The RND-family transporter, HpnN, is required for hopanoid localization to the outer membrane of Rhodopseudomonas palustris TIE-1

Abstract

Rhodopseudomonas palustris TIE-1 is a Gram-negative bacterium that produces structurally diverse hopanoid lipids that are similar to eukaryotic steroids. Its genome encodes several homologues to proteins involved in eukaryotic steroid trafficking. In this study, we explored the possibility that two of these proteins are involved in intracellular hopanoid transport. R. palustris has a sophisticated membrane system comprising outer, cytoplasmic, and inner cytoplasmic membranes. It also divides asymmetrically, producing a mother and swarmer cell. We deleted genes encoding two putative hopanoid transporters that belong to the resistance–nodulation–cell division superfamily. Phenotypic analyses revealed that one of these putative transporters (HpnN) is essential for the movement of hopanoids from the cytoplasmic to the outer membrane, whereas the other (Rpal_4267) plays a minor role. C 30 hopanoids, such as diploptene, are evenly distributed between mother and swarmer cells, whereas hpnN is required for the C 35 hopanoid, bacteriohopanetetrol, to remain localized to the mother cell type. Mutant cells lacking HpnN grow like the WT at 30 °C but slower at 38 °C. Following cell division at 38 °C, the ΔhpnN cells remain connected by their cell wall, forming long filaments. This phenotype may be attributed to hopanoid mislocalization because a double mutant deficient in both hopanoid biosynthesis and transport does not form filaments. However, the lack of hopanoids severely compromises cell growth at higher temperatures more generally. Because hopanoid mutants only manifest a strong phenotype under certain conditions, R. palustris is an attractive model organism in which to study their transport and function.

Hopanoids as functional analogues of cholesterol in bacterial membranes

“…Hopanoids have been identified in the OM of diverse bacteria (29,(43)(44)(45)(46), and two recent studies have reported a covalently linked hopanoid-lipid A compound in rhizobial plantassociated bacteria (47,48). These observations suggest that hopanoid-lipid A ordering may be widespread among hopanoid-producing bacteria.…”

The General Stress Response Factor EcfG Regulates Expression of the C-2 Hopanoid Methylase HpnP in Rhodopseudomonas palustris TIE-1

“…Interestingly, within the putative hopanoid biosynthesis cluster, the consensus (or minimal) motif was only found upstream of the C-2 hopanoid methylase gene hpnP. In addition, two motifs with imperfect spacing were identified upstream of Rpal_4266, which encodes a hypothetical protein and the hopanoid transporter, hpnN (25). Although the hpnP EcfG motif is conserved among all R. palustris strains (see Fig.…”

A Post-Genomic View of the Ecophysiology, Catabolism and Biotechnological Relevance of Sulphate-Reducing Prokaryotes

“…Delivery of the Tn5-RL27 transposon (Larsen et al, 2002) (Fig. 17) by conjugation from the Dap À E. coli BW29427 (Doughty et al, 2011) was shown to produce 2.5 À 10 5 unique insertion mutants overnight. Because the Petri plates needed to obtain this number of mutants would be a significant challenge to handle in an anaerobic chamber, the transposon mutants were not identified as colonies but were enriched in liquid cultures with overnight growth (Fels et al, 2013).…”