A Genome-Wide CRISPR Interference Screen Reveals an StkP-Mediated Connection between Cell Wall Integrity and Competence in Streptococcus salivarius.

Competence is one of the most efficient bacterial evolutionary and adaptative strategies by synchronizing production of antibacterial compounds and integration of DNA released by dead cells. In most streptococci, this tactic is orchestrated by the ComRS system, a pheromone communication device providing a short time window of activation in which only part of the population is responsive. Understanding how this developmental process integrates multiple inputs to fine-tune the adequate response is a long-standing question. However, essential genes involved in the regulation of ComRS have been challenging to study. In this work, we built a conditional mutant library using CRISPR interference and performed three complementary screens to investigate competence genetic regulation in the human commensal Streptococcus salivarius. We show that initiation of competence increases upon cell wall impairment, suggesting a connection between cell envelope stress and competence activation. Notably, we report a key role for StkP, a serine-threonine kinase known to regulate cell wall homeostasis. We show that StkP controls competence by a mechanism that reacts to peptidoglycan fragments. Together, our data suggest a key cell wall sensing mechanism coupling competence to cell envelope integrity. IMPORTANCE Survival of human commensal streptococci in the digestive tract requires efficient strategies which must be tightly and collectively controlled for responding to competitive pressure and drastic environmental changes. In this context, the autocrine signaling system ComRS controlling competence for natural transformation and predation in salivarius streptococci could be seen as a multi-input device integrating a variety of environmental stimuli. In this work, we revealed novel positive and negative competence modulators by using a genome-wide CRISPR interference strategy. Notably, we highlighted an unexpected connection between bacterial envelope integrity and competence activation that involves several cell wall sensors. Together, these results showcase how commensal streptococci can fine-tune the pheromone-based competence system by responding to multiple inputs affecting their physiological status in order to calibrate an appropriate collective behavior.

Competence shut-off by intracellular pheromone degradation in salivarius streptococci.

Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci.

Expanding natural transformation to improve beneficial lactic acid bacteria.

Nowadays, the growing human population exacerbates the need for sustainable resources. Inspiration and achievements in nutrient production or human/animal health might emanate from microorganisms and their adaptive strategies. Here, we exemplify the benefits of lactic acid bacteria (LAB) for numerous biotechnological applications and showcase their natural transformability as a fast and robust method to hereditarily influence their phenotype/traits in fundamental and applied research contexts. We described the biogenesis of the transformation machinery and we analyzed the genome of hundreds of LAB strains exploitable for human needs to predict their transformation capabilities. Finally, we provide a stepwise rational path to stimulate and optimize natural transformation with standard and synthetic biology techniques. A comprehensive understanding of the molecular mechanisms driving natural transformation will facilitate and accelerate the improvement of bacteria with properties that serve broad societal interests.

The CovRS Environmental Sensor Directly Controls the ComRS Signaling System To Orchestrate Competence Bimodality in Salivarius Streptococci.

In bacteria, phenotypic heterogeneity in an isogenic population compensates for the lack of genetic diversity and allows concomitant multiple survival strategies when choosing only one is too risky. This powerful tactic is exploited for competence development in streptococci where only a subset of the community triggers the pheromone signaling system ComR-ComS, resulting in a bimodal activation. However, the regulatory cascade and the underlying mechanisms of this puzzling behavior remained partially understood. Here, we show that CovRS, a well-described virulence regulatory system in pathogenic streptococci, directly controls the ComRS system to generate bimodality in the gut commensal Streptococcus salivarius and the closely related species Streptococcus thermophilus. Using single-cell analysis of fluorescent reporter strains together with regulatory mutants, we revealed that the intracellular concentration of ComR determines the proportion of competent cells in the population. We also showed that this bimodal activation requires a functional positive-feedback loop acting on ComS production, as well as its exportation and reinternalization via dedicated permeases. As the intracellular ComR concentration is critical in this process, we hypothesized that an environmental sensor could control its abundance. We systematically inactivated all two-component systems and identified CovRS as a direct repression system of comR expression. Notably, we showed that the system transduces its negative regulation through CovR binding to multiple sites in the comR promoter region. Since CovRS integrates environmental stimuli, we suggest that it is the missing piece of the puzzle that connects environmental conditions to (bimodal) competence activation in salivarius streptococci. IMPORTANCE Combining production of antibacterial compounds and uptake of DNA material released by dead cells, competence is one of the most efficient survival strategies in streptococci. Yet, this powerful tactic is energy consuming and reprograms the metabolism to such an extent that cell proliferation is transiently impaired. To circumvent this drawback, competence activation is restricted to a subpopulation, a process known as bimodality. In this work, we explored this phenomenon in salivarius streptococci and elucidated the molecular mechanisms governing cell fate. We also show that an environmental sensor controlling virulence in pathogenic streptococci is diverted to control competence in commensal streptococci. Together, those results showcase how bacteria can sense and transmit external stimuli to complex communication devices for fine-tuning collective behaviors.

