Random Mutagenesis Applied to Reveal Factors Involved in Oxidative Tolerance and Biofilm Formation in Foodborne Cronobacter malonaticus

Cronobacter species are linked with life-treating diseases in neonates and show strong tolerances to environmental stress. However, the information about factors involved in oxidative tolerance in Cronobacter remains elusive. Here, factors involved in oxidative tolerance in C. malonaticus were ident...

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Main Authors: Maofeng Zhang, Xiyan Zhang, Liaowang Tong, Dexin Ou, Yaping Wang, Jumei Zhang, Qingping Wu, Yingwang Ye
Format: Article
Language:English
Published: Frontiers Media S.A. 2019-05-01
Series:Frontiers in Microbiology
Subjects:
Online Access:https://www.frontiersin.org/article/10.3389/fmicb.2019.00877/full
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author Maofeng Zhang
Maofeng Zhang
Xiyan Zhang
Liaowang Tong
Dexin Ou
Yaping Wang
Jumei Zhang
Qingping Wu
Yingwang Ye
Yingwang Ye
author_facet Maofeng Zhang
Maofeng Zhang
Xiyan Zhang
Liaowang Tong
Dexin Ou
Yaping Wang
Jumei Zhang
Qingping Wu
Yingwang Ye
Yingwang Ye
author_sort Maofeng Zhang
collection DOAJ
description Cronobacter species are linked with life-treating diseases in neonates and show strong tolerances to environmental stress. However, the information about factors involved in oxidative tolerance in Cronobacter remains elusive. Here, factors involved in oxidative tolerance in C. malonaticus were identified using a transposon mutagenesis. Eight mutants were successfully screened based on a comparison of the growth of strains from mutant library (n = 215) and wild type (WT) strain under 1.0 mM H2O2. Mutating sites including thioredoxin 2, glutaredoxin 3, pantothenate kinase, serine/threonine protein kinase, pyruvate kinase, phospholipase A, ferrous iron transport protein A, and alanine racemase 2 were successfully identified by arbitrary PCR and sequencing alignment. Furthermore, the comparison about quantity and structure of biofilms formation among eight mutants and WT was determined using crystal violet staining (CVS), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Results showed that the biofilms of eight mutants significantly decreased within 48 h compared to that of WT, suggesting that mutating genes play important roles in biofilm formation under oxidative stress. The findings provide valuable information for deeply understanding molecular mechanism about oxidative tolerance of C. malonaticus.
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spelling doaj.art-3a25660547f54746a3d6227d38117e682022-12-21T19:12:27ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-05-011010.3389/fmicb.2019.00877424709Random Mutagenesis Applied to Reveal Factors Involved in Oxidative Tolerance and Biofilm Formation in Foodborne Cronobacter malonaticusMaofeng Zhang0Maofeng Zhang1Xiyan Zhang2Liaowang Tong3Dexin Ou4Yaping Wang5Jumei Zhang6Qingping Wu7Yingwang Ye8Yingwang Ye9School of Food Science and Engineering, Hefei University of Technology, Hefei, ChinaState Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, ChinaSchool of Food Science and Engineering, Hefei University of Technology, Hefei, ChinaSchool of Food Science and Engineering, Hefei University of Technology, Hefei, ChinaSchool of Food Science and Engineering, Hefei University of Technology, Hefei, ChinaSchool of Food Science and Engineering, Hefei University of Technology, Hefei, ChinaState Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, ChinaState Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, ChinaSchool of Food Science and Engineering, Hefei University of Technology, Hefei, ChinaState Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, ChinaCronobacter species are linked with life-treating diseases in neonates and show strong tolerances to environmental stress. However, the information about factors involved in oxidative tolerance in Cronobacter remains elusive. Here, factors involved in oxidative tolerance in C. malonaticus were identified using a transposon mutagenesis. Eight mutants were successfully screened based on a comparison of the growth of strains from mutant library (n = 215) and wild type (WT) strain under 1.0 mM H2O2. Mutating sites including thioredoxin 2, glutaredoxin 3, pantothenate kinase, serine/threonine protein kinase, pyruvate kinase, phospholipase A, ferrous iron transport protein A, and alanine racemase 2 were successfully identified by arbitrary PCR and sequencing alignment. Furthermore, the comparison about quantity and structure of biofilms formation among eight mutants and WT was determined using crystal violet staining (CVS), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Results showed that the biofilms of eight mutants significantly decreased within 48 h compared to that of WT, suggesting that mutating genes play important roles in biofilm formation under oxidative stress. The findings provide valuable information for deeply understanding molecular mechanism about oxidative tolerance of C. malonaticus.https://www.frontiersin.org/article/10.3389/fmicb.2019.00877/fullCronobacter malonaticusrandom mutagenesisarbitrary PCRoxidative stressbiofilm formation
spellingShingle Maofeng Zhang
Maofeng Zhang
Xiyan Zhang
Liaowang Tong
Dexin Ou
Yaping Wang
Jumei Zhang
Qingping Wu
Yingwang Ye
Yingwang Ye
Random Mutagenesis Applied to Reveal Factors Involved in Oxidative Tolerance and Biofilm Formation in Foodborne Cronobacter malonaticus
Frontiers in Microbiology
Cronobacter malonaticus
random mutagenesis
arbitrary PCR
oxidative stress
biofilm formation
title Random Mutagenesis Applied to Reveal Factors Involved in Oxidative Tolerance and Biofilm Formation in Foodborne Cronobacter malonaticus
title_full Random Mutagenesis Applied to Reveal Factors Involved in Oxidative Tolerance and Biofilm Formation in Foodborne Cronobacter malonaticus
title_fullStr Random Mutagenesis Applied to Reveal Factors Involved in Oxidative Tolerance and Biofilm Formation in Foodborne Cronobacter malonaticus
title_full_unstemmed Random Mutagenesis Applied to Reveal Factors Involved in Oxidative Tolerance and Biofilm Formation in Foodborne Cronobacter malonaticus
title_short Random Mutagenesis Applied to Reveal Factors Involved in Oxidative Tolerance and Biofilm Formation in Foodborne Cronobacter malonaticus
title_sort random mutagenesis applied to reveal factors involved in oxidative tolerance and biofilm formation in foodborne cronobacter malonaticus
topic Cronobacter malonaticus
random mutagenesis
arbitrary PCR
oxidative stress
biofilm formation
url https://www.frontiersin.org/article/10.3389/fmicb.2019.00877/full
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