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The MM2060 ( cobD ) gene from Methanosarcina mazei strain Gö1 encodes a protein ( Mm CobD) with l-threonine kinase (PduX) and l-threonine- O -3-phosphate decarboxylase (CobD) activities. In addition to the unexpected l-Thr kinase activity, Mm CobD has an extended carboxy-terminal (C-terminal) region annotated as a putative metal-binding zinc finger-like domain. Here, we demonstrate that the C-terminus of Mm CobD is a ferroprotein containing ∼25 non-heme iron atoms per monomer of protein. The absence of the C-terminus substantially reduces, but does not abolish, enzymatic activities in vitro and in vivo . Single-residue substitutions of C-terminal putative Fe-binding cysteinyl and histidinyl residues resulted in the loss of Fe and changes in enzyme activity levels. Salmonella enterica Δ pduX and Δ cobD strains were used as heterologous hosts to assess coenzyme B 12 biosynthesis as a function of 17 Mm CobD variants tested. Some of the latter displayed 5-fold higher enzymatic activity in vitro and enhanced the growth rate of the S. enterica strains that synthesized them. Most of the Mm CobD variants tested were up to 6-fold less active in vitro and supported slow growth rates of the S. enterica strains that synthesized them; some substitutions abolished enzyme activity. Mm CobD exhibited an ultraviolet-visible absorption spectrum consistent with [4Fe-4S] clusters that appeared to be susceptible to oxidation by H 2 O 2 and reduction by sodium dithionite. The presence of FeS clusters in Mm CobD was corroborated by electron paramagnetic resonance and magnetic circular dichroism studies. Collectively, our results suggest that Mm CobD contains one or more diamagnetic [4Fe-4S] 2+ center(s) that may play a structural or regulatory role.

Several of the enzymes involved in the conversion of adenosylcobyric acid (AdoCby) to adenosylcobamide (AdoCba) are yet to be identified and characterized in some cobamide (Cba)-producing prokaryotes. Using a bioinformatics approach, we identified the bluE gene (locus tag RSP_0788) of Rhodobacter sphaeroides 2.4.1 as a putative functional homolog of the L-threonine kinase enzyme (PduX, EC 2.7.1.177) of S. enterica. In AdoCba, (R)-1-aminopropan-2-ol O-phosphate (AP-P) links the nucleotide loop to the corrin ring; most known AdoCba producers derive AP-P from L-Thr-O-3-phosphate (L-Thr-P). Here, we show that RsBluE has L-Thr-independent ATPase activity in vivo and in vitro. We used 31 P-NMR spectroscopy to show that RsBluE generates L-Thr-P at the expense of ATP and is unable to use L-Ser as a substrate. BluE from R. sphaeroides or Rhodobacter capsulatus restored AdoCba biosynthesis in S. enterica ΕpduX and R. sphaeroides ΕbluE mutant strains. R. sphaeroides ΕbluE strains exhibited a decreased pigment phenotype that was restored by complementation with BluE. Finally, phylogenetic analyses revealed that bluE was restricted to the genomes of a few Rhodobacterales that appear to have a preference for a specific form of Cba, namely Coᴽ-(ᴽ-5,6-dimethylbenzimidazolyl-Coᵦ-adenosylcobamide (a.k.a. adenosylcobalamin, AdoCbl; coenzyme B 12 , CoB 12 )., (© 2018 John Wiley & Sons Ltd.)

Cobamides (Cbas) are synthesized by many archaea, but some aspects of Cba biosynthesis in these microorganisms remain unclear. Here, we demonstrate that open reading frame MM2060 in the archaeum Methanosarcina mazei strain Gö1 encodes a bifunctional enzyme with l-threonine- O-3-phosphate (l-Thr-P) decarboxylase (EC 4.1.1.81) and l-Thr kinase activities (EC 2.7.1.177). In Salmonella enterica, where Cba biosynthesis has been extensively studied, the activities mentioned above are encoded by separate genes, namely, cobD and pduX, respectively. The activities associated with the MM2060 protein ( MmCobD) were validated in vitro and in vivo. In vitro, MmCobD used ATP and l-Thr as substrates and generated ADP, l-Thr-P, and ( R)-1-aminopropan-2-ol O-phosphate as products. Notably, MmCobD has a 111-amino acid C-terminal extension of unknown function, which contains a putative metal-binding motif. This C-terminal domain alone did not display activity either in vivo or in vitro. Although the C-terminal MmCobD domain was not required for l-Thr-P decarboxylase or l-Thr kinase activities in vivo, its absence negatively affected both activities. In vitro results suggested that this domain may have a regulatory or substrate-gating role. When purified under anoxic conditions, MmCobD displayed Michaelis-Menten kinetics and had a 1000-fold higher affinity for ATP and a catalytic efficiency 1300-fold higher than that of MmCobD purified under oxic conditions. To the best of our knowledge, MmCobD is the first example of a new class of l-Thr-P decarboxylases that also have l-Thr kinase activity. An archaeal protein with l-Thr kinase activity had not been identified prior to this work.

