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.
Hopanes preserved in both modern and ancient sediments are recognized as the molecular fossils of bacteriohopanepolyols, pentacyclic hopanoid lipids. Based on the phylogenetic distribution of hopanoid production by extant bacteria, hopanes have been used as indicators of specific bacterial groups and/or their metabolisms. However, our ability to interpret them ultimately depends on understanding the physiological roles of hopanoids in modern bacteria. Toward this end, we set out to identify genes required for hopanoid biosynthesis in the anoxygenic phototroph Rhodopseudomonas palustris TIE-1 to enable selective control of hopanoid production. We attempted to delete 17 genes within a putative hopanoid biosynthetic gene cluster to determine their role, if any, in hopanoid biosynthesis. Two genes, hpnH and hpnG, are required to produce both bacteriohopanetetrol and aminobacteriohopanetriol, whereas a third gene, hpnO, is required only for aminobacteriohopanetriol production. None of the genes in this cluster are required to exclusively synthesize bacteriohopanetetrol, indicating that at least one other hopanoid biosynthesis gene is located elsewhere on the chromosome. Physiological studies with the different deletion mutants demonstrated that unmethylated and C(30) hopanoids are sufficient to maintain cytoplasmic but not outer membrane integrity. These results imply that hopanoid modifications, including methylation of the A-ring and the addition of a polar head group, may have biologic functions beyond playing a role in membrane permeability.
Abstract2-Methylhopanes, molecular fossils of 2-methylbacteriohopanepolyol (2-MeBHP) lipids, have been proposed as biomarkers for cyanobacteria, and by extension, oxygenic photosynthesis. However, the robustness of this interpretation is unclear, as 2-methylhopanoids occur in organisms besides cyanobacteria and their physiological functions are unknown. As a first step towards understanding the role of 2-MeBHP in cyanobacteria, we examined the expression and intercellular localization of hopanoids in the three cell types of Nostoc punctiforme: vegetative cells, akinetes, and heterocysts. Cultures in which N. punctiforme had differentiated into akinetes contained approximately 10-fold higher concentrations of 2-methylhopanoids than did cultures that contained only vegetative cells. In contrast, 2-methylhopanoids were only present at very low concentrations in heterocysts. Hopanoid production initially increased 3-fold in cells starved of nitrogen but returned to levels consistent with vegetative cells within two weeks. Vegetative and akinete cell types were separated into cytoplasmic, thylakoid, and outer membrane fractions; the increase in hopanoid expression observed in akinetes was due to a 34-fold enrichment of hopanoid content in their outer membrane relative to vegetative cells. Akinetes formed in response either to low light or phosphorus limitation, exhibited the same 2-methylhopanoid localization and concentration, demonstrating that 2-methylhopanoids are associated with the akinete cell type per se. Because akinetes are resting cells that are not photosynthetically active, 2-methylhopanoids cannot be functionally linked to oxygenic photosyntheis in N. punctiforme.
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 are triterpenoids produced mainly by bacteria, are ubiquitous in the environment, and have many important applications as biological markers. A wide variety of related hopanoid structures exists, many of which are polyfunctionalized. These modifications render the hopanoids too involatile for conventional gas chromatography (GC) separation, so require either laborious oxidative cleavage of the functional groups or specialized high temperature (HT) columns. Here we describe the systematic evaluation and optimization of a HT-GC method for the analysis of polyfunctionalized hopanoids and their methylated homologs. Total lipid extracts are derivatized with acetic anhydride and no further treatment or workup is required. We show that acid or base hydrolysis to remove di-and triacylglycerides leads to degradation of several BHP structures. DB-XLB type columns can elute hopanoids up to bacteriohopane-tetrol at 350 °C, with baseline separation of all 2-methyl/desmethyl homologs. DB-5HT type columns can additionally elute bacteriohopaneaminotriol and bacteriohopaneaminotetrol, but do not fully separate 2-methyl/ desmethyl homologs. The method gave 2-to 7-fold higher recovery of hopanoids than oxidative cleavage and can provide accurate quantification of all analytes including 2-methyl hopanoids. By comparing data from mass spectra with those from a flame ionization detector, we show that the mass spectromet (MS) response factors for different hopanoids using either total ion counts or m/z 191 vary substantially. Similarly, 2-methyl ratios estimated from selected-ion data are lower than those from FID by 10-30% for most hopanoids, but higher by ca. 10% for bacteriohopanetetrol. Mass spectra for a broad suite of hopanoids, including 2-methyl homologs, from Rhodopseudomonas palustris are presented, together with the tentative assignment of several new hopanoid degradation products.
