Heterologous Biosynthesis of Five New Class II Bacteriocins From Lactobacillus paracasei CNCM I-5369 With Antagonistic Activity Against Pathogenic Escherichia coli Strains

Abstract

Lactobacillus paracasei CNCM I-5369 isolated from a traditional Algerian dairy product produces extracellular inhibitory substances, namely, bacteriocins, which are active against a panel of pathogenic Escherichia coli strains. This activity was observed only at a narrow pH 4.5-5, and resulted to be heat stable and sensitive to the action of proteolytic enzymes, which indicate a proteinaceous nature. This new strain has a genome of 2,752,975 bp, with a 46.6% G + C ratio and contains at least 2664 coding sequences. The Bagel software analysis identified five open reading frames (ORFs) that are translated to new class II bacteriocin. Each ORF was cloned in frame with a Histag tail and expressed in E. coli BL21 (DE3) (pLysS) strain. Of note, each fusion protein carrying any of these ORFs at the C-or N-terminal position resulted to be active against E. coli 184 strain used as target organism. This manuscript reports the first multibacteriocinogenic strain producing five new class II bacteriocins with activity against Gram-negative bacilli (GNB), namely, E. coli. Heterologous expression and activity of each new class II bacteriocin were demonstrated.

Expression of five class II bacteriocins with activity against Escherichia coli in Lacticaseibacillus paracasei CNCM I-5369, and in a heterologous host

“…The nucleotide and protein sequences for the five bacteriocins of L. paracasei CNCM I-5369 strain were identified using Bagel 4 software [ 16 ]. The putative sequences of promoter regions are depicted for each gene in Fig.…”

“…The nucleotide sequences of the bacteriocin genes of L. paracasei CNCM I-5369 were previously determined with Bagel 4 software [ 16 ]. The house-keeping genes used to standardize the expression of bacteriocin genes were the gyrA and 16S rRNA genes [ 19 ].…”

“…Each gene was cloned under the control of the inducible T7 promoter as recently reported [ 16 ], using e-Zyvec technology. Each recombinant plasmid ( Table 1 ) was recovered and used to transform the E. coli Rosetta (DE3) and E. coli BL21 (DE3) competent cells by a heat shock method (42 °C).…”

“…In regard to characterization of LAB-bacteriocins endowed with activity against Gram-negative bacteria, our previous work reported the isolation of Lacticaseibacillus paracasei CNCM I-5369 strain carrying five genes ( orf010 , orf012 , orf023 , orf030 and orf038 ) coding for class II bacteriocins [ 16 ]. These bacteriocins were heterologously expressed in E. coli BL21 and shown to be active against E. coli ATCC 8739 [ 16 ]. The present study is a continuation, which is aimed at evaluating the expression of each gene coding for these bacteriocins and defining the appropriate conditions for their effective purification.…”

Probiotics at War Against Viruses: What Is Missing From the Picture?

“… Tagg et al (1976) have defined bacteriocins as peptides active against closely related bacteria. However, some of the more recently described bacteriocins are also active against relatively distantly related bacteria ( Gupta et al, 2016 ; Belguesmia et al, 2020 ). The sensitivity of a target bacterium to bacteriocins depends on the physico-chemical characteristics of the environment, of which pH, ionic strength, and the presence of neutralizing or membrane-disrupting molecules all play a major role ( Belguesmia et al, 2010 ).…”