The Enzymatic Activities of the <i>Escherichia coli</i> Basic Aliphatic Amino Acid Decarboxylases Exhibit a pH Zone of Inhibition

The stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a “zone of inhibition” between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH

The Enzymatic Activities of the <i>Escherichia coli</i> Basic Aliphatic Amino Acid Decarboxylases Exhibit a pH Zone of Inhibition

The stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a “zone of inhibition” between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH

Analysis of the Evolution of the MoxR ATPases

MoxR proteins comprise a family of ATPases Associated with diverse cellular Activities (AAA+). These proteins are widespread and found across the diversity of prokaryotic species. Despite their ubiquity, members of the group remain poorly characterized. Only a few examples of MoxR proteins have been associated with cellular roles, where they have been shown to perform chaperone-like functions. A characteristic feature of MoxR proteins is their association with proteins containing the von Willebrand factor type A (VWA) domain. In an effort to understand the spread and diversity of the MoxR family, an evolutionary approach was undertaken. Phylogenetic techniques were used to define nine major subfamilies within the MoxR family. A combination of phylogenetic and genomic approaches was utilized to explore the extent of the partnership between the MoxR and VWA domain containing proteins (VWA proteins). These analyses led to the clarification of genetic linkages between MoxR and VWA proteins. A significant partnership is described here, as seven of nine MoxR subfamilies were found to be linked to VWA proteins. Available genomic data were also used to assess the intraprotein diversification of MoxR and VWA protein sequences. Data clearly indicated that, in MoxR proteins, the ATPase domain is maintained with high conservation while the remaining protein sequence evolves at a faster rate; a similar pattern was observed for the VWA domain in VWA proteins. Overall, our data present insights into the modular evolution of MoxR ATPases

Analysis of the Evolution of the MoxR ATPases

MoxR proteins comprise a family of ATPases Associated with diverse cellular Activities (AAA+). These proteins are widespread and found across the diversity of prokaryotic species. Despite their ubiquity, members of the group remain poorly characterized. Only a few examples of MoxR proteins have been associated with cellular roles, where they have been shown to perform chaperone-like functions. A characteristic feature of MoxR proteins is their association with proteins containing the von Willebrand factor type A (VWA) domain. In an effort to understand the spread and diversity of the MoxR family, an evolutionary approach was undertaken. Phylogenetic techniques were used to define nine major subfamilies within the MoxR family. A combination of phylogenetic and genomic approaches was utilized to explore the extent of the partnership between the MoxR and VWA domain containing proteins (VWA proteins). These analyses led to the clarification of genetic linkages between MoxR and VWA proteins. A significant partnership is described here, as seven of nine MoxR subfamilies were found to be linked to VWA proteins. Available genomic data were also used to assess the intraprotein diversification of MoxR and VWA protein sequences. Data clearly indicated that, in MoxR proteins, the ATPase domain is maintained with high conservation while the remaining protein sequence evolves at a faster rate; a similar pattern was observed for the VWA domain in VWA proteins. Overall, our data present insights into the modular evolution of MoxR ATPases

Analysis of the Evolution of the MoxR ATPases

MoxR proteins comprise a family of ATPases Associated with diverse cellular Activities (AAA+). These proteins are widespread and found across the diversity of prokaryotic species. Despite their ubiquity, members of the group remain poorly characterized. Only a few examples of MoxR proteins have been associated with cellular roles, where they have been shown to perform chaperone-like functions. A characteristic feature of MoxR proteins is their association with proteins containing the von Willebrand factor type A (VWA) domain. In an effort to understand the spread and diversity of the MoxR family, an evolutionary approach was undertaken. Phylogenetic techniques were used to define nine major subfamilies within the MoxR family. A combination of phylogenetic and genomic approaches was utilized to explore the extent of the partnership between the MoxR and VWA domain containing proteins (VWA proteins). These analyses led to the clarification of genetic linkages between MoxR and VWA proteins. A significant partnership is described here, as seven of nine MoxR subfamilies were found to be linked to VWA proteins. Available genomic data were also used to assess the intraprotein diversification of MoxR and VWA protein sequences. Data clearly indicated that, in MoxR proteins, the ATPase domain is maintained with high conservation while the remaining protein sequence evolves at a faster rate; a similar pattern was observed for the VWA domain in VWA proteins. Overall, our data present insights into the modular evolution of MoxR ATPases

