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

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. C30 hopanoids, such as diploptene, are evenly distributed between mother and swarmer cells, whereas hpnN is required for the C35 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

Cloning, expression, and sequencing of squalene-hopene cyclase, a key enzyme in triterpenoid metabolism.

The pentacyclic hopanoids, a class of eubacterial lipids, are synthesized by squalene-hopene cyclase and side chain-elongating enzymes. With the aid of DNA probes based on the amino-terminal sequence of purified squalene-hopene cyclase from Bacillus acidocaldarius, clones of Escherichia coli that express this enzyme in the cytoplasmic membrane were isolated. According to the DNA sequence, the cyclase contained 627 amino acids with a molecular mass of 69,473 Da. A high percentage of the amino acids were basic. No significant similarity to existing sequenced proteins was found