Identification of Key Residues in the NisK Sensor Region for Nisin Biosynthesis Regulation.

Xiaoxuan Ge,Fangyuan Zhao,Kunling Teng,Jin Zhong,Jie Zhang,Jian Wang

doi:10.3389/fmicb.2017.00106

Abstract

Histidine kinase (HK) NisK is well known to sense lantibiotic nisin for regulating the biosynthesis of nisin. NisK possesses two trans-membrane segments and a large extracellular region and nisin contains 34 amino acids with five lanthionine rings. Unlike most peptide sensing HK with multi trans-membrane segments, NisK is a representative of a group of rarely reported HK that sense peptide as ligand. To reveal how NisK senses nisin molecule to regulate nisin biosynthesis, we constructed a reporter Lactococcus lactis strain with nisRK constitutively expressed and a reporter gene lacZ expressed under the control of promoter PnisA. We showed that the extracellular region of NisK was involved in recognizing nisin. Conserved residues in this group of HK were found in the extracellular region of NisK and mutagenesis of these residues in the reporter strain revealed that several hydrophobic residues including two aromatic residues are crucial for NisK sensing nisin and regulating nisin biosynthesis. Substitutions of hydrophobic regions in NisK extracellular domain showed that the first strand that was rich of hydrophobic amino acids was involved in regulating nisin biosynthesis. A negatively charged residue in the first βstrand also contributed to nisin biosynthesis. Protein binding analyses demonstrated that nisin could not interact with key NisK mutants, indicating these site in the extracellular region of NisK was involved in recognizing nisin.

Highlights

Nisin is the most representative lantibiotic produced by Lactococcus lactis and is widely used as a natural and safe food preservative in many countries (Gharsallaoui et al, 2015a,b)
We found that there was a group of histidine kinase (HK) with two trans-membrane segments and an extracellular region (>100 amino acids) which is likely the sensor domain
The mutants 69–73A5 and 74–76A3 which were located in the first strand showed greatly reduced or no β-galactosidase activities, while other mutants still maintained similar regulation abilities to NisK (Figure 4B). These results indicated that the first strand which was rich of hydrophobic amino acids in NisK extracellular region was necessary to regulate nisin biosynthesis

Summary

Introduction

Nisin is the most representative lantibiotic produced by Lactococcus lactis and is widely used as a natural and safe food preservative in many countries (Gharsallaoui et al, 2015a,b). The gene cluster related to nisin biosynthesis includes 11 genes nisABTCIPRKFEG. NisA encodes nisin precursor in which serine and threonine residues are dehydrated to dehydroalanine and dehydrobutyrine by NisB and cyclized with cysteines by NisC to form thioether bridges. Nisin belongs to the group of AI lantibiotics and its structure has been elucidated as a linear peptide containing five intermolecular lanthionine rings (Chatterjee et al, 2005). Nisin is the first found bifunctional lantibiotic which could work as antimicrobial agent, and act as signal to induce its own biosynthesis through the quorum sensing system NisK/R two-component regulatory system (Kuipers et al, 1995)

Methods

Results

Conclusion