Divergent functional roles of D-amino acids secreted by Vibrio cholerae

The L-forms of amino acids are used in all kingdoms of life to synthesize proteins. However, the bacterium Vibriocholerae, the causative agent of cholera, produces D-amino acids which are released to the environment at millimolar concentrations.We baptized these D-amino acids as non-canonical D-amino acids (NCDAAs) since they are different from those (i.e.D-alanine and D-glutamate) normally present in the bacterial cell wall. In V. cholerae, production of NCDAAs relies on the BsrVenzyme, a periplasmic broad spectrum racemase. BsrV multispecific activity, produces of a wide range of distinct D-amino acids.Using a combination of genetics and molecular physiology approaches we have demonstrated that NCDAAs target different cellularprocesses which may function as part of a cooperative strategy in vibrio communities to protect non-producing members fromcompeting bacteria. Because NCDAA production is widespread in bacteria, we anticipate that NCDAAs are relevant modulatorsof microbial subpopulations in diverse ecosystems

The role of conserved proteins DrpA and DrpB in nitrate respiration of Thermus thermophilus

In many Thermus thermophilus strains, nitrate respiration is encoded in mobile genetic regions, along with regulatory circuits that modulate its expression based on anoxia and nitrate presence. The oxygen-responsive system has been identified as the product of the dnrST (dnr) operon located immediately upstream of the nar operon (narCGHJIKT), which encodes the nitrate reductase (NR) and nitrate/nitrite transporters. In contrast, the nature of the nitrate sensory system is not known. Here, we analyse the putative nitrate-sensing role of the bicistronic drp operon (drpAB) present downstream of the nar operon in most denitrifying Thermus spp. Expression of drp was found to depend on the master regulator DnrT, whereas the absence of DrpA or DrpB increased the expression of both DnrS and DnrT and, concomitantly, of the NR. Absence of both proteins made expression from the dnr and nar operons independent of nitrate. Polyclonal antisera allowed us to identify DrpA as a periplasmic protein and DrpB as a membrane protein, with capacity to bind to the cytoplasmic membrane. Here, we propose a role for DrpA/DrpB as nitrate sensors during denitrification.This work was supported by grant BIO2016-77031-R from the Spanish Ministry of Economy and Competitiveness and grant 685474 from the H2020 research and innovation program of the European Union. An institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa (CBMSO) is also acknowledge

Control del metabolismo respiratorio en thermus thermophilus por el elemento genético conjugativo NCE

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular, Fecha de lectura: 19-10-200

Structural basis for the broad specificity of a new family of amino-acid racemases

Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical d-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members. © 2014 International Union of Crystallography.Peer Reviewe

D-amino acids govern stationary phase cell wall remodeling in bacteria

4 pages, 4 figures.-- PMID: 19762646 [PubMed].-- Supporting information available at: http://www.sciencemag.org/cgi/content/full/sci;325/5947/1552/DC1In all known organisms, amino acids are predominantly thought to be synthesized and used as their L-enantiomers. Here, we found that bacteria produce diverse D-amino acids as well, which accumulate at millimolar concentrations in supernatants of stationary phase cultures. In Vibrio cholerae, a dedicated racemase produced D-Met and D-Leu, whereas Bacillus subtilis generated D-Tyr and D-Phe. These unusual D-amino acids appear to modulate synthesis of peptidoglycan, a strong and elastic polymer that serves as the stress-bearing component of the bacterial cell wall. D-Amino acids influenced peptidoglycan composition, amount, and strength, both by means of their incorporation into the polymer and by regulating enzymes that synthesize and modify it. Thus, synthesis of D-amino acids may be a common strategy for bacteria to adapt to changing environmental conditions.This work was supported by Howard Hughes Medical Institute (HHMI); NIH AI-R37-42347 (M.K.W.) and CA24487 and GM086258 (J.C.); Ministry of Education and Science, Spain (MEC) BFU2006-04574 and Fundación Ramón Areces (M.A.P.); Jane Coffin Childs Fellowship (H.L.); MEC Fellowship (F.C.); and HHMI Exceptional Research Opportunities (EXROP) (C.N.T.).Peer reviewe

Hydroxyl radical overproduction in the envelope : an achilles' heel in peptidoglycan synthesis

While many mechanisms governing bacterial envelope homeostasis have been identified, others remain poorly understood. To decipher these processes, we previously developed an assay in the Gram-negative model Escherichia coli to identify genes involved in maintenance of envelope integrity. One such gene was ElyC, which was shown to be required for envelope integrity and peptidoglycan synthesis at room temperature. ElyC is predicted to be an integral inner membrane protein with a highly conserved domain of unknown function (DUF218). In this study, and stemming from a further characterization of the role of ElyC in maintaining cell envelope integrity, we serendipitously discovered an unappreciated form of oxidative stress in the bacterial envelope. We found that cells lacking ElyC overproduce hydroxyl radicals (HO ) in their envelope compartment and that HO overproduction is directly or indirectly responsible for the peptidoglycan synthesis arrest, cell envelope integrity defects, and cell lysis of the Δ mutant. Consistent with these observations, we show that the Δ mutant defect is suppressed during anaerobiosis. HO is known to cause DNA damage but to our knowledge has not been shown to interfere with peptidoglycan synthesis. Thus, our work implicates oxidative stress as an important stressor in the bacterial cell envelope and opens the door to future studies deciphering the mechanisms that render peptidoglycan synthesis sensitive to oxidative stress. Oxidative stress is caused by the production and excessive accumulation of oxygen reactive species. In bacterial cells, oxidative stress mediated by hydroxyl radicals is typically associated with DNA damage in the cytoplasm. Here, we reveal the existence of a pathway for oxidative stress in the envelope of Gram-negative bacteria. Stemming from the characterization of a poorly characterized gene, we found that HO overproduction specifically in the envelope compartment causes inhibition of peptidoglycan synthesis and eventually bacterial cell lysis

Peptidoglycan editing in non-proliferating intracellular Salmonella as source of interference with immune signaling

This work was funded by grants PID2020-112971GB-I00/10.13039/501100011033 (F.G-dP.) and PID2019-104070RB-C21 (S.V.) of the Spanish Ministry of Science and Innovation, VR2018-02823 of the Swedish Research Council (F.C.), KAW2012.0184 of the Knut and Alice Wallenberg Foundation (F.C.), and SMK2062 of the Kempe Foundation (F.C.

Structural basis for the broad specificity of a new family of amino-acid racemases

Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical d-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family membersResearch in the Cava laboratory is supported by the MINECO, Spain (RYC-2010-06241), Universidad Autonoma de Madrid (UAM-38) and by the Knut and Alice Wallenberg Foundation (KAW). Additionally, this work was supported by the BFU2011-25326 MEC grant (JAH), by the S2010/BMD-2457 grant from CAM (JAH) and by HHMI (MKW