Inferring pathways leading to organic-sulfur mineralization in the Bacillales.

Microbial organic sulfur mineralization to sulfate in terrestrial systems is poorly understood. The process is often missing in published sulfur cycle models. Studies on microbial sulfur cycling have been mostly centered on transformations of inorganic sulfur, mainly on sulfate-reducing and inorganic sulfur-oxidizing bacteria. Nevertheless, organic sulfur constitutes most sulfur in soils. Recent reports demonstrate that the mobilization of organic-bound-sulfur as sulfate in terrestrial environments occurs preferentially under high temperatures and thermophilic Firmicutes bacteria play a major role in the process, carrying out dissimilative organic-sulfur oxidation. So far, the determinant metabolic reactions of such activity have not been evaluated. Here, in silico analysis was performed on the genomes of sulfate-producing thermophilic genera and mesophilic low-sulfate producers, revealing that highest sulfate production is related to the simultaneous presence of metabolic pathways leading to sulfite synthesis, similar to the ones found in mammalian cells. Those pathways include reverse transsulfuration reactions (tightly associated with methionine cycling), and the presence of aspartate aminotransferases (ATs) with the potential of 3-sulfinoalanine AT and cysteine AT activity, which ultimately leads to sulfite production. Sulfite is oxidized to sulfate by sulfite oxidase, this enzyme is determinant in sulfate synthesis, and it is absent in many mesophiles.

Influence of Temperature and Copper on Oxalobacteraceae in Soil Enrichments.

ß-Proteobacteria is one of the most abundant phylum in soils, including autotrophic and heterotrophic ammonium-consumers with relevance in N circulation in soils. The effects of high-temperature events and phytosanitary treatments, such as copper amendments, on soil bacterial communities relevant to N-cycling remain to be studied. As an example, South Portugal soils are seasonally exposed to high-temperature periods, the temperature at the upper soil layers can reach over 40 °C. Here, we evaluated the dynamics of mesophilic and thermophilic bacteria from a temperate soil, in particular of heterotrophic ß-Proteobacteria, regarding the ammonium equilibrium, as a function of temperature and copper treatment. Soil samples were collected from an olive orchard in southern Portugal. Selective enrichments were performed from samples under different conditions of temperature (30 and 50 °C) and copper supplementation (100 and 500 µM) in order to mime seasonal variations and phytosanitary treatments. Changes in the microbial communities under these conditions were examined by denaturing gradient gel electrophoresis, a molecular fingerprint technique. At moderate temperature--30 °C--either without or with copper addition, dominant members were identified as different strains belonging to genus Massilia, a genus of the Oxalobacteraceae (ß-Proteobacteria), while at 50 °C, members of the Brevibacillus genus, phylum Firmicutes were also represented. Ammonium production during bacterial growth at moderate and high temperatures was not affected by copper addition. Results indicate that both copper and temperature selected specific tolerant bacterial strains with consequences for N-cycling in copper-treated orchards.

Soil Thermophiles and Their Extracellular Enzymes: A Set of Capabilities Able to Provide Significant Services and Risks.

During this century, a number of reports have described the potential roles of thermophiles in the upper soil layers during high-temperature periods. This study evaluates the capabilities of these microorganisms and proposes some potential consequences and risks associated with the activity of soil thermophiles. They are active in organic matter mineralization, releasing inorganic nutrients (C, S, N, P) that otherwise remain trapped in the organic complexity of soil. To process complex organic compounds in soils, these thermophiles require extracellular enzymes to break down large polymers into simple compounds, which can be incorporated into the cells and processed. Soil thermophiles are able to adapt their extracellular enzyme activities to environmental conditions. These enzymes can present optimum activity under high temperatures and reduced water content. Consequently, these microorganisms have been shown to actively process and decompose substances (including pollutants) under extreme conditions (i.e., desiccation and heat) in soils. While nutrient cycling is a highly beneficial process to maintain soil service quality, progressive warming can lead to excessive activity of soil thermophiles and their extracellular enzymes. If this activity is too high, it may lead to reduction in soil organic matter, nutrient impoverishment and to an increased risk of aridity. This is a clear example of a potential effect of future predicted climate warming directly caused by soil microorganisms with major consequences for our understanding of ecosystem functioning, soil health and the risk of soil aridity.

The Plant Growth-Promoting Potential of Halotolerant Bacteria Is Not Phylogenetically Determined: Evidence from Two Bacillus megaterium Strains Isolated from Saline Soils Used to Grow Wheat.