The installation of dams causes changes to the integrity of rivers and to the water cycle, performing an instrumental role in the organization of biological communities, including that of phytoplankton. In the present study, we analyzed the taxonomic composition of phytoplankton in two hydroelectric reservoirs on the São Francisco River, Itaparica and Xingó reservoirs. Samples were collected at quarterly intervals between December 2007 and September 2009, at 12 sampling stations in each reservoir, totaling 92 samples. We identified 110 species in the Itaparica reservoir and 136 in the Xingó reservoir, of which diatoms followed by green algae, played a major contribution to both reservoirs. Most of the species is rare and/or occasional. In the Itaparica reservoir, there were no very frequent species, although in the Xingó this category was represented by the diatoms Aulacoseira granulata (Ehrenberg) Simonsen and Fragilaria crotonensis Kitton. These results show that, despite the similarity in the composition of phytoplankton, the reservoirs studied certainly differed regarding their environmental conditions.

Brazil has a history of blooms and contamination of freshwater systems by cyanobacterial toxins. The monitoring relevance of toxins from cyanobacteria in reservoirs for public supply is notorious given its high toxicity to mammals, included humans beings. The most recurrent toxins in Brazilian water bodies are microcystins (MC). However, the recent record of cylindrospermopsin (CYN) in northeastern Brazil, Pernambuco state, alerts us to the possibility that this could be escalating. This study reports occurrence of MC and CYN, quantified with ELISA, in 10 reservoirs, devoted to public drinking supply in northeastern Brazil. The composition and quantification of the cyanobacteria community associated with these water bodies is also presented. From 23 samples investigated for the presence of MC, and CYN, 22 and 8 out were positive, respectively. Considering the similarity of the cyanobacteria communities found in reservoirs from Pernambuco, including toxin-producing species associated to MC and CYN, we suggest that geographic spreading can be favored by these factors. These issues emphasize the need for increased monitoring of MC and CYN in drinking supply reservoirs in Brazil.

This study aims to analyse the influence of a cascade of reservoirs on the density, richness and functional groups of phytoplankton in the Contas River, a tropical river of Brazil. This river has two dams along its course, forming the Pedra and Funil reservoirs. Samples were collected over three consecutive years (Dec., 2007 to Dec., 2010) at 28 sampling stations along the river. We identified 198 species and the stretches downstream from the reservoirs showed greater richness. Chlorophyceae, followed by Bacillariophyceae and Cyanophyceae were the dominant groups and highest density was recorded during the rainy season. Overall, a longitudinal pattern in algal densities was found for both seasons, with the lowest values recorded in sections of the Pedra and Funil reservoirs and the highest densities in the downstream sections. Nine functional groups were identified (C, F, J, MP, S1, Sn, Td, Y, Ws); of these, F and J grouped the species with the highest relative abundance during the dry season, while the S1 group, represented by the cyanobacterium Planktothrix agardhii, was dominant in the rainy season. The present study showed a high longitudinal variation in the phytoplankton richness and density, attributed to the hydrological change between the lotic and lentic stretches. Furthermore, the effects of the cascade of reservoirs on phytoplankton, such as reduced density, increased richness and changes in algal associations, were strongly influenced by habitat heterogeneity found in this environment, as well as the rainfall in the region.

Bacteria and archaea use distinct pathways for salvaging exogenous cobinamide (Cbi), a precursor of adenosylcobalamin (coenzyme B(12)). The bacterial pathway depends on a bifunctional enzyme with kinase and guanylyltransferase activities (CobP in aerobic adenosylcobalamin synthesizers) to convert adenosylcobinamide (AdoCbi) to AdoCbi-guanosine diphosphate (AdoCbi-GDP) via an AdoCbi-phosphate intermediate. Archaea lack CobP, and use a different strategy for the synthesis of AdoCbi-GDP. Archaea cleave off the aminopropanol group of AdoCbi using the CbiZ AdoCbi amidohydrolase to generate adenosylcobyric acid, which is converted to AdoCbi-phosphate by the CbiB synthetase, and to AdoCbi-GDP by the CobY guanylyltransferase. We report phylogenetic, in vivo and in vitro evidence that the genome of Rhodobacter sphaeroides encodes functional enzymes for Cbi salvaging systems of both bacterial and archaeal origins. Products of the reactions were identified by high-performance liquid chromatography, UV-visible spectroscopy and bioassay. The cbiZ genes of several bacteria and archaea restored Cbi salvaging in a strain of Salmonella enterica unable to salvage Cbi. Phylogenetic data led us to conclude that CbiZ is an enzyme of archaeal origin that was horizontally transferred to bacteria. Reasons why some bacteria may contain both types of Cbi salvaging systems are discussed.