Coevolution of the bacterial pheromone ComS and sensor ComR fine-tunes natural transformation in streptococci.

Competence for natural transformation extensively contributes to genome evolution and the rapid adaptability of bacteria dwelling in challenging environments. In most streptococci, this process is tightly controlled by the ComRS signaling system, which is activated through the direct interaction between the (R)RNPP-type ComR sensor and XIP pheromone (mature ComS). The overall mechanism of activation and the basis of pheromone selectivity have been previously reported in Gram-positive salivarius streptococci; however, detailed 3D-remodeling of ComR leading up to its activation remains only partially understood. Here, we identified using a semirational mutagenesis approach two residues in the pheromone XIP that bolster ComR sensor activation by interacting with two aromatic residues of its XIP-binding pocket. Random and targeted mutagenesis of ComR revealed that the interplay between these four residues remodels a network of aromatic-aromatic interactions involved in relaxing the sequestration of the DNA-binding domain. Based on these data, we propose a comprehensive model for ComR activation based on two major conformational changes of the XIP-binding domain. Notably, the stimulation of this newly identified trigger point by a single XIP substitution resulted in higher competence and enhanced transformability, suggesting that pheromone-sensor coevolution counter-selects for hyperactive systems in order to maintain a trade-off between competence and bacterial fitness. Overall, this study sheds new light on the ComRS activation mechanism and how it could be exploited for biotechnological and biomedical purposes.

Uncovering a superfamily of nickel-dependent hydroxyacid racemases and epimerases.

Isomerization reactions are fundamental in biology. Lactate racemase, which isomerizes L- and D-lactate, is composed of the LarA protein and a nickel-containing cofactor, the nickel-pincer nucleotide (NPN). In this study, we show that LarA is part of a superfamily containing many different enzymes. We overexpressed and purified 13 lactate racemase homologs, incorporated the NPN cofactor, and assayed the isomerization of different substrates guided by gene context analysis. We discovered two malate racemases, one phenyllactate racemase, one α-hydroxyglutarate racemase, two D-gluconate 2-epimerases, and one short-chain aliphatic α-hydroxyacid racemase among the tested enzymes. We solved the structure of a malate racemase apoprotein and used it, along with the previously described structures of lactate racemase holoprotein and D-gluconate epimerase apoprotein, to identify key residues involved in substrate binding. This study demonstrates that the NPN cofactor is used by a diverse superfamily of α-hydroxyacid racemases and epimerases, widely expanding the scope of NPN-dependent enzymes.

Molecular dissection of pheromone selectivity in the competence signaling system ComRS of streptococci.

Competence allows bacteria to internalize exogenous DNA fragments for the acquisition of new phenotypes such as antibiotic resistance or virulence traits. In most streptococci, competence is regulated by ComRS signaling, a system based on the mature ComS pheromone (XIP), which is internalized to activate the (R)RNPP-type ComR sensor by triggering dimerization and DNA binding. Cross-talk analyses demonstrated major differences of selectivity between ComRS systems and raised questions concerning the mechanism of pheromone-sensor recognition and coevolution. Here, we decipher the molecular determinants of selectivity of the closely related ComRS systems from Streptococcus thermophilus and Streptococcus vestibularis Despite high similarity, we show that the divergence in ComR-XIP interaction does not allow reciprocal activation. We perform the structural analysis of the ComRS system from S. vestibularis. Comparison with its ortholog from S. thermophilus reveals an activation mechanism based on a toggle switch involving the recruitment of a key loop by the XIP C terminus. Together with a broad mutational analysis, we identify essential residues directly involved in peptide binding. Notably, we generate a ComR mutant that displays a fully reversed selectivity toward the heterologous pheromone with only five point mutations, as well as other ComR variants featuring XIP bispecificity and/or neofunctionalization for hybrid XIP peptides. We also reveal that a single XIP mutation relaxes the strictness of ComR activation, suggesting fast adaptability of molecular communication phenotypes. Overall, this study is paving the way toward the rational design or directed evolution of artificial ComRS systems for a range of biotechnological and biomedical applications.

Subtle selectivity in a pheromone sensor triumvirate desynchronizes competence and predation in a human gut commensal.

