Packing of the cyclic nucleotides in the crystal structure of 3’,5’ cGMP provides with better steric conditions for the transphophorylation leading to oligonucleotides than that of 3’,5’ cAMP [10,11].

<p>Packing of the cyclic nucleotides in the crystal structure of 3’,5’ cGMP provides with better steric conditions for the transphophorylation leading to oligonucleotides than that of 3’,5’ cAMP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165723#pone.0165723.ref010" target="_blank">10</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165723#pone.0165723.ref011" target="_blank">11</a>].</p

Hydrolysis of A₂₄ by water or formamide.

<p>Panel A: 20% acrylamide PAGE analysis of 5’ labeled A₂₄, hydrolyzed in water (Lane 1, see text) or formamide (Lane 2, see text). The product formed from A₂₄ (not phosphorylated at 5’) upon digestion in formamide was analyzed by MALDI in the negative ion mode. Panel B shows, as examples, the MALDI profiles corresponding to the trimer and tetramer families produced by formamide hydrolysis. The hydrolysis produces: (i) a species with <i>m/z</i> = 986.21 corresponding to the primary product of the cleavage at 3’ = ApApA>p, (ii) a species with <i>m/z</i> = 1004.2 (986 + 18) derived from its opening to ApApA_p, (iii) a species with <i>m/z</i> = 924.25 (1004–80) derived by the phosphate-loss of this latter = ApApA. Analogous products can be derived for the tetramer: 1315.27 (ApApApA>p) opened to 1333.28 (ApApApA_p) and dephosphorylated to 1253.31 (ApApApA). The fully dephosphorylated species migrates in the gel faster than the phoshorylated ones. Likewise, in MALDI this species gives a signal at lower <i>m/z</i> value. Longer oligomers behave similarly. Thus, using a reference ladder produced this way for the interpretation of the 3’,5’ cAMP oligomerization products is fully justified. Note that in contrast to the oligomers detected by MALDI, the oligomers detected by PAGE always carry a ³²P-phosphate group at 5’.</p

Non-Enzymatic Oligomerization of 3’, 5’ Cyclic AMP

<div><p>Recent studies illustrate that short oligonucleotide sequences can be easily produced from nucleotide precursors in a template-free non-enzymatic way under dehydrating conditions, i.e. using essentially dry materials. Here we report that 3’,5’ cyclic AMP may also serve as a substrate of the reaction, which proceeds under moderate conditions yet with a lower efficiency than the previously reported oligomerization of 3’,5’ cyclic GMP. Optimally the oligomerization requires (i) a temperature of 80°C, (ii) a neutral to alkaline environment and (iii) a time on the order of weeks. Differences in the yield and required reaction conditions of the oligomerizations utilizing 3’,5’ cGMP and cAMP are discussed in terms of the crystal structures of the compounds. Polymerization of 3’,5’ cyclic nucleotides, whose paramount relevance in a prebiotic chemistry context has been widely accepted for decades, supports the possibility that the origin of extant genetic materials might have followed a direct uninterrupted path since its very beginning, starting from non-elaborately pre-activated monomer compounds and simple reactions.</p></div

Comparison of the bond distances and angles characterizing the steric conditions for the in-line attack of O3’ at the next phosphate in the crystal structure of 3’,5’ cGMP and 3’,5’ cAMP [10,11].

<p>Comparison of the bond distances and angles characterizing the steric conditions for the in-line attack of O3’ at the next phosphate in the crystal structure of 3’,5’ cGMP and 3’,5’ cAMP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165723#pone.0165723.ref010" target="_blank">10</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165723#pone.0165723.ref011" target="_blank">11</a>].</p

MALDI analysis of the reaction products obtained by reacting 3’,5’ cAMP for 60 days, pH 7.0, 80°C.

<p>Panel A: the overall pattern recorded for the reacted 3’,5’ cAMP sample. Panel B: spectrum of the unreacted sample. Panels C and D show the blow-ups of the relevant areas from panels A and B, respectively.</p

3’, 5’ cAMP oligomerization as a function of temperature and of pH.

<p>Panel A: 3’,5’ cAMP oligomerization as a function of temperature, from 25 to 85°C (lanes 2 to 10, respectively), pH 7.0, 24 hrs. Panel B: 3’,5’ cAMP oligomerization as a function of pH, from 2.7 to 10.6 (lanes 2 to 9, respectively). The dry buffered material (for preparation see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165723#sec004" target="_blank">Methods</a> section), was reacted at 80°Cfor 6 hrs, purposely in sub-optimal time conditions. U = untreated.</p

PAGE-analysis of the 3’, 5’ cAMP oligomerization products as a function of time at pH 10.6.

<p>Panel A. The material obtained by drying the initial 3’,5’ cAMP solution (pH 10.6) was reacted at 80°C for the indicated times (between 1 and 312 hours, see lanes 3 to 6). U = untreated (see lane 2). Panel B shows a blow-up analysis of the time periods encompassed between 1 and 1440 minutes (lanes from 2 to 9). The plot shows that the reaction rate slows down after 5 hours. Markers (M): formamide-digested 5’-labelled A₂₄. For the attribution of the molecular species involved, see the text. Gels with few samples were routinely preferred in order to avoid “gel smiling” effects due to the relatively large concentrations of materials loaded, present in the oligomerization assays. Therefore, the analysis was usually split in small-number groups. Acrylamide = 20%. The salts migration front, below which no attribution is possible, is indicated. The interpolating line in the plot is drawn as a guide to the eye here and in the following figures.</p

In the chain extension step of the oligomerization of 2’,3’ and 3’,5’ cyclic nucleotides an intramolecular phosphodiester linkage is replaced with an intermolecular one.