Analysis of the Evolution of the MoxR ATPases

MoxR proteins comprise a family of ATPases Associated with diverse cellular Activities (AAA+). These proteins are widespread and found across the diversity of prokaryotic species. Despite their ubiquity, members of the group remain poorly characterized. Only a few examples of MoxR proteins have been associated with cellular roles, where they have been shown to perform chaperone-like functions. A characteristic feature of MoxR proteins is their association with proteins containing the von Willebrand factor type A (VWA) domain. In an effort to understand the spread and diversity of the MoxR family, an evolutionary approach was undertaken. Phylogenetic techniques were used to define nine major subfamilies within the MoxR family. A combination of phylogenetic and genomic approaches was utilized to explore the extent of the partnership between the MoxR and VWA domain containing proteins (VWA proteins). These analyses led to the clarification of genetic linkages between MoxR and VWA proteins. A significant partnership is described here, as seven of nine MoxR subfamilies were found to be linked to VWA proteins. Available genomic data were also used to assess the intraprotein diversification of MoxR and VWA protein sequences. Data clearly indicated that, in MoxR proteins, the ATPase domain is maintained with high conservation while the remaining protein sequence evolves at a faster rate; a similar pattern was observed for the VWA domain in VWA proteins. Overall, our data present insights into the modular evolution of MoxR ATPases

The MoxR ATPase RavA and Its Cofactor ViaA Interact with the NADH:Ubiquinone Oxidoreductase I in <i>Escherichia coli</i>

<div><p>MoxR ATPases are widespread throughout bacteria and archaea. The experimental evidence to date suggests that these proteins have chaperone-like roles in facilitating the maturation of dedicated protein complexes that are functionally diverse. In <i>Escherichia coli</i>, the MoxR ATPase RavA and its putative cofactor ViaA are found to exist in early stationary-phase cells at 37°C at low levels of about 350 and 90 molecules per cell, respectively. Both proteins are predominantly localized to the cytoplasm, but ViaA was also unexpectedly found to localize to the cell membrane. Whole genome microarrays and synthetic lethality studies both indicated that RavA-ViaA are genetically linked to Fe-S cluster assembly and specific respiratory pathways. Systematic analysis of mutant strains of <i>ravA</i> and <i>viaA</i> indicated that RavA-ViaA sensitizes cells to sublethal concentrations of aminoglycosides. Furthermore, this effect was dependent on RavA's ATPase activity, and on the presence of specific subunits of NADH:ubiquinone oxidoreductase I (Nuo Complex, or Complex I). Importantly, both RavA and ViaA were found to physically interact with specific Nuo subunits. We propose that RavA-ViaA facilitate the maturation of the Nuo complex.</p></div

List of bacterial strains and plasmids used in this study.

<p><i>cat</i> = chloramphenicol acetyltransferase gene; confers resistance to chloramphenicol.</p><p><i>kan</i> = kanamycin resistance gene.</p><p>ViaA expression is increased in <i>ΔravA</i>::<i>cat</i> compared to WT (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085529#pone.0085529.s002" target="_blank">Figure S2</a>).</p

Growth profiles of cells in the presence of sublethal concentrations of aminoglycosides.

<p>Growth profiles for MG1655 WT and the KO mutants <i>ΔravA</i>, <i>ΔviaA</i> and <i>ΔravAviaA</i> grown aerobically in LB at 37°C over 24 hours. Growth of cells was monitored using OD₆₀₀ readings at the designated time points. The cultures were supplemented as follows: (A) no antibiotics; (B) 4 µg/mL kanamycin; (C) 6 µg/mL streptomycin; (D) 0.5 µg/mL tetracycline; and (E) 1.2 µg/mL chloramphenicol. To confirm the phenotypes observed, <i>ΔravA</i> (F), <i>ΔravAviaA</i> (G) and WT cells (H) were complemented with the plasmids p11 (empty vector control), pR, pRV, pR_K52Q, or pR_K52QV. All cultures in the complementation experiments were supplemented with 4 µg/mL kanamycin for stress induction, and 100 µg/mL ampicillin for plasmid maintenance. Error bars were derived from three independent cultures for each strain and for each condition. Details on the <i>E. coli</i> strains and plasmids used are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085529#pone-0085529-t001" target="_blank">Table 1</a>.</p

Up-regulated gene in PIH1D1-knockdown MDA-MB-231 cells.

<p>Up-regulated gene in PIH1D1-knockdown MDA-MB-231 cells.</p