Constantly surrounded by kin or alien organisms in nature, eukaryotes and prokaryotes developed various communication systems to coordinate adaptive multi-entity behavior. In complex and overcrowded environments, they require to discriminate relevant signals in a myriad of pheromones to execute appropriate responses. In the human gut commensal Streptococcus salivarius, the cytoplasmic Rgg/RNPP regulator ComR couples competence to bacteriocin-mediated predation. Here, we describe a paralogous sensor duo, ScuR and SarF, which circumvents ComR in order to disconnect these two physiological processes. We highlighted the recurring role of Rgg/RNPP in the production of antimicrobials and designed a robust genetic screen to unveil potent/optimized peptide pheromones. Further mutational and biochemical analyses dissected the modifiable selectivity toward their pheromone and operating sequences at the subtle molecular level. Additionally, our results highlight how we might mobilize antimicrobial molecules while silencing competence in endogenous populations of human microflora and temper gut disorders provoked by bacterial pathogens.

Mobilization of Microbiota Commensals and Their Bacteriocins for Therapeutics.

With the specter of resurgence of pathogens due to the propagation of antibiotic-resistance genes, innovative antimicrobial strategies are needed. In this review, we summarize the beneficial aspects of bacteriocins, a set of miscellaneous peptide-based bacterium killers, compared with classical antibiotics, and emphasize their use in cocktails to curb the emergence of new resistance. We highlight that their prey spectrum, their molecular malleability, and their multiple modes of production might lead to specific and personalized treatments to prevent systemic disorders. Complementarily, we discuss how we might exploit prevailing bacterial commensals, such as Streptococcus salivarius, and deliberately mobilize their bacteriocin arsenal 'on site' to cure multiresistant infections or finely reshape the endogenous microbiota for prophylaxis purposes.

Distinct and Specific Role of NlpC/P60 Endopeptidases LytA and LytB in Cell Elongation and Division of Lactobacillus plantarum.

Peptidoglycan (PG) is an essential lattice of the bacterial cell wall that needs to be continuously remodeled to allow growth. This task is ensured by the concerted action of PG synthases that insert new material in the pre-existing structure and PG hydrolases (PGHs) that cleave the PG meshwork at critical sites for its processing. Contrasting with Bacillus subtilis that contains more than 35 PGHs, Lactobacillus plantarum is a non-sporulating rod-shaped bacterium that is predicted to possess a minimal set of 12 PGHs. Their role in morphogenesis and cell cycle remains mostly unexplored, except for the involvement of the glucosaminidase Acm2 in cell separation and the NlpC/P60 D, L-endopeptidase LytA in cell shape maintenance. Besides LytA, L. plantarum encodes three additional NlpC/P60 endopeptidases (i.e., LytB, LytC and LytD). The in silico analysis of these four endopeptidases suggests that they could have redundant functions based on their modular organization, forming two pairs of paralogous enzymes. In this work, we investigate the role of each Lyt endopeptidase in cell morphogenesis in order to evaluate their distinct or redundant functions, and eventually their synthetic lethality. We show that the paralogous LytC and LytD enzymes are not required for cell shape maintenance, which may indicate an accessory role such as in PG recycling. In contrast, LytA and LytB appear to be key players of the cell cycle. We show here that LytA is required for cell elongation while LytB is involved in the spatio-temporal regulation of cell division. In addition, both PGHs are involved in the proper positioning of the division site. The absence of LytA activity is responsible for the asymmetrical positioning of septa in round cells while the lack of LytB results in a lateral misplacement of division planes in rod-shaped cells. Finally, we show that the co-inactivation of LytA and LytB is synthetically affecting cell growth, which confirms the key roles played by both enzymes in PG remodeling during the cell cycle of L. plantarum. Based on the large distribution of NlpC/P60 endopeptidases in low-GC Gram-positive bacteria, these enzymes are attractive targets for the discovery of novel antimicrobial compounds.

Intensive targeting of regulatory competence genes by transposable elements in streptococci.