Physiological and regulatory mechanisms that allow the alkane-oxidizing bacterium Pseudomonas butanovora to consume C 2 to C 8 alkane substrates via butane monooxygenase (BMO) were examined. Striking differences were observed in response to even-versus odd-chain-length alkanes. Propionate, the downstream product of propane oxidation and of the oxidation of other odd-chain-length alkanes following -oxidation, was a potent repressor of BMO expression. The transcriptional activity of the BMO promoter was reduced with as little as 10 M propionate, even in the presence of appropriate inducers. Propionate accumulated stoichiometrically when 1-propanol and propionaldehyde were added to butane-and ethane-grown cells, indicating that propionate catabolism was inactive during growth on even-chain-length alkanes. In contrast, propionate consumption was induced (about 80 nmol propionate consumed · min ؊1 · mg protein ؊1 ) following growth on the odd-chain-length alkanes, propane and pentane. The induction of propionate consumption could be brought on by the addition of propionate or pentanoate to the growth medium. In a reporter strain of P. butanovora in which the BMO promoter controls -galactosidase expression, only even-chain-length alcohols (C 2 to C 8 ) induced -galactosidase following growth on acetate or butyrate. In contrast, both even-and odd-chain-length alcohols (C 3 to C 7 ) were able to induce -galactosidase following the induction of propionate consumption by propionate or pentanoate.Considerable research has been carried out on the biochemistry and physiology associated with the catabolism of intermediate-chain-length n-alkanes (1,13,14,22,23,35). However, much less is known about the transcriptional regulation of these pathways (27,28,29,34). Insights into the complexity of the transcriptional regulation of alkane utilization have been obtained by studying Pseudomonas putida GPo1 that grows on liquid alkanes (C 5 to C 12 ). The alkane monooxygenase of this bacterium is induced during growth on alkanes and repressed during growth on either complex medium or minimal medium containing simple organic acids (10,30,37,38). The deletion of the gene encoding the Crc protein that is involved in the repression of alkane hydroxylase in complex medium does not affect repression exerted by organic acids (37, 38). To date, the signaling pathway involved in the catabolite repression of the alkane hydroxylase in P. putida GPo1 by complex medium has been well studied (37,38). In contrast, catabolite repression by organic acids has received less attention (10).Recent work from our laboratory has shown that genes coding for a broad-substrate-range alkane monooxygenase, commonly referred to as butane monooxygenase (BMO), are responsible for the ability of Pseudomonas butanovora to grow on alkanes C 2 to C 9 (29). The region immediately 5Ј of the BMO operon in P. butanovora contains a putative sigma 54-dependent promoter (29). Sigma 54-dependent promoters are subject to positive control mediated by enhancer-binding proteins,...
We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium "Pseudomonas butanovora." Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of ϳ20 nmol mg protein ؊1 of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein ؊1 . Oxidation of similar amounts of each DCE isomer (ϳ20 nmol mg protein ؊1 ) produced different negative effects on lactic aciddependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown "P. butanovora" did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.Chlorinated ethenes (CEs), such as perchloroethene (PCE) and trichloroethene (TCE), are common groundwater contaminants that have been linked to liver and kidney damage and are suspected carcinogens (1). Although the microbially driven process of reductive dechlorination can effectively reduce the concentrations of both PCE and TCE under anaerobic groundwater conditions (5,23,27,28), partially dechlorinated products, such as dichloroethenes (DCEs), often persist and disperse in groundwater plumes (35). Recent evidence has emerged for the existence of bacteria that will grow aerobically on 1,2-cis DCE as a sole C source, yet their enrichment is difficult and growth is extremely slow (8). In contrast, it is well documented that hydrocarbon-oxidizing microorganisms are able to degrade DCEs via reductant-driven cooxidative mechanisms (4,6,9,15,17,19,38,39). Unfortunately, the need for the natural hydrocarbon to serve both as enzyme inducer and source of reductant results in competition between the natural substrate and CEs, and cooxidation efficiency is compromised (6,11,21,24,25,37).In this connection, several studies have shown that a combination of TCE and nonhydrocarbon substrates simultaneously induce the genes for toluene oxygenases and provide reductant for TCE oxidation by some toluene-degrading bacterial strains (26,30,36,41). Conceptually, this observation has some appeal for bioremediation because it implies that ...
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