(1) Background: Increasing salinity, further potentiated by climate change and soil degradation, will jeopardize food security even more. Therefore, there is an urgent need for sustainable agricultural practices capable of maintaining high crop yields despite adverse conditions. Here, we tested if wheat, a salt-sensitive crop, could be a good reservoir for halotolerant bacteria with plant growth-promoting (PGP) capabilities. (2) Methods: We used two agricultural soils from Algeria, which differ in salinity but are both used to grow wheat. Soil halotolerant bacterial strains were isolated and screened for 12 PGP traits related to phytohormone production, improved nitrogen and phosphorus availability, nutrient cycling, and plant defence. The four 'most promising' halotolerant PGPB strains were tested hydroponically on wheat by measuring their effect on germination, survival, and biomass along a salinity gradient. (3) Results: Two halotolerant bacterial strains with PGP traits were isolated from the non-saline soil and were identified as Bacillus subtilis and Pseudomonas fluorescens, and another two halotolerant bacterial strains with PGP traits were isolated from the saline soil and identified as B. megaterium. When grown under 250 mM of NaCl, only the inoculated wheat seedlings survived. The halotolerant bacterial strain that displayed all 12 PGP traits and promoted seed germination and plant growth the most was one of the B. megaterium strains isolated from the saline soil. Although they both belonged to the B. megaterium clade and displayed a remarkable halotolerance, the two bacterial strains isolated from the saline soil differed in two PGP traits and had different effects on plant performance, which clearly shows that PGP potential is not phylogenetically determined. (4) Conclusions: Our data highlight that salt-sensitive plants and non-saline soils can be reservoirs for halotolerant microbes with the potential to become effective and sustainable strategies to improve plant tolerance to salinity. However, these strains need to be tested under field conditions and with more crops before being considered biofertilizer candidates.

On a Non-Discrete Concept of Prokaryotic Species.

The taxonomic concept of species has received continuous attention. A microbial species as a discrete box contains a limited number of highly similar microorganisms assigned to that taxon, following a polyphasic approach. In the 21st Century, with the advancements of sequencing technologies and genomics, the existence of a huge prokaryotic diversity has become well known. At present, the prokaryotic species might no longer have to be understood as discrete values (such as 1 or 2, by homology to Natural numbers); rather, it is expected that some microorganisms could be potentially distributed (according to their genome features and phenotypes) in between others (such as decimal numbers between 1 and 2; real numbers). We propose a continuous species concept for microorganisms, which adapts to the current knowledge on the huge diversity, variability and heterogeneity existing among bacteria and archaea. Likely, this concept could be extended to eukaryotic microorganisms. The continuous species concept considers a species to be delimited by the distance between a range of variable features following a Gaussian-type distribution around a reference organism (i.e., its type strain). Some potential pros and cons of a continuous concept are commented on, offering novel perspectives on our understanding of the highly diversified prokaryotic world, thus promoting discussion and further investigation in the field.

Nitric Oxide Accumulation: The Evolutionary Trigger for Phytopathogenesis.

Many publications highlight the importance of nitric oxide (NO) in plant-bacteria interactions, either in the promotion of health and plant growth or in pathogenesis. However, the role of NO in the signaling between bacteria and plants and in the fate of their interaction, as well as the reconstruction of their interactive evolution, remains largely unknown. Despite the complexity of the evolution of life on Earth, we explore the hypothesis that denitrification and aerobic respiration were responsible for local NO accumulation, which triggered primordial antagonistic biotic interactions, namely the first phytopathogenic interactions. N-oxides, including NO, could globally accumulate via lightning synthesis in the early anoxic ocean and constitute pools for the evolution of denitrification, considered an early step of the biological nitrogen cycle. Interestingly, a common evolution may be proposed for components of denitrification and aerobic respiration pathways, namely for NO and oxygen reductases, a theory compatible with the presence of low amounts of oxygen before the great oxygenation event (GOE), which was generated by Cyanobacteria. During GOE, the increase in oxygen caused the decrease of Earth's temperature and the consequent increase of oxygen dissolution and availability, making aerobic respiration an increasingly dominant trait of the expanding mesophilic lifestyle. Horizontal gene transfer was certainly important in the joint expansion of mesophily and aerobic respiration. First denitrification steps lead to NO formation through nitrite reductase activity, and NO may further accumulate when oxygen binds NO reductase, resulting in denitrification blockage. The consequent transient NO surplus in an oxic niche could have been a key factor for a successful outcome of an early denitrifying prokaryote able to scavenge oxygen by NO/oxygen reductase or by an independent heterotrophic aerobic respiration pathway. In fact, NO surplus could result in toxicity causing "the first disease" in oxygen-producing Cyanobacteria. We inspected in bacteria the presence of sequences similar to the NO-producing nitrite reductase nirS gene of Thermus thermophilus, an extreme thermophilic aerobe of the Thermus/Deinococcus group, which constitutes an ancient lineage related to Cyanobacteria. In silico analysis revealed the relationship between the presence of nirS genes and phytopathogenicity in Gram-negative bacteria.

