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. 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.

Product and product-independent induction of butane oxidation in Pseudomonas butanovora.

Pseudomonas butanovora grows on butane by means of an inducible soluble alkane monooxygenase (sBMO). The induction of sBMO was studied using the wild type and a sBMO reporter strain. The reporter strain has the lacZ::kan cassette inserted into bmoX, the gene that encodes the alpha-subunit of the hydroxylase of sBMO. The beta-galactosidase activity in the reporter strain was not induced by butane, but was induced by 1-butanol and butyraldehyde. P. butanovora expressed sBMO product-independent activity at 3.0+/-1 nmol ethylene oxide min(-1) mg protein(-1) in stationary phase. The sBMO product-independent activity likely primes the expression of sBMO by butane.

Probing the subcellular localization of hopanoid lipids in bacteria using NanoSIMS.

The organization of lipids within biological membranes is poorly understood. Some studies have suggested lipids group into microdomains within cells, but the evidence remains controversial due to non-native imaging techniques. A recently developed NanoSIMS technique indicated that sphingolipids group into microdomains within membranes of human fibroblast cells. We extended this NanoSIMS approach to study the localization of hopanoid lipids in bacterial cells by developing a stable isotope labeling method to directly detect subcellular localization of specific lipids in bacteria with ca. 60 nm resolution. Because of the relatively small size of bacterial cells and the relative abundance of hopanoid lipids in membranes, we employed a primary (2)H-label to maximize our limit of detection. This approach permitted the analysis of multiple stable isotope labels within the same sample, enabling visualization of subcellular lipid microdomains within different cell types using a secondary label to mark the growing end of the cell. Using this technique, we demonstrate subcellular localization of hopanoid lipids within alpha-proteobacterial and cyanobacterial cells. Further, we provide evidence of hopanoid lipid domains in between cells of the filamentous cyanobacterium Nostoc punctiforme. More broadly, our method provides a means to image lipid microdomains in a wide range of cell types and test hypotheses for their functions in membranes.

The continuing puzzle of the great oxidation event.

The rise of atmospheric O(2) was a milestone in the history of life. Although O(2) itself is not a climate-active gas, its appearance would have removed a methane greenhouse present on the early Earth and potentially led to dramatic cooling. Moreover, by fundamentally altering the biogeochemical cycles of C, N, S and Fe, its rise first in the atmosphere and later in the oceans would also have had important indirect effects on Earth's climate. Here, we summarize major lines of evidence from the geological literature that pertain to when and how O(2) first appeared in significant amounts in the atmosphere. On the early Earth, atmospheric O(2) would initially have been very low, probably <10(-5) of the present atmospheric level. Around 2.45 billion years ago, atmospheric O(2) rose suddenly in what is now termed the Great Oxidation Event. While the rise of oxygen has been the subject of considerable attention by Earth scientists, several important aspects of this problem remain unresolved. Our goal in this review is to provide a short summary of the current state of the field, and make the case that future progress towards solving the riddle of oxygen will benefit greatly from the involvement of molecular biologists.

Involvement of BmoR and BmoG in n-alkane metabolism in 'Pseudomonas butanovora'.

'Pseudomonas butanovora' uses an alcohol-inducible alkane monooxygenase (BMO) to grow on C(2)-C(9) n-alkanes. Five ORFs were identified flanking the BMO structural genes. Two of the ORFs, bmoR, encoding a putative sigma(54)-transcriptional regulator BmoR, and bmoG, encoding a putative GroEL chaperonin BmoG, were analysed by gene-inactivation experiments. The BmoR-deficient mutant grew at slower growth rates than the wild-type on C(2)-C(5) n-alkanes and showed little to no growth on C(6)-C(8) n-alkanes within 7 days. A BmoR-deficient mutant was constructed in the 'P. butanovora' bmoX : : lacZ reporter strain and used to test whether bmoR was involved in bmoX induction after growth on C(2)-C(8) carbon sources. In acetate- or lactate-grown cells, C(2)-C(8) n-alcohols failed to induce beta-galactosidase activity. In contrast, in propionate-, butyrate- or pentanoate-grown cells, n-butanol induced approximately 45 % of the beta-galactosidase activity observed in the control bmoX : : lacZ strain. In propionate-grown cells, C(2)-C(5) n-alcohols induced beta-galactosidase activity, whereas C(7) and C(8) n-alcohols did not. BmoR may act as a sigma(54)-transcriptional regulator of bmo that is controlled by the n-alcohol produced in the alkane oxidation. During growth on short-chain-length fatty acids, however, another BMO regulatory system seems to be activated to promote transcription of bmo by short-chain-length alcohols (i.e.

Evidence for modified mechanisms of chloroethene oxidation in Pseudomonas butanovora mutants containing single amino acid substitutions in the hydroxylase alpha-subunit of butane monooxygenase.

The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the alpha-subunit of butane monooxygenase hydroxylase (BMOH-alpha) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain G113N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain G113N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent O(2) uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH-alpha mutants might allow insights into the catalytic mechanism of BMO to be obtained.