ArcA-P target genes only in the absence of ArcB. Therefore, the acetyl phosphate dependent ArcA phosphorylation route does not seem to play a significant role under physiological conditions.

Research Support, U.S. Gov't, Non-P.H.S.

Identification of Two-Component Regulatory Genes Involved in o-Xylene Degradation by Rhodococcus sp. Strain DK17: Dockyu Kim , Jong-Chan Chae , Gerben J. Zylstra , Ho-Yong Sohn , Gi-Seok Kwon , Eungbin Kim; J. Microbiol. 2005;43(1):49-53.; DOI: https://doi.org/2138 [pii]

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Abstract: Putative genes for a two-component signal transduction system (akbS and akbT) were detected near the alkylbenzene-degrading operon of Rhodococcus sp. DK17. Sequence analysis indicates that AkbS possesses potential ATP-binding and histidine autophosphorylation sites in the N- and C-terminal regions, respectively, and that AkbT has a typical response regulator domain. Mutant analysis combined with RT-PCR experiments further shows that AkbS is required to induce the expression of o-xylene dioxygenase in DK17.

Human Cytomegalovirus Inhibition of Interferon Signal Transduction: Daniel M. Miller , Colleen M. Cebulla , Daniel D. Sedmak; J. Microbiol. 2000;38(4):203-208.

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Abstract: Cytomegalovirus (CMV), a beta-herpesvirus with worldwide distribution, exhibits host persistence, a distinguishing characteristic of all herpesviruses. This persistence is dependent upon restricted gene expression in infected cells as well as the ability of productively infected cells to escape from normal cell-mediated anti-viral immunosurveillance. Type I (IFN-[alpha]/[beta]) and type II (IFN-[gamma]) interferons are major components of the innate defense system against viral infection. They are potent inducers of MHC class I and II antigens and of antigen processing proteins. Additionally, IFNs mediate direct anti-viral effects through induction of effector molecules that block viral infection and replication, such as 2', 5-oligoadenylate synthetase (2, 5-OAS). IFNs function through activation of well-defined signal transduction pathways that involve phosphorylation of constituent proteins and ultimate formation of active transcription factors. Recent studies have shown that a number of diverse viruses, including CMV, EBV, HPV, mumps and Ebola, are capable of inhibiting IFN-mediated signal transduction through a variety of mechanisms. As an example, CMV infection inhibits the ability of infected cells to transcribe HLA class I and II antigens as well as the antiviral effector molecules 2, 5-OAS and MxA I. EMSA studies have shown that IFN-[alpha] and IFN-[gamma] are unable to induce complete signal transduction in the presence of CMV infection, phenomena that are associated with specific decreases in JAK1 and p48. Viral inhibition of IFN signal transduction represents a new mechanistic paradigm for increased viral survival, a paradigm predicting widespread consequences in the case of signal transduction factors common to multiple cytokine pathways.

Regulation of Class II Bacteriocin Production by Cell-Cell Signaling: Luis E. N. Quadri; J. Microbiol. 2003;41(3):175-182.

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Abstract: Production of ribosomally synthesized antimicrobial peptides usually referred to as bacteriocins is an inducible trait in several gram positive bacteria, particularly in those belonging to the group of lactic acid bacteria. In many of these organisms, production of bacteriocins is inducible and induction requires secretion and extracellular accumulation of peptides that act as chemical messengers and trigger bacteriocin production. These inducer peptides are often referred to as autoinducers and are believed to permit a quorum sensing-based regulation of bacteriocin production. Notably, the peptides acting as autoinducers are dedicated peptides with or without antimicrobial activity or the bacteriocins themselves. The autoinducer-dependent induction of bacteriocin production requires histidine protein kinases and response regulator proteins of two-component signal transduction systems. The current working model for the regulation of class II bacteriocin production in lactic acid bacteria and the most relevant direct and indirect pieces of evidence supporting the model are discussed in this minireview.

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