title: A single substitution in 5'-untranslated region of plcB is involved in enhanced broad-range phospholipase C activity in Listeria monocytogenes strain H4.
abstract: To examine whether the in vitro phospholipase activity in Listeria monocytogenes strain H4 was due to two nucleotide mutations (C to T at position -26 and A to G at position +1) in plcB or resulted from regulatory activation, two mutants H4-plcB-m1 (single mutation at position -26) and H4-plcB-m2 (substitution at both positions) were constructed by site-directed mutagenesis. It was found that the two mutants had significantly lower transcription of plcB than their parent strain H4 and did not show phospholipase activity on the egg yolk agar, implying that the apparent phospholipase activity of strain H4 could be related to single substitution at position -26 of plcB, most probably by its 5'-untranslated region (5'-UTR) regulation mechanism. Tn917-based transposon mutagenesis generated eight L. monocytogenes mutants lacking phospholipase activity among 560 mutant candidates. Seven mutants had transposon insertion into prfA (encoding positive regulatory factor A) open reading frame, whereas only one mutant (WF-L127) was inserted into the P1 promoter region of prfA (prfAP1). Transcription of major virulence genes was significantly lower in both types of mutants than in their parent strain H4. Disruption of prfAP1 in WF-L127 abolished its phospholipase C activity but did not change its hemolytic phenotype, indicating that plcB was more dependent on prfA regulation than hly. Taken together, this study presents some evidence for the regulation of plcB expression by its 5'-UTR mechanism.
authors.affiliation: Institute of Preventive Veterinary Medicine, Zhejiang Provincial Key Laboratory for Preventive Veterinary Medicine, Zhejiang University, Hangzhou, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Bai
authors.forename: Jianshun
authors.initials: J
authors.lastname: Chen
authors.forename: Qiaomiao
authors.initials: Q
authors.lastname: Chen
authors.forename: Xiaokai
authors.initials: X
authors.lastname: Luo
authors.forename: Weihuan
authors.initials: W
authors.lastname: Fang
authors.forename: Lingli
authors.initials: L
authors.lastname: Jiang
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006461:Hemolysis / Q000235:genetics; D008089:Listeria monocytogenes / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D010738:Type C Phospholipases / Q000235:genetics* / Q000378:metabolism; D014774:Virulence
chemical_list: D020121:5' Untranslated Regions; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D010454:Peptide Termination Factors; C545356:PrfA protein, Listeria monocytogenes; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C