Hopanoids Confer Robustness to Physicochemical Variability in the Niche of the Plant Symbiont Bradyrhizobium diazoefficiens.

Rhizobia are a group of bacteria that increase soil nitrogen content through symbiosis with legume plants. The soil and symbiotic host are potentially stressful environments, and the soil will likely become even more stressful as the climate changes. Many rhizobia within the clade, like Bradyrhizobium diazoefficiens, possess the genetic capacity to synthesize hopanoids, steroid-like lipids similar in structure and function to cholesterol.

Investigating Abiotic and Biotic Mechanisms of Pyrite Reduction.

Pyrite (FeS) has a very low solubility and therefore has historically been considered a sink for iron (Fe) and sulfur (S) and unavailable to biology in the absence of oxygen and oxidative weathering. Anaerobic methanogens were recently shown to reduce FeS and assimilate Fe and S reduction products to meet nutrient demands. However, the mechanism of FeS mineral reduction and the forms of Fe and S assimilated by methanogens remained unclear.

Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase.

The methanogenic archaeon encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations.

Energy Conservation via Hydrogen Cycling in the Methanogenic Archaeon Methanosarcina barkeri.

Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon is capable of energy conservation via H cycling, based on genetic data that suggest that H is a preferred, but nonessential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H cycling.

Specific hopanoid classes differentially affect free-living and symbiotic states of Bradyrhizobium diazoefficiens.

Unlabelled: A better understanding of how bacteria resist stresses encountered during the progression of plant-microbe symbioses will advance our ability to stimulate plant growth. Here, we show that the symbiotic system comprising the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens and the legume Aeschynomene afraspera requires hopanoid production for optimal fitness. While methylated (2Me) hopanoids contribute to growth under plant-cell-like microaerobic and acidic conditions in the free-living state, they are dispensable during symbiosis.

Covalently linked hopanoid-lipid A improves outer-membrane resistance of a Bradyrhizobium symbiont of legumes.

Lipopolysaccharides (LPSs) are major components of the outer membrane of Gram-negative bacteria and are essential for their growth and survival. They act as a structural barrier and play an important role in the interaction with eukaryotic hosts. Here we demonstrate that a photosynthetic Bradyrhizobium strain, symbiont of Aeschynomene legumes, synthesizes a unique LPS bearing a hopanoid covalently attached to lipid A.

The general stress response factor EcfG regulates expression of the C-2 hopanoid methylase HpnP in Rhodopseudomonas palustris TIE-1.

Lipid molecules preserved in sedimentary rocks facilitate the reconstruction of events that have shaped the evolution of the Earth's biosphere. A key limitation for the interpretation of many of these molecular fossils is that their biological roles are still poorly understood. Here, we use Rhodopseudomonas palustris TIE-1 to identify factors that induce biosynthesis of 2-methyl hopanoids (2-MeBHPs), progenitors of 2-methyl hopanes, one of the most abundant biomarkers in the rock record.

Genetic methods for methanosarcina species.

Unlike most methanogenic microorganisms, Methanosarcina species are capable of utilizing a variety of growth substrates, a trait that greatly simplifies genetic analysis of the methanogenic process. The genetic tools and techniques discussed in this chapter form the basis for all genetic experiments in Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro, two methanogens that are routinely used as model organisms for genetic experiments. Based on a number of reports, it is likely that they are portable to other Methanosarcina species, and perhaps to other methanogens as well.

Hydrogen is a preferred intermediate in the energy-conserving electron transport chain of Methanosarcina barkeri.

Methanogens use an unusual energy-conserving electron transport chain that involves reduction of a limited number of electron acceptors to methane gas. Previous biochemical studies suggested that the proton-pumping F(420)H(2) dehydrogenase (Fpo) plays a crucial role in this process during growth on methanol. However, Methanosarcina barkeri Delta fpo mutants constructed in this study display no measurable phenotype on this substrate, indicating that Fpo plays a minor role, if any.

Regulation of putative methyl-sulphide methyltransferases in Methanosarcina acetivorans C2A.

The regulation of the Methanosarcina acetivorans mtsD, mtsF and mtsH genes, which encode putative corrinoid/methyltransferase isozymes involved in methylsulphide metabolism, was examined by a variety of methods, suggesting that their expression is regulated at both the transcriptional and post-transcriptional levels. Transcripts of all three genes, measured by quantitative reverse transcription PCR, were shown to be most abundant during growth on methanol with dimethylsulphide (DMS). Transcript levels were also high in media with CO or methylamines, but much lower with methanol.

Differences in hydrogenase gene expression between Methanosarcina acetivorans and Methanosarcina barkeri.

Methanosarcina acetivorans C2A encodes three putative hydrogenases, including one cofactor F(420)-linked (frh) and two methanophenazine-linked (vht) enzymes. Comparison of the amino acid sequences of these putative hydrogenases to those of Methanosarcina barkeri and Methanosarcina mazei shows that each predicted subunit contains all the known residues essential for hydrogenase function. The DNA sequences upstream of the genes in M.

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species.

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage phiC31. Host strains expressing the phiC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors.