Characterization of a Regulatory Network of Peptide Antibiotic Detoxification Modules in <em>Lactobacillus casei</em> BL23

Two-component systems (TCS) are major signal transduction pathways that allow bacteria to detect and respond to environmental and intracellular changes. A group of TCS has been shown to be involved in the response against antimicrobial peptides (AMPs). These TCS are characterized by the possession of intramembrane-sensing histidine kinases, and they are usually associated with ABC transporters of the peptide-7 exporter family (Pep7E). Lactobacillus casei BL23 encodes two TCS belonging to this group (TCS09 and TCS12) that are located next to two ABC transporters (ABC09 and ABC12), as well as a third Pep7E ABC transporter not genetically associated with any TCS (orphan ABC). This study addressed the involvement of modules TCS09/ABC09 and TCS12/ABC12 in AMP resistance. Results showed that both systems contribute to L. casei resistance to AMPs, and that each TCS constitutes a functional unit with its corresponding ABC transporter. Analysis of transcriptional levels showed that module 09 is required for the induction of ABC09 expression in response to nisin. In contrast, module 12 controls a wider regulon that encompasses the orphan ABC, the dlt operon (d-alanylation of teichoid acids), and the mprF gene (l-lysinylation of phospholipids), thereby controlling properties of the cell envelope. Furthermore, the characterization of a dltA mutant showed that Dlt plays a major role in AMP resistance in L. casei. This is the first report on the regulation of the response of L. casei to AMPs, giving insight into its ability to adapt to the challenging environments that it encounters as a probiotic microorganism

NADP+ binding to the regulatory subunit of methionine adenosyltransferase II increases intersubunit binding affinity in the hetero-trimer

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP+ with a 1:1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP+ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed on the light of the recent identification of several splicing forms of this subunit in hepatoma cells.Ministerio de Economía y Competitividad (BFU2005-00050, BFU2008-00666 and BFU2009-08977 to MAP; BIO2010-20508-C04-03 to JS-A; BFU2011-24982 to BG; BIO2010-14983 to MM-J; BFU2010-19451 to AV-C) and Comunidad de Madrid (GR/SAL/0833/2004 to JS-A).B. G. had a J&C appointment from the Spanish Government. R.O. had a Graduate Fellowship from CSIC and A. R-G. was a student of the Universidad Autónoma de Madrid with a scholarship of the Ministerio de Educación y Ciencia.Peer reviewe

Participación de los sistemas de dos componentes TC09 y TC12 de Lactobacillus casei en la respuesta a estrés por péptidos antimicrobianos y en el control de la integridad de la pared celular

Comunicación presentadas en la 6ª reunión de la Red temática BAL (Participación de las Bacterias Lácticas en la Salud Humana y en la Calidad Alimentaria), celebrada el 28 y 29 de junio de 2012 en Tarragona.Peer Reviewe

Comparison of the structures of human β subunits in free and NADP⁺ bound states.

<p>The figure depicts the structure of free β subunits (white; 2YDY) and that of the NADP⁺ bound state (red; 2YDX). Cofactor and resveratrol molecules are shown as sticks with coloured carbon (black), nitrogen (red), phosphorus (orange) and oxygen (blue) atoms. Three regions not visible in the apoenzyme and ordered upon NADP⁺ binding are highlighted: F60-A77 (A), A95-N113 (B), and D325-F333 (C); only A and B regions are linked to NADP⁺. On the right the surface of the β dimer is shown as found in the NADP⁺ bound crystal structure, illustrating location of A and C regions in the dimer interface.</p

Production of recombinant proteins and purification steps.

<p>The upper part of the figure shows a scheme of the purification steps followed to prepare the three types of hetero-oligomers obtained in this work. The numbers indicate the corresponding lane of the representative stained gels shown below. Panel A shows a representative SDS-PAGE gel used for molecular mass estimation, including two sets of standards as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050329#s2" target="_blank">Materials and Methods</a> section. Samples (30 µl) of key purification steps to produce wild type (B), mutated (C) and truncated MAT II (D) were prepared under standard reducing conditions for SDS-PAGE electrophoresis. Gel lanes correspond to: (1) refolded α2 (10 µg); (2) Q-Sepharose collected peak (10 µg); (3) concentrated Q-Sepharose peak (25 µg); (4) standards; (5) purified hetero-oligomer (10 µg); and (6) purified wild type (10 µg), mutant (5 µg) or truncated β subunit (10 µg). Panels B–D show only the relevant sections of the stained gels for each type of purification; the positions for the 45 and 31 kDa protein standards are indicated on the side of the gels. Dots indicate places where gel lanes have been cropped for clarity.</p

ATP kinetics for α2 homo- and hetero-oligomers.

<p>The table shows the calulated S_0.5 values for ATP of α2 homo-dimers and hetero-oligomers composed by: α2 and wild type β (MAT II); α2 and Y159F/K163A-β (mutant MAT II); and α2 and a ΔS16 β subunit (truncated MAT II). S_0.5 refers to the concentration of substrate at which half the V_max is achieved. The results shown are the mean ± SD of a minimum of three independent experiments carried out in triplicate and were considered significant only when p≤0.05 (*).</p

Gel filtration chromatography of the β subunit.

<p>The purified wild type β subunit was analyzed on a Superdex 200 16/60 gel filtration column and the elution followed by A₂₈₀. Vertical bars indicate the elution position of relevant standards. The elution volume of all the markers used was: dextran blue (2000 kDa) 39.4 ml; apoferritin (443 kDa) 52.3 ml; β-amylase (200 kDa) 59.5 ml; alcohol dehydrogenase (150 kDa) 64.3 ml; bovine serum albumin (66.2 kDa) 71.6 ml; and cytochrome <i>c</i> (12.4 kDa) 93 ml.</p

Methionine kinetics of α2 homo- and hetero-oligomers.

<p>The table shows the calulated S_0.5 values for methionine of α2 homo-dimers and hetero-oligomers composed by: α2 and wild type β (MAT II); α2 and Y159F/K163A-β (mutant MAT II); and α2 and a ΔS16 β subunit (truncated MAT II). S_0.5 refers to the concentration of substrate at which half the V_max is achieved. The results shown are the mean ± SD of a minimum of three independent experiments carried out in triplicate and were considered significant only when p≤0.05 (*).</p

Results of peptide mass fingerprint of MAT α2 bands.

<p>Purified refolded-α2 was loaded in SDS-PAGE gels, the two bands (α2 and α2′) observed after staining were excised and digested with trypsin. Peptides were desalted and subjected to MALDI-TOF to obtain the corresponding mass fingerprint. Database searches were performed with MASCOT and successful protein identification was considered when p<0.05.</p