<p>In the chain extension step of the oligomerization of 2’,3’ and 3’,5’ cyclic nucleotides an intramolecular phosphodiester linkage is replaced with an intermolecular one.</p

Image_5_Description and Comparative Genomics of Macrococcus caseolyticus subsp. hominis subsp. nov., Macrococcus goetzii sp. nov., Macrococcus epidermidis sp. nov., and Macrococcus bohemicus sp. nov., Novel Macrococci From Human Clinical Material With Virulence Potential and Suspected Uptake of Foreign DNA by Natural Transformation.PDF

<p>The genus Macrococcus is a close relative of the genus Staphylococcus. Whilst staphylococci are widespread as human pathogens, macrococci have not yet been reported from human clinical specimens. Here we investigated Gram-positive and catalase-positive cocci recovered from human clinical material and identified as Macrococcus sp. by a polyphasic taxonomic approach and by comparative genomics. Relevant phenotypic, genotypic and chemotaxonomic methods divided the analyzed strains into two separate clusters within the genus Macrococcus. Comparative genomics of four representative strains revealed enormous genome structural plasticity among the studied isolates. We hypothesize that high genomic variability is due to the presence of a com operon, which plays a key role in the natural transformation of bacilli and streptococci. The possible uptake of exogenous DNA by macrococci can contribute to a different mechanism of evolution from staphylococci, where phage-mediated horizontal gene transfer predominates. The described macrococcal genomes harbor novel plasmids, genomic islands and islets, as well as prophages. Capsule gene clusters, intracellular protease, and a fibronectin-binding protein enabling opportunistic pathogenesis were found in all four strains. Furthermore, the presence of a CRISPR-Cas system with 90 spacers in one of the sequenced genomes corresponds with the need to limit the burden of foreign DNA. The highly dynamic genomes could serve as a platform for the exchange of virulence and resistance factors, as was described for the methicillin resistance gene, which was found on the novel composite SCCmec-like element containing a unique mec gene complex that is considered to be one of the missing links in SCC evolution. The phenotypic, genotypic, chemotaxonomic and genomic results demonstrated that the analyzed strains represent one novel subspecies and three novel species of the genus Macrococcus, for which the names Macrococcus caseolyticus subsp. hominis subsp. nov. (type strain CCM 7927^T = DSM 103682^T), Macrococcus goetzii sp. nov. (type strain CCM 4927^T = DSM 103683^T), Macrococcus epidermidis sp. nov. (type strain CCM 7099^T = DSM 103681^T), and Macrococcus bohemicus sp. nov. (type strain CCM 7100^T = DSM 103680^T) are proposed. Moreover, a formal description of Macrococcus caseolyticus subsp. caseolyticus subsp. nov. and an emended description of the genus Macrococcus are provided.</p

Table_1_Description and Comparative Genomics of Macrococcus caseolyticus subsp. hominis subsp. nov., Macrococcus goetzii sp. nov., Macrococcus epidermidis sp. nov., and Macrococcus bohemicus sp. nov., Novel Macrococci From Human Clinical Material With Virulence Potential and Suspected Uptake of Foreign DNA by Natural Transformation.DOCX

<p>The genus Macrococcus is a close relative of the genus Staphylococcus. Whilst staphylococci are widespread as human pathogens, macrococci have not yet been reported from human clinical specimens. Here we investigated Gram-positive and catalase-positive cocci recovered from human clinical material and identified as Macrococcus sp. by a polyphasic taxonomic approach and by comparative genomics. Relevant phenotypic, genotypic and chemotaxonomic methods divided the analyzed strains into two separate clusters within the genus Macrococcus. Comparative genomics of four representative strains revealed enormous genome structural plasticity among the studied isolates. We hypothesize that high genomic variability is due to the presence of a com operon, which plays a key role in the natural transformation of bacilli and streptococci. The possible uptake of exogenous DNA by macrococci can contribute to a different mechanism of evolution from staphylococci, where phage-mediated horizontal gene transfer predominates. The described macrococcal genomes harbor novel plasmids, genomic islands and islets, as well as prophages. Capsule gene clusters, intracellular protease, and a fibronectin-binding protein enabling opportunistic pathogenesis were found in all four strains. Furthermore, the presence of a CRISPR-Cas system with 90 spacers in one of the sequenced genomes corresponds with the need to limit the burden of foreign DNA. The highly dynamic genomes could serve as a platform for the exchange of virulence and resistance factors, as was described for the methicillin resistance gene, which was found on the novel composite SCCmec-like element containing a unique mec gene complex that is considered to be one of the missing links in SCC evolution. The phenotypic, genotypic, chemotaxonomic and genomic results demonstrated that the analyzed strains represent one novel subspecies and three novel species of the genus Macrococcus, for which the names Macrococcus caseolyticus subsp. hominis subsp. nov. (type strain CCM 7927^T = DSM 103682^T), Macrococcus goetzii sp. nov. (type strain CCM 4927^T = DSM 103683^T), Macrococcus epidermidis sp. nov. (type strain CCM 7099^T = DSM 103681^T), and Macrococcus bohemicus sp. nov. (type strain CCM 7100^T = DSM 103680^T) are proposed. Moreover, a formal description of Macrococcus caseolyticus subsp. caseolyticus subsp. nov. and an emended description of the genus Macrococcus are provided.</p