Characterization of Two Component Systems of Lactobacillus casei BL23 and their involvement in stress response

Lactobacillus casei es una bacteria del ácido láctico de interés aplicado por su uso como cultivo iniciador en la industria alimentaria y por el carácter probiótico de algunas cepas. Como probiótico, L. casei debe sobrevivir a las condiciones de producción industrial y a su paso por el tracto gastrointestinal manteniendo sus propiedades. Para ello, L. casei posee rutas de reconocimiento de señales ambientales específicas y convierten esta información en una respuesta fisiológica adecuada. Un mecanismo comúnmente encontrado en bacterias para la transducción de señal son los sistemas de dos componentes (Two Components Systems, TCS). Los TCS están formados por una proteína sensora con actividad histidina quinasa (HK) y un regulador de respuesta (RR). La detección de un estímulo específico por la proteína sensora induce su autofosforilación y la transferencia del fosfato al regulador de respuesta, produciéndose la activación del mismo. Los sistemas de dos componentes median la respuesta adaptativa a una amplia gama de estímulos ambientales en bacterias. En el laboratorio de Bacterias Lácticas del Instituto de Agroquímica y Tecnología de Alimentos se ha iniciado el estudio de los TCS codificados por L. casei BL23 dentro del cual se incluye el presente proyecto de tesis doctoral.Revilla Guarinos, A. (2014). Characterization of Two Component Systems of Lactobacillus casei BL23 and their involvement in stress response [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/43589TESI

Bacterial Biohybrid Microswimmers

Over millions of years, Nature has optimized the motion of biological systems at the micro and nanoscales. Motor proteins to motile single cells have managed to overcome Brownian motion and solve several challenges that arise at low Reynolds numbers. In this review, we will briefly describe naturally motile systems and their strategies to move, starting with a general introduction that surveys a broad range of developments, followed by an overview about the physical laws and parameters that govern and limit motion at the microscale. We characterize some of the classes of biological microswimmers that have arisen in the course of evolution, as well as the hybrid structures that have been constructed based on these, ranging from Montemagno's ATPase motor to the SpermBot. Thereafter, we maintain our focus on bacteria and their biohybrids. We introduce the inherent properties of bacteria as a natural microswimmer and explain the different principles bacteria use for their motion. We then elucidate different strategies that have been employed for the coupling of a variety of artificial microobjects to the bacterial surface, and evaluate the different effects the coupled objects have on the motion of the 'biohybrid.' Concluding, we give a short overview and a realistic evaluation of proposed applications in the field

Peptide and amino acid metabolism is controlled by an OmpR-family response regulator in Lactobacillus casei

A Lactobacillus casei BL23 strain defective in an OmpR-family response regulator encoded by LCABL_18980 (PrcR, RR11), showed enhanced proteolytic activity caused by overexpression of the gene encoding the proteinase PrtP. Transcriptomic analysis revealed that, in addition to prtP expression, PrcR regulates genes encoding peptide and amino acid transporters, intracellular peptidases and amino acid biosynthetic pathways, among others. Binding of PrcR to twelve promoter regions of both upregulated and downregulated genes, including its own promoter, was demonstrated by electrophoretic mobility shift assays showing that PrcR can act as a transcriptional repressor or activator. Phosphorylation of PrcR increased its DNA binding activity and this effect was abolished after replacement of the phosphorylatable residue Asp-52 by alanine. Comparison of the transcript levels in cells grown in the presence or absence of tryptone in the growth medium revealed that PrcR activity responded to the presence of a complex amino acid source in the growth medium. We conclude that the PrcR plays a major role in the control of the peptide and amino acid metabolism in L. casei BL23. Orthologous prcR genes are present in most members of the Lactobacillaceae and Leuconostocaceae families. We hypothesize that they play a similar role in these bacterial groups.This work was financed by funds from the former Spanish Ministry of Science and Innovation (AGL2007-60975 and AGL2010-15679) and Generalitat Valenciana (ACOMP2012/137). A. Revilla-Guarinos was the recipient of a research fellowship from the Instituto Danone.Peer reviewe

The ABC transporter DerAB of <i>Lactobacillus casei</i> mediates resistance against insect-derived defensins

