Microbial Hauberks: Composition and Function of Surface Layer Proteins in Gammaproteobacterial Methanotrophs

Abstract

Many species of proteobacterial methane-consuming bacteria (methanotrophs) form a hauberk-like envelope represented by a surface (S-) layer protein matrix. While several proteins were predicted to be associated with the cell surface, the composition and function of the hauberk matrix remained elusive. Here we report the identification of the genes encoding the hauberk-forming protein in two gamma-proteobacterial (Type I) methanotrophs,Methylotuvimicrobium buryatense5GB1 (EQU24_15540) andMethylotuvimicrobium alcaliphilum20ZR(MEALZ_0971 and MEALZ_0972). The proteins share 40% AA identity with each other and are distantly related to the RsaA proteins fromCaulobacter crescentus(20% AA identity).Deletion of these genes resulted in loss of the characteristic hauberk pattern on the cell surface. A set of transcriptional fusions between the MEALZ_0971 and a superfolder green fluorescent protein (sfGFP) further confirmed its surface localization. The functional roles of the hauberk and cell-surface associated proteins, including MEALZ_0971, MEALZ_0972, EQU24_15540, and a copper-induced CorA protein, were further investigated via gene expression studies and phenotypic tests. The hauberk core protein shows constitutive expression across 18 growth conditions. TheM. alcaliphilum20ZRΔ0971 showed reduced growth at high salinity, high methanol and metal-limited conditions, suggesting a role in cell-envelope stability and metal scavenging.Overall, understanding the genetics, composition and cellular functions of the S-layers contributes to our knowledge of methanotroph adaptation to environmental perturbations and opens a promising prospect for (nano)biotechnology applications.