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 (SLP) 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 proteins in two gamma-proteobacterial (Type I) methanotrophs, Methylotuvimicrobium buryatense 5GB1 (EQU24_15540) and Methylotuvimicrobium alcaliphilum 20ZR (MEALZ_0971 and MEALZ_0972). The proteins share 40% amino acid (AA) sequence identity with each other and are distantly related to the RsaA proteins from Caulobacter crescentus (20% AA sequence 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 of M. alcaliphilum 20ZR showed constitutive expression across 18 growth conditions with 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 S-layers contributes to our knowledge of methanotroph adaptation to environmental perturbations and opens a promising prospect for (nano)biotechnology applications.