Plantaricin 423

Plantaricin 423 is produced by Lactobacillus plantarum 423 using the pla biosynthetic operon located on the 8,188-bp plasmid pPLA4. As with many class IIa bacteriocin operons, the pla operon carries biosynthetic genes (plaA, precursor peptide; plaB, immunity; plaC, accessory; and plaD, ABC transporter) but does not carry local regulatory genes. Little is known about the regulatory mechanisms involved in the expression of the apparently regulationless class IIa bacteriocins, such as plantaricin 423. In this study, phylogenetic analysis of class IIa immunity proteins indicated that at least three distinct clades exist, which were then used to subgroup the class IIa operons. It became evident that the absence of classical quorum-sensing genes on mobile bacteriocin-encoding elements is a predisposition of the subgroup that includes plantaricin 423, pediocin AcH/PA-1, divercin V41, enterocin A, leucocin-A and -B, mesentericin Y105, and sakacin G. Further analysis of the subgroup suggested that the regulation of these class IIa operons is linked to transition metal homeostasis in the host. By using a fluorescent promoter-reporter system in Lactobacillus plantarum 423, transcriptional regulation of plantaricin 423 was shown to be upregulated in response to manganese privation. IMPORTANCE Lactic acid bacteria hold huge industrial application and economic value, especially bacteriocinogenic strains, which further aids in the exclusion of specific foodborne pathogens. Since bacteriocinogenic strains are sought after, it is equally important to understand the mechanism of bacteriocin regulation. This is currently an understudied aspect of class IIa operons. Our research suggests the existence of a previously undescribed mode of class IIa bacteriocin regulation, whereby bacteriocin expression is linked to management of the producer's transition metal homeostasis. This delocalized metalloregulatory model may fundamentally affect the selection of culture conditions for bacteriocin expression and change our understanding of class IIa bacteriocin gene transfer dynamics in a given microbiome.

Plantaricin 423 is a class IIa bacteriocin produced by Lactobacillus plantarum isolated from sorghum beer. It has been previously determined that plantaricin 423 is encoded by a plasmid designated pPLA4, which is now completely sequenced. The plantaricin 423 operon shares high sequence similarity with the operons of coagulin, pediocin PA-1, and pediocin AcH, with small differences in the DNA sequence encoding the mature bacteriocin peptide and the immunity protein. Apart from the bacteriocin operon, no significant sequence similarity could be detected between the DNA or translated sequence of pPLA4 and the available DNA or translated sequences of the plasmids encoding pediocin AcH, pediocin PA-1, and coagulin, possibly indicating a different origin. In addition to the bacteriocin operon, sequence analysis of pPLA4 revealed the presence of two open reading frames (ORFs). ORF1 encodes a putative mobilization (Mob) protein that is homologous to the pMV158 superfamily of mobilization proteins. Highest sequence similarity occurred between this protein and the Mob protein of L. plantarum NCDO 1088. ORF2 encodes a putative replication protein that revealed low sequence similarity to replication proteins of plasmids pLME300 from Lactobacillus fermentum and pYIT356 from Lactobacillus casei. The immunity protein of plantaricin 423 contains 109 amino acids. Although plantaricin 423 shares high sequence similarity with the pediocin PA-1 operon, no cross-reactivity was recorded between the immunity proteins of plantaricin 423 and pediocin PA-1.

The antilisterial class IIa bacteriocins, plantaricin 423 and mundticin ST4SA, have previously been purified from the cell-free supernatants of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, respectively. Here, we present the fusions of mature plantaricin 423 and mundticin ST4SA to His-tagged green fluorescent protein (GFP) for respective heterologous expression in Escherichia coli. Fusion of plantaricin 423 and mundticin ST4SA to His-tagged GFP produced the fusion proteins GFP-PlaX and GFP-MunX, respectively. Both fusion proteins were autofluorescent, circumvented inclusion body formation and lowered the toxicity of class IIa bacteriocins during heterologous expression. Not only did GFP-class IIa fusion stabilize heterologous expression and boost yields, the fluorescent intensity of GFP-PlaX and GFP-MunX could be monitored quantitatively and qualitatively throughout expression and purification. This robust fluorometric property allowed rapid optimization of conditions for expression and bacteriocin liberation from GFP via the incorporated WELQut protease cleavage sequence. Incubation temperature and IPTG concentration had a significant effect on bacteriocin yield, and was optimal at 18°C and 0.1-0.2 mM, respectively. GFP-MunX was approximately produced at a yield of 153.30 mg/L culture which resulted in 12.4 mg/L active mundticin ST4SA after liberation and HPLC purification. While GFP-PlaX was produced at a yield of 121.29 mg/L culture, evidence suggests heterologous expression resulted in conformation isomers of WELQut liberated plantaricin 423.