Structure at 1.3 A resolution of Rhodothermus marinus caa(3) cytochrome c domain.

The cytochrome c domain of subunit II from the Rhodothermus marinus caa(3) HiPIP:oxygen oxidoreductase, a member of the superfamily of heme-copper-containing terminal oxidases, was produced in Escherichia coli and characterised. The recombinant protein, which shows the same optical absorption and redox properties as the corresponding domain in the holo enzyme, was crystallized and its structure was determined to a resolution of 1.3 A by the multiwavelength anomalous dispersion (MAD) technique using the anomalous dispersion of the heme iron atom. The model was refined to final R(cryst) and R(free) values of 13.9% and 16.7%, respectively. The structure reveals the insertion of two short antiparallel beta-strands forming a small beta-sheet, an interesting variation of the classical all alpha-helical cytochrome c fold. This modification appears to be common to all known caa(3)-type terminal oxidases, as judged by comparative modelling and by analyses of the available amino acid sequences for these enzymes. This is the first high-resolution crystal structure reported for a cytochrome c domain of a caa(3)-type terminal oxidase. The R.marinus caa(3) uses HiPIP as the redox partner. The calculation of the electrostatic potential at the molecular surface of this extra C-terminal domain provides insights into the binding to its redox partner on one side and its interaction with the remaining subunit II on the other side.

The adaptive genome of Desulfovibrio vulgaris Hildenborough.

Peculiar attributes revealed by sequencing the genome of Desulfovibrio vulgaris Hildenborough are analyzed, particularly in relation to the presence of a phosphotransferase system (PTS). The PTS is a typical bacterial carbohydrate transport system functioning via group translocation. Novel avenues for investigations are proposed emphasizing the metabolic diversity of D. vulgaris Hildenborough, especially the likely utilization of mannose-type sugars. Comparative analysis with PTS from other Gram-negative and Gram-positive bacteria indicates regulatory functions for the PTS of D. vulgaris Hildenborough, including catabolite repression and inducer exclusion. Chemotaxis towards PTS substrates is considered. Evidence suggests that this organism may not be a strict anaerobic sulfate reducer typical of the ocean, but a versatile organism capable of bidirectional transmigration and adaptation to both water and terrestrial environments.

A cloning strategy to obtain recombinant proteins with identical primary structure to the native forms.

Recombinant gene strategies using fusion tags for purification are essential procedures to obtain large protein quantities. However, most cloning systems result in recombinant proteins with added amino acids inexistent in their native forms which can lead to significant changes in protein properties. An original and simple cloning strategy is proposed to obtain proteins identical in amino acid sequence to the native proteins.

Presence and expression of terminal oxygen reductases in strictly anaerobic sulfate-reducing bacteria isolated from salt-marsh sediments.

In the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough genes were found encoding membrane terminal oxygen reductases of two types: a cytochrome c oxidase and a cytochrome bd oxidase, both enzymes are terminal oxidases typical of facultative or aerobic microorganisms (Heidelberg JF, et al., The genome sequence of the anaerobic, sulfate-reducing bacterium D. vulgaris Hildenborough. Nat Biotechnol 2004; 22: 554-9). To apprehend the presence of both oxidases in other sulfate-reducing bacteria (SRB), several assays were performed on isolates recovered from salt-marsh sediments in Portugal, representative of the different phylogenetic groups identified. Hybridization and PCR experiments for DNA sequencing were performed on the chosen isolates. Primers were selected to amplify conserved regions of cytochrome c oxidases and cytochrome bd oxidases taking into consideration alignment of corresponding subunit I sequences. The results showed that both oxidase genes are present on the chromosome of several isolates characterized as Desulfovibrio. These genes were shown to be transcribed, as demonstrated by Reverse Transcriptase-PCR experiments on total RNA. In order to assess the relative contribution of each oxidase to oxygen consumption, oxygen uptake was measured for each isolate and further characterized by the effect of cyanide on oxygen consumption. It was concluded that cytochrome bd oxidase was the terminal membrane oxygen reductase allowing oxygen consumption. In addition, it was observed that isolates containing cytochrome bd oxidase had higher resistance to air exposure, suggesting an important role of this enzyme in survival to air exposure. The pattern for the presence of oxygen reductase genes was compared to the physiological pattern of substrate use, which was determined for each isolate. Salinity tolerance, pH and temperature growth of each isolate were also analyzed.