Microbial membrane transport proteins and their biotechnological applications.

Because of the hydrophobic nature of the membrane lipid bilayer, the majority of the hydrophilic solutes require special transportation mechanisms for passing through the cell membrane. Integral membrane transport proteins (MTPs), which belong to the Major Intrinsic Protein Family, facilitate the transport of these solutes across cell membranes. MTPs including aquaporins and carrier proteins are transmembrane proteins spanning across the cell membrane. The easy handling of microorganisms enabled the discovery of a remarkable number of transport proteins specific to different substances. It has been realized that these transporters have very important roles in the survival of microorganisms, their pathogenesis, and antimicrobial resistance. Astonishing features related to the solute specificity of these proteins have led to the acceleration of the research on the discovery of their properties and the development of innovative products in which these unique properties are used or imitated. Studies on microbial MTPs range from the discovery and characterization of a novel transporter protein to the mining and screening of them in a large transporter library for particular functions, from simulations and modeling of specific transporters to the preparation of biomimetic synthetic materials for different purposes such as biosensors or filtration membranes. This review presents recent discoveries on microbial membrane transport proteins and focuses especially on formate nitrite transport proteins and aquaporins, and advances in their biotechnological applications.

Comparison of ion selectivities of nitrite channel NirC and water channel aquaporin.

nirC gene coding for the nitrite channel of E. coli K12 was cloned into the pET28a vector and expressed in E. coli BL21 cells. 28.5 kDa NirC monomer was purified from membrane components of E. coli. Selectivity of NirC for different ions including nitrite, nitrate, sulfate, formate, and acetate anions, and a divalent cation, magnesium, was compared with that of bacterial aquaporin from Halomonas elongata. Water and ion permeability values were determined by measuring the light scattering rates of proteoliposomes containing NirC and aquaporins during their water loss and gain. NirC shows a selective permeability to nitrite and is more resistant to the entry of other anions as compared to aquaporin. The single channel permeability of NirC for nitrite is about 10-fold that of a single aquaporin channel. Both aquaporin and NirC channel proteins were impermeable to MgCl2 and (NH4)2SO4 and their permeability to other tested ions was remarkably lower as compared to nitrite ions. The study also presents the 3D model and channel characteristics of NirC. The translocation channel of E. coli NirC is determined to be larger, and its length is shorter than aquaporin channels. Although the NirC channel throat is more hydrophobic than aquaporin, its water permeability is almost equal to that of aquaporin. The hydrophobic nature of the NirC channel might play an important role in the selective permeability of the channel for nitrite ions.

Nitrite is reduced by nitrite reductase NirB without small subunit NirD in Escherichia coli.

The assimilatory nitrite reductase enzyme NirB and small subunit NirD genes encoded in nir operon in Escherichia coli were cloned into the pET28a vector, and the recombinant enzyme was characterized for the first time. Docking of NirB with NirD, NADH, NO2-, NO3-, and CHO2- was performed using docking modeling programs. Methyl viologen and sodium dithionite were used as electron couples, and the amount of reduced nitrite was measured to calculate enzyme activity. NirB is the main enzyme and shows high activity with or without NirD. However, the inclusion of NirD into the enzyme solution at a ratio of 1NirD:2NirB resulted in 10% higher nitrite reductase activity. The enzyme tends to aggregate in the absence of ß-mercaptoethanol, which causes the conversion of tetrameric NirB to monomeric form, and the NirB enzyme shows its highest activity in monomeric form. The optimum temperature for enzyme activity was 37 °C and the optimum pH was found to be 7.0. Km and Vmax values of NirB were calculated as 9833 µM and 416.67 µmol NO2- reduced min-1 mg-1. Enzyme activity decreased by 55% and 50% in the presence of 100 mM nitrate and formate, respectively. The presence of 25 mM Cd2+ protected the enzyme at room temperature and the enzyme showed 10% higher activity in the presence of cadmium.