HU binds and folds single-stranded DNA

The nucleoid-associated protein HU plays an important role in bacterial nucleoid organization and is involved in numerous processes including transposition, recombination and DNA repair. We show here that HU binds specifically DNA containing mismatched region longer than 3 bp as well as DNA bulges. HU binds single-stranded DNA (ssDNA) in a binding mode that is reminiscent but different from earlier reported specific HU interactions with double-helical DNA lesions. An HU dimer requires 24 nt of ssDNA for initial binding, and 12 nt of ssDNA for each additional dimer binding. In the presence of equimolar amounts of HU dimer and DNA, the ssDNA molecule forms an U-loop (hairpin-like) around the protein, providing contacts with both sides of the HU body. This mode differs from the binding of the single-strand-binding protein (SSB) to ssDNA: in sharp contrast to SSB, HU binds ssDNA non-cooperatively and does not destabilize double-helical DNA. Furthermore HU has a strong preference for poly(dG), while binding to poly(dA) is the weakest. HU binding to ssDNA is probably important for its capacity to cover and protect bacterial DNA both intact and carrying lesions

Metabolomic Analysis of Three Mollicute Species

<div><p>We present a systematic study of three bacterial species that belong to the class Mollicutes, the smallest and simplest bacteria, <i>Spiroplasma melliferum</i>, <i>Mycoplasma gallisepticum</i>, and <i>Acholeplasma laidlawii</i>. To understand the difference in the basic principles of metabolism regulation and adaptation to environmental conditions in the three species, we analyzed the metabolome of these bacteria. Metabolic pathways were reconstructed using the proteogenomic annotation data provided by our lab. The results of metabolome, proteome and genome profiling suggest a fundamental difference in the adaptation of the three closely related Mollicute species to stress conditions. As the transaldolase is not annotated in Mollicutes, we propose variants of the pentose phosphate pathway catalyzed by annotated enzymes for three species. For metabolite detection we employed high performance liquid chromatography coupled with mass spectrometry. We used liquid chromatography method - hydrophilic interaction chromatography with silica column - as it effectively separates highly polar cellular metabolites prior to their detection by mass spectrometer.</p></div

Reconstructed metabolic map of <i>M. gallisepticum</i>.

<p>The pathways common for three Mollicute species are represented. Metabolites are shown as circles; compounds identified by LC-MS are marked in green, other predicted compounds are marked in red. Proteins that catalyze metabolic reactions are shown as diamonds, and their ID numbers are indicated. Enzymatic activities, which are not associated with annotated proteins, are indicated in italics. Abbreviations: D-F-6-P - D-fructose-6-phosphate; D-F-1,6-PP - D-fructose-1,6-bisphosphate; D-S-1,7-PP - D-sedoheptulose-1,7-bisphosphate; D-S-7-P - D-sedoheptulose-7-phosphate; GAP - glyceraldehyde-3-P).</p

Overlap of the detected metabolites for three Mollicute species.

<p>Yellow circle represents metabolites of <i>M. gallisepticum</i>, violet circle represents metabolites of <i>S. melliferum</i>, and green circle represents metabolites of <i>A. laidlawii</i>. The list of identified metabolites of <i>S. melliferum</i>, <i>M. gallisepticum</i> and <i>A. laidlawii</i> is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089312#pone-0089312-t001" target="_blank">Table 1</a>.</p

Terpenoid backbone biosynthesis pathway.

<p>Metabolites involved in this pathway are shown as circles; enzymes are shown as diamonds; and compounds that we detected are marked in green.</p

Reconstructed metabolic map of <i>A. laidlawii</i>.

<p>The pathways common for three Mollicute species are represented. Abbreviations and symbols are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089312#pone-0089312-g001" target="_blank">figure 1</a>.</p

Fragmentation spectra results for the detected metabolites of <i>S. melliferum</i>, <i>M. gallisepticum</i> and <i>A. laidlawii</i>, and fragmentation spectra of the analyzed ion standards from the Metlin database [23] in the same experimental conditions (fixed collision energy of 20 eV).

<p>Fragmentation spectra results for the detected metabolites of <i>S. melliferum</i>, <i>M. gallisepticum</i> and <i>A. laidlawii</i>, and fragmentation spectra of the analyzed ion standards from the Metlin database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089312#pone.0089312-Yus1" target="_blank">[23]</a> in the same experimental conditions (fixed collision energy of 20 eV).</p

Metabolite peaks counting and pairwise comparison of three Mollicute species.

<p>Results are summarized for the positive and negative acquisition modes.</p

Identified metabolites of <i>S. melliferum</i>, <i>M. gallisepticum</i> and <i>A. laidlawii</i>.

<p>Identified metabolites of <i>S. melliferum</i>, <i>M. gallisepticum</i> and <i>A. laidlawii</i>.</p