Competence for natural transformation is a widespread developmental process of streptococci. By allowing the uptake and recombination of exogenous naked DNA into the genome, natural transformation, as transposable elements, plays a key role in the plasticity of bacterial genomes. We previously analysed the insertion sites of IS1548, an insertion sequence present in Streptococcus agalactiae and S. pyogenes, and showed that some targeted loci are involved in competence induction. In this work, we investigated on a large scale if loci coding for early competence factors (ComX and the two pheromone-dependent signalling systems ComCDE and ComRS) of streptococci are especially targeted by transposable elements. The transposable elements inserted in regions surrounding these genes and housekeeping genes used for Multilocus Sequence Typing (MLST) were systematically searched for. We found numerous insertion events in the close vicinity of early competence genes, but only very few into the MLST loci. The incidence of transposable elements, mainly insertion sequences, is particularly high in the intergenic regions surrounding comX alleles in numerous species belonging to most streptococcal groups. The identification of scarce disruptive insertions inside early competence genes indicates that the maintenance of competence is essential for streptococci. The specific association of transposable elements with intergenic regions bordering the main regulatory genes of competence may impact on the induction of transformability and so, on the genome plasticity and adaptive evolution of streptococci. This widespread phenomenon brings new perspectives on our understanding of competence regulation and its role in the bacterial life cycle.

Biosynthesis of the nickel-pincer nucleotide cofactor of lactate racemase requires a CTP-dependent cyclometallase.

Bacterial lactate racemase is a nickel-dependent enzyme that contains a cofactor, nickel pyridinium-3,5-bisthiocarboxylic acid mononucleotide, hereafter named nickel-pincer nucleotide (NPN). The LarC enzyme from the bacterium Lactobacillus plantarum participates in NPN biosynthesis by inserting nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide. This reaction, known in organometallic chemistry as a cyclometalation, is characterized by the formation of new metal-carbon and metal-sulfur σ bonds. LarC is therefore the first cyclometallase identified in nature, but the molecular mechanism of LarC-catalyzed cyclometalation is unknown. Here, we show that LarC activity requires Mn2+-dependent CTP hydrolysis. The crystal structure of the C-terminal domain of LarC at 1.85 Å resolution revealed a hexameric ferredoxin-like fold and an unprecedented CTP-binding pocket. The loss-of-function of LarC variants with alanine variants of acidic residues leads us to propose a carboxylate-assisted mechanism for nickel insertion. This work also demonstrates the in vitro synthesis and purification of the NPN cofactor, opening new opportunities for the study of this intriguing cofactor and of NPN-utilizing enzymes.

PBP2b plays a key role in both peripheral growth and septum positioning in Lactococcus lactis.

Lactococcus lactis is an ovoid bacterium that forms filaments during planktonic and biofilm lifestyles by uncoupling cell division from cell elongation. In this work, we investigate the role of the leading peptidoglycan synthase PBP2b that is dedicated to cell elongation in ovococci. We show that the localization of a fluorescent derivative of PBP2b remains associated to the septal region and superimposed with structural changes of FtsZ during both vegetative growth and filamentation indicating that PBP2b remains intimately associated to the division machinery during the whole cell cycle. In addition, we show that PBP2b-negative cells of L. lactis are not only defective in peripheral growth; they are also affected in septum positioning. This septation defect does not simply result from the absence of the protein in the cell growth machinery since it is also observed when PBP2b-deficient cells are complemented by a catalytically inactive variant of PBP2b. Finally, we show that round cells resulting from ß-lactam treatment are not altered in septation, suggesting that shape elongation as such is not a major determinant for selection of the division site. Altogether, we propose that the specific PBP2b transpeptidase activity at the septum plays an important role for tagging future division sites during L. lactis cell cycle.

Role of cell surface composition and lysis in static biofilm formation by Lactobacillus plantarum WCFS1.

Next to applications in fermentations, Lactobacillus plantarum is recognized as a food spoilage organism, and its dispersal from biofilms in food processing environments might be implicated in contamination or recontamination of food products. This study provides new insights into biofilm development by L. plantarum WCFS1 through comparative analysis of wild type and mutants affected in cell surface composition, including mutants deficient in the production of Sortase A involved in the covalent attachment of 27 predicted surface proteins to the cell wall peptidoglycan (ΔsrtA) and mutants deficient in the production of capsular polysaccharides (CPS1-4, Δcps1-4). Surface adhesion and biofilm formation studies revealed none of the imposed cell surface modifications to affect the initial attachment of cells to polystyrene while biofilm formation based on Crystal Violet (CV) staining was severely reduced in the ΔsrtA mutant and significantly increased in mutants lacking the cps1 cluster, compared to the wild-type strain. Fluorescence microscopy analysis of biofilm samples pointed to a higher presence of extracellular DNA (eDNA) in cps1 mutants and this corresponded with increased autolysis activity. Subsequent studies using Δacm2 and ΔlytA derivatives affected in lytic behaviour revealed reduced biofilm formation measured by CV staining, confirming the relevance of lysis for the build-up of the biofilm matrix with eDNA.