Bce-like systems mediate resistance against antimicrobial peptides in Firmicutes bacteria. Lactobacillus casei BL23 encodes an “orphan” ABC transporter that, based on homology to BceAB-like systems, was proposed to contribute to antimicrobial peptide resistance. A mutant lacking the permease subunit was tested for sensitivity against a collection of peptides derived from bacteria, fungi, insects, and humans. Our results show that the transporter specifically conferred resistance against insect-derived cysteine-stabilized αβ defensins, and it was therefore renamed DerAB for defensin resistance ABC transporter. Surprisingly, cells lacking DerAB showed a marked increase in resistance against the lantibiotic nisin. This could be explained by significantly increased expression of the antimicrobial peptide resistance determinants regulated by the Bce-like systems PsdRSAB (formerly module 09) and ApsRSAB (formerly module 12). Bacterial two-hybrid studies in Escherichia coli showed that DerB could interact with proteins of the sensory complex in the Psd resistance system. We therefore propose that interaction of DerAB with this complex in the cell creates signaling interference and reduces the cell’s potential to mount an effective nisin resistance response. In the absence of DerB, this negative interference is relieved, leading to the observed hyperactivation of the Psd module and thus increased resistance to nisin. Our results unravel the function of a previously uncharacterized Bce-like orphan resistance transporter with pleiotropic biological effects on the cell.This work was financially supported by DFG grant MA2837/3-2 (to T.M.) and by funds from the former Spanish Ministry of Science and Innovation and FEDER (grant AGL2010-15679) and the Generalitat Valenciana (grant ACOMP2012/137) (to M.Z.). A.R.-G. thanks the Federation of European Microbiological Societies for research grant FEMS-RG-2014-0067. Q.Z. is financially supported by a stipend from the China Scholarship Council (CSC).Peer reviewe

Synthesis and mechanism-of-action of a novel synthetic antibiotic based on a dendritic system with bow-tie topology

Over the course of the last decades, the continuous exposure of bacteria to antibiotics—at least in parts due to misprescription, misuse, and misdosing—has led to the widespread development of antimicrobial resistances. This development poses a threat to the available medication in losing their effectiveness in treating bacterial infections. On the drug development side, only minor advances have been made to bring forward novel therapeutics. In addition to increasing the efforts and approaches of tapping the natural sources of new antibiotics, synthetic approaches to developing novel antimicrobials are being pursued. In this study, BDTL049 was rationally designed using knowledge based on the properties of natural antibiotics. BDTL049 is a carbosilane dendritic system with bow-tie type topology, which has antimicrobial activity at concentrations comparable to clinically established natural antibiotics. In this report, we describe its mechanism of action on the Gram-positive model organism Bacillus subtilis. Exposure to BDTL049 resulted in a complex transcriptional response, which pointed toward disturbance of the cell envelope homeostasis accompanied by disruption of other central cellular processes of bacterial metabolism as the primary targets of BDTL049 treatment. By applying a combination of whole-cell biosensors, molecular staining, and voltage sensitive dyes, we demonstrate that the mode of action of BDTL049 comprises membrane depolarization concomitant with pore formation. As a result, this new molecule kills Gram-positive bacteria within minutes. Since BDTL049 attacks bacterial cells at different targets simultaneously, this might decrease the chances for the development of bacterial resistances, thereby making it a promising candidate for a future antimicrobial agent

Influence of Two-Component Signal Transduction Systems of Lactobacillus casei BL23 on Tolerance to Stress Conditions ▿ †

Lactobacillus casei BL23 carries 17 two-component signal transduction systems. Insertional mutations were introduced into each gene encoding the cognate response regulators, and their effects on growth under different conditions were assayed. Inactivation of systems TC01, TC06, and TC12 (LCABL_02080-LCABL_02090, LCABL_12050-LCABL_12060, and LCABL_19600-LCABL_19610, respectively) led to major growth defects under the conditions assayed

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

Cross-regulation of Aps-promoters in Lacticaseibacillus paracasei by the PsdR response regulator in response to lantibiotics

Abstract The PsdRSAB and ApsRSAB detoxification modules, together with the antimicrobial peptides (AMPs)-resistance determinants Dlt system and MprF protein, play major roles in the response to AMPs in Lacticaseibacillus paracasei BL23. Sensitivity assays with a collection of mutants showed that the PsdAB ABC transporter and the Dlt system are the main subtilin resistance determinants. Quantification of the transcriptional response to subtilin indicate that this response is exclusively regulated by the two paralogous systems PsdRSAB and ApsRSAB. Remarkably, a cross-regulation of the derAB, mprF and dlt-operon genes—usually under control of ApsR—by PsdR in response to subtilin was unveiled. The high similarity of the predicted structures of both response regulators (RR), and of the RR-binding sites support this possibility, which we experimentally verified by protein-DNA binding studies. ApsR-P shows a preferential binding in the order P derA  > P dlt  > P mprF  > P psdA . However, PsdR-P bound with similar apparent affinity constants to the four promoters. This supports the cross-regulation of derAB, mprF and the dlt-operon by PsdR. The possibility of cross-regulation at the level of RR-promoter interaction allows some regulatory overlap with two RRs controlling the expression of systems involved in maintenance of critical cell membrane functions in response to lantibiotics