Phylogenetic tree based on 16S rDNA sequences showing the phylogenetic distribution of the TD enzyme.

<p>The phylogenetic tree was constructed with MEGA 5 software using sequences from RDP database. Because it is too big to show in a single page, the structure of the phylogenetic tree is divided into two panels (A and B). The connecting point of the tree segments in the two panels is marked with a broken line. The scale bar indicates 0.02 change per nucleotide. The arrows at the right represent the TDs that could exist in the bacterium and the numbers next to the arrow show the number of genes that might encode the TD.</p

The primers used for the PCR amplifications in this work.

<p>The recognition sites for restriction enzymes are underlined.</p

The two different pathways BTD and CTD involved in bacteria.

<p>A. BTD catalyzes the first reaction in the biosynthesis of L-isoleucine in bacteria under aerobic conditions. BTD is feedback inhibited by L-isioleucine. B. CTD degrades L-threonine to propionate in bacteria under anaerobic conditions to generate ATP. CTD is activated by AMP and CMP.</p

Stabillity of TDs from different bacteria.

<p>Stabillity of TDs from different bacteria.</p

Evolutionary model proposed for the evolution of genes encoding TDs in bacteria.

<p>Genes encoding the enzymes are represented by arrows.</p

Information of TDs from different bacteria.

<p>Information of TDs from different bacteria.</p

Sequence alignment of CTDs and BTDs and structure alignment of BTD2 (1TDJ) and CTD (2GN2).

<p>A. The sequence alignment of CTDs from 328 species of bacteria. B. The sequence alignment of BTDs from 546 species of bacteria. The PLP binding sites and the substrate binding sites are labelled by purple and blue dots, respectively. The other highly conserved residues are labelled by black dots. C. The aligned structure of PLP binding sites of BTD2 and CTD. D. The aligned structure of substrate binding sites of BTD2 and CTD. The amino acid residues directly involved in PLP binding sites and the substrate binding sites are shown in sticks. Residues from CTD are shown in blue and residues from BTD are shown in red. The residues are labled accoding to the sequence of CTD coded by <i>tdcB</i> in <i>S. typhimurium </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080750#pone.0080750-Simanshu2" target="_blank">[4]</a>.</p

Structure comparison of BTD2 (1TDJ) in <i>E. coli</i>, BTD1 in <i>B. subtilis</i> and CTD (2GN2) in <i>S. typhimurium</i>.

<p>Two domains in BTD1 and BTD2 are separated by a middle linker. The larger domain on the left is the catalytic domain, and the smaller one on the right is the regulatory domain composed of ACT-like subdomains. CTD (shown in green) contains only the catalytic domain; BTD1 (shown in red) contains the catalytic domain and one ACT-like subdomain; BTD2 (shown in blue) contains the catalytic domain and two ACT-like subdomains.</p

SDS-PAGE of the nine purified TDs.

<p>Lane M, molecular mass marker; lane 1, CgCTD purified from BL21(DE3)/pET28a-<i>CgtdcB</i>; lane 2, EcCTD purified from BL21(DE3)/pET28a-<i>EctdcB</i>; lane 3, SaCTD purified from BL21(DE3)/pET28a-<i>SatdcB</i>; lane 4, CgBTD1 purified from BL21(DE3)/pET28a-<i>CgilvA</i>; lane 5, SaBTD1 purified from BL21(DE3)/pET28a-<i>SailvA</i>; lane 6, SgBTD1 purified from BL21(DE3)/pET28a-<i>SgilvA</i>; lane 7, BsBTD1 purified from BL21(DE3)/pET28a-<i>BsilvA</i>; lane 8, EcBTD2 purified from BL21(DE3)/pET28a-<i>EcilvA</i>; lane 9, PpBTD2 purified from BL21(DE3)/pET28a-<i>PpilvA</i>.</p

The effect of temperature on the activity of nine different TDs.

<p>Error bars indicate the standard deviations from three parallel samples.</p