Circuitry Rewiring Directly Couples Competence to Predation in the Gut Dweller Streptococcus salivarius.

Small distortions in transcriptional networks might lead to drastic phenotypical changes, especially in cellular developmental programs such as competence for natural transformation. Here, we report a pervasive circuitry rewiring for competence and predation interplay in commensal streptococci. Canonically, in streptococci paradigms such as Streptococcus pneumoniae and Streptococcus mutans, the pheromone-based two-component system BlpRH is a central node that orchestrates the production of antimicrobial compounds (bacteriocins) and incorporates signal from the competence activation cascade. However, the human commensal Streptococcus salivarius does not contain a functional BlpRH pair, while the competence signaling system ComRS directly couples bacteriocin production and competence commitment. This network shortcut might underlie an optimal adaptation against microbial competitors and explain the high prevalence of S. salivarius in the human digestive tract. Moreover, the broad spectrum of bacteriocin activity against pathogenic bacteria showcases the commensal and genetically tractable S. salivarius species as a user-friendly model for competence and bacterial predation.

Unexpected complexity in the lactate racemization system of lactic acid bacteria.

Analysis of lactate racemase (Lar) in lactic acid bacteria (LAB) has been a scientific challenge for many years, as indicated by the numerous contradictory reports on this activity. Recently, genetic and biochemical studies of the Lar system of Lactobacillus plantarum have unveiled the complexity of this particular enzymatic system. Lar activity is associated with LarA and its nickel-containing cofactor, synthesized from nicotinic acid adenine dinucleotide by the three biosynthetic enzymes: LarB, LarC, and LarE. In addition to these core Lar enzymes, a nickel transporter (Lar(MN)QO), a lactic acid channel (LarD) and a transcriptional regulator (LarR) which promotes expression of the lar genes in the presence of excess L-lactate are also part of the Lar system of Lb. plantarum and of many other LAB. These proteins promote racemization of external L-lactate, in addition to carrying out intracellular racemization. This additional outcome suggests that racemization of L-lactate is not only required for cell wall biosynthesis, as reported before, but may have additional roles in lactate production and utilization in LAB. Finally, bioinformatics analyses indicate that some Lar homologs probably catalyze reactions other than lactate racemization.

Natural DNA Transformation Is Functional in Lactococcus lactis subsp. cremoris KW2.

Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

Correction: Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS.

[This corrects the article DOI: 10.1371/journal.ppat.1005980.].

Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS.

In Gram-positive bacteria, cell-to-cell communication mainly relies on extracellular signaling peptides, which elicit a response either indirectly, by triggering a two-component phosphorelay, or directly, by binding to cytoplasmic effectors. The latter comprise the RNPP family (Rgg and original regulators Rap, NprR, PrgX and PlcR), whose members regulate important bacterial processes such as sporulation, conjugation, and virulence. RNPP proteins are increasingly considered as interesting targets for the development of new antibacterial agents. These proteins are characterized by a TPR-type peptide-binding domain, and except for Rap proteins, also contain an N-terminal HTH-type DNA-binding domain and display a transcriptional activity. Here, we elucidate the structure-function relationship of the transcription factor ComR, a new member of the RNPP family, which positively controls competence for natural DNA transformation in streptococci. ComR is directly activated by the binding of its associated pheromone XIP, the mature form of the comX/sigX-inducing-peptide ComS. The crystal structure analysis of ComR from Streptococcus thermophilus combined with a mutational analysis and in vivo assays allows us to propose an original molecular mechanism of the ComR regulation mode. XIP-binding induces release of the sequestered HTH domain and ComR dimerization to allow DNA binding. Importantly, we bring evidence that this activation mechanism is conserved and specific to ComR orthologues, demonstrating that ComR is not an Rgg protein as initially proposed, but instead constitutes a new member of the RNPP family. In addition, identification of XIP and ComR residues important for competence activation constitutes a crucial step towards the design of antagonistic strategies to control gene exchanges among streptococci.

Yeast Gdt1 is a Golgi-localized calcium transporter required for stress-induced calcium signaling and protein glycosylation.

Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. We have recently reported that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and might form a new group of Golgi-localized cation/Ca(2+) exchangers. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis, we demonstrated the calcium transport activity of Gdt1p. We observed a Ca(2+) uptake activity in cells expressing GDT1, which was dependent on the external pH, indicating that Gdt1p may act as a Ca(2+)/H(+) antiporter. In yeast, we found that Gdt1p controls cellular calcium stores and plays a major role in the calcium response induced by osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external calcium, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Finally we showed that glycosylation process is restored by providing more Mn(2+) to the cells.