Altered residues in key proteins influence the expression and activity of the nitrogenase complex in an adaptive CO2 fixation-deficient mutant strain of Rhodobacter sphaeroides

Abstract

Previously, the RubisCO-compromised spontaneous adaptive Rhodobacter sphaeroides mutant, strain 16PHC, was shown to derepress the expression of genes that encode the nitrogenase complex under normal repressive conditions. As a result of this adaptation, the active nitrogenase complex restored redox balance, thus allowing strain 16PHC to grow under photoheterotrophic conditions in the absence of an exogenous electron acceptor. A combination of whole genome pyrosequencing and whole genome microarray analyses was employed to identify possible loci responsible for the observed phenotype. Mutations were found in two genes, glnA and nifA, whose products are involved in the regulatory cascade that controls nitrogenase complex gene expression. In addition, a nucleotide reversion within the nifK gene, which encodes a subunit of the nitrogenase complex, was also identified. Subsequent genetic, physiological and biochemical studies revealed alterations that led to derepression of the synthesis of an active nitrogenase complex in strain 16PHC.

Carbon substrate re‐orders relative growth of a bacterium using Mo‐, V‐, or Fe‐nitrogenase for nitrogen fixation

“…). Nase functions in both nitrogen acquisition and electron balance, while the primary role of CO 2 fixation during photoheterotrophic growth is as an electron sink (Hallenbeck et al ., ; Falcone and Tabita, ; Hädicke et al ., ; Farmer et al ., ; Gordon and McKinlay, ). Total electron flux through Nase and Rubisco increases with the growth rate for the wild type, Mo‐Nase and V‐Nase strains, but is always high for the Fe‐Nase strain (Fig.…”

“…1, Arrow E), such that electrons from the organic substrate are fully allocated between biomass, H 2 and CO 2 . Under these conditions, Nase is important both for nutrient acquisition and as an electron sink (Muller, 1933;Hillmer and Gest, 1977;Harwood, 2010a, 2011;Farmer et al, 2014;Gordon and McKinlay, 2014;McCully and McKinlay, 2016). To determine how simultaneous nitrogen acquisition and electron balancing constraints influence growth based on each Nase isoform, we cultured wild type R. palustris and mutant strains known to utilize only a single isoform (hereafter Mo-Nase, V-Nase and Fe-Nase strains, Oda et al, 2005) in diazotrophic photoheterotrophic media with organic substrates of varying oxidation states and assimilation pathways (Table 1) and measured growth, N 2 and CO 2 fixation, H 2 production, biomass composition and global protein abundance patterns.…”

Recent advances in hydrogen production by photosynthetic bacteria

“…In another study, a mutant of Rhodobacter capsulatus having a decreased pigment content was created by inserting pufQ, a bacteriochlorophyll regulatory factor, under the control of the cbb3 promotor. Although it was claimed that total cumulative hydrogen production was increased 27% (from 2955 ml to 3753 ml), substrated conversion efficiency ony increase 5% (from 56.5% to 59.2%) and the maximum rate of hydrogen production actually showed an enormous decrease of 64% (fromm 28.9 to 18.6 ml H2 L -1 h -1 ) [41]. Moreover, in this study the strain that was created had also lost cbb3 function and any effect of this secondary mutation was ignored, ].…”

Phosphoribulokinase mediates nitrogenase-induced carbon dioxide fixation gene repression in Rhodobacter sphaeroides

“…Routine maintenance of Escherichia coli and R. sphaeroides was performed as described previously ( Farmer et al , 2014 ); in some cases Ormerod's media lacked the normal complement of molybdenum and was supplemented with 5 μM sodium tungstate.…”

“…Acetylene reduction assays and Western immunoblot determinations were performed as previously described ( Farmer et al , 2014 ).…”