Journal of Microbiology

Journal Article

Characterization of a novel phage depolymerase specific to Escherichia coli O157:H7 and biofilm control on abiotic surfaces: Do-Won Park , Jong-Hyun Park; J. Microbiol. 2021;59(11):1002-1009. Published online October 6, 2021; DOI: https://doi.org/10.1007/s12275-021-1413-0

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The increasing prevalence of foodborne diseases caused by Escherichia coli O157:H7 as well as its ability to form biofilms poses major threats to public health worldwide. With increasing concerns about the limitations of current disinfectant treatments, phage-derived depolymerases may be used as promising biocontrol agents. Therefore, in this study, the characterization, purification, and application of a novel phage depolymerase, Dpo10, specifically targeting the lipopolysaccharides of E. coli O157, was performed. Dpo10, with a molecular mass of 98 kDa, was predicted to possess pectate lyase activity via genome analysis and considered to act as a receptor- binding protein of the phage. We confirmed that the purified Dpo10 showed O-polysaccharide degrading activity only for the E. coli O157 strains by observing its opaque halo. Dpo10 maintained stable enzymatic activities across a wide range of temperature conditions under 55°C and mild basic pH. Notably, Dpo10 did not inhibit bacterial growth but significantly increased the complement-mediated serum lysis of E. coli O157 by degrading its O-polysaccharides. Moreover, Dpo10 inhibited the biofilm formation against E. coli O157 on abiotic polystyrene by 8-fold and stainless steel by 2.56 log CFU/coupon. This inhibition was visually confirmed via fieldemission scanning electron microscopy. Therefore, the novel depolymerase from E. coli siphophage exhibits specific binding and lytic activities on the lipopolysaccharide of E. coli O157 and may be used as a promising anti-biofilm agent against the E. coli O157:H7 strain.

Citations

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Review

Microbial phenomics linking the phenotype to function: The potential of Raman spectroscopy: Jin-Kyung Hong , Soo Bin Kim , Eun Sun Lyou , Tae Kwon Lee; J. Microbiol. 2021;59(3):249-258. Published online January 26, 2021; DOI: https://doi.org/10.1007/s12275-021-0590-1

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Abstract

Raman spectroscopy is a promising tool for identifying microbial phenotypes based on single cell Raman spectra reflecting cellular biochemical biomolecules. Recent studies using Raman spectroscopy have mainly analyzed phenotypic changes caused by microbial interactions or stress responses (e.g., antibiotics) and evaluated the microbial activity or substrate specificity under a given experimental condition using stable isotopes. Lack of labelling and the nondestructive pretreatment and measurement process of Raman spectroscopy have also aided in the sorting of microbial cells with interesting phenotypes for subsequently conducting physiology experiments through cultivation or genome analysis. In this review, we provide an overview of the principles, advantages, and status of utilization of Raman spectroscopy for studies linking microbial phenotypes and functions. We expect Raman spectroscopy to become a next-generation phenotyping tool that will greatly contribute in enhancing our understanding of microbial functions in natural and engineered systems.

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

Expression and Purification of Lacticin Q by Small Ubiquitin-Related Modifier Fusion in Escherichia coli: Qingshan Ma , Zhanqiao Yu , Bing Han , Qing Wang , Rijun Zhang; J. Microbiol. 2012;50(2):326-331. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1425-x

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Abstract: Lacticin Q is a broad-spectrum class II bacteriocin with potential as an alternative to conventional antibiotics. The objective of this study was to produce recombinant lacticin Q using a small ubiquitin-related modifier (SUMO) fusion protein expression system. The 168-bp lacticin Q gene was cloned into the expression vector pET SUMO and transformed into Escherichia coli BL21(DE3). The soluble fusion protein was recovered with a Ni-NTA Sepharose column (95% purity); 130 mg protein was obtained per liter of fermentation culture. The SUMO tag was then proteolytically cleaved from the protein, which was re-applied to the column. Finally, about 32 mg lacticin Q (≥96% purity) was obtained. The recombinant protein exhibited antimicrobial properties similar to that of the native protein, demonstrating that lacticin Q had been successfully expressed by the SUMO fusion system.

Promoter Analysis of Bombyx mori Nucleopolyhedrovirus Ubiquitin Gene: Xu’ai Lin , Yin Chen , Yongzhu Yi , Jie Yan , Zhifang Zhang; J. Microbiol. 2008;46(4):429-435. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-007-0163-y

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Abstract: The aim of this study was to analyze the characteristics of Bombyx mori nucleopolyhedrovirus (BmNPV) ubiquitin gene promoter and the effects of conserved motifs, such as TAAG, TATA, and CAAT, along with baculovirus enhancer homologous region 3 (hr3), on promoter activity. Ubiquitin gene of BmNPV was expressed during the late phase of virus infection. In the presence of viral factors, significant reduction of promoter activity was observed by deletion of -382 to -124 bp upstream of ATG. The fragment between -187 and -383 bp upstream of ATG, including distal TAAG, CAAT motif, and TATA box, could also drive expression of the reporter gene. The mutation of cis-elements TATA boxes and TAAG motifs significantly decreased the promoter’s activity, while CAAT mutations enhanced promoter activity by 2- or 3-fold, as compared with the native promoter. In the presence of BmNPV, hr3, both located downstream of the reporter gene of the same vector, and separate vector, could significantly enhance transcription activity of ubiquitin promoter as compared to the control. We concluded that BmNPV ubiquitin gene might be regulated by dual sets of promoter elements, where TAAG and TATA box may positively regulate the expression of ubiquitin, while CAAT motif functions as a negative regulator. Viral factor(s) play an important role in the co-activation of hr3 and promoter.

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