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

ABSTRACT 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.

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ZpdN, a Plasmid-Encoded Sigma Factor Homolog, Induces pBS32-Dependent Cell Death in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00213-16 ◽

2016 ◽

Vol 198 (21) ◽

pp. 2975-2984 ◽

Cited By ~ 6

Author(s):

B.-E. Myagmarjav ◽

M. A. Konkol ◽

J. Ramsey ◽

S. Mukhopadhyay ◽

D. B. Kearns

Keyword(s):

Cell Death ◽

Bacillus Subtilis ◽

Mitomycin C ◽

Sigma Factor ◽

Natural Transformation ◽

Potent Inhibitor ◽

Dna Uptake ◽

Content Type ◽

Dependent Cell ◽

Necessary And Sufficient

ABSTRACTThe ancestralBacillus subtilisstrain 3610 contains an 84-kb plasmid called pBS32 that was lost during domestication of commonly used laboratory derivatives. Here we demonstrate that pBS32, normally present at 1 or 2 copies per cell, increases in copy number nearly 100-fold when cells are treated with the DNA-damaging agent mitomycin C. Mitomycin C treatment also caused cell lysis dependent on pBS32-borne prophage genes. ZpdN, a sigma factor homolog encoded by pBS32, was required for the plasmid response to DNA damage, and artificial expression of ZpdN was sufficient to induce pBS32 hyperreplication and cell death. Plasmid DNA released by cell death was protected by the capsid protein ZpbH, suggesting that the plasmid was packaged into a phagelike particle. The putative particles were further indicated by CsCl sedimentation but were not observed by electron microscopy and were incapable of killingB. subtiliscells extracellularly. We hypothesize that pBS32-mediated cell death releases a phagelike particle that is defective and unstable.IMPORTANCEProphages are phage genomes stably integrated into the host bacterium's chromosome and less frequently are maintained as extrachromosomal plasmids. Here we report that the extrachromosomal plasmid pBS32 ofBacillus subtilisencodes a prophage that, when activated, kills the host. pBS32 also encodes both the sigma factor homolog ZpdN that is necessary and sufficient for prophage induction and the protein ComI, which is a potent inhibitor of DNA uptake by natural transformation. We provide evidence that the entire pBS32 sequence may be part of the prophage and thus that competence inhibition may be linked to lysogeny.

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Lysis of a Lactococcus lactis Dipeptidase Mutant and Rescue by Mutation in the Pleiotropic Regulator CodY

Applied and Environmental Microbiology ◽

10.1128/aem.02937-19 ◽

2020 ◽

Vol 86 (8) ◽

Cited By ~ 2

Author(s):

Chenxi Huang ◽

Jhonatan A. Hernandez-Valdes ◽

Oscar P. Kuipers ◽

Jan Kok

Keyword(s):

Cell Wall ◽

Amino Acid ◽

Nitrogen Metabolism ◽

Lactococcus Lactis ◽

Cell Shape ◽

Single Point Mutation ◽

Binding Motif ◽

Acid Uptake ◽

Cell Wall Synthesis ◽

Content Type

ABSTRACT Lactococcus lactis subsp. cremoris MG1363 is a model for the lactic acid bacteria (LAB) used in the dairy industry. The proteolytic system, consisting of a proteinase, several peptide and amino acid uptake systems, and a host of intracellular peptidases, plays a vital role in nitrogen metabolism and is of eminent importance for flavor formation in dairy products. The dipeptidase PepV functions in the last stages of proteolysis. A link between nitrogen metabolism and peptidoglycan (PG) biosynthesis was underlined by the finding that deletion of the dipeptidase gene pepV (creating strain MGΔpepV) resulted in a prolonged lag phase when the mutant strain was grown with a high concentration of glycine. In addition, most MGΔpepV cells lyse and have serious defects in their shape. This phenotype is due to a shortage of alanine, since adding alanine can rescue the growth and shape defects. Strain MGΔpepV is more resistant to vancomycin, an antibiotic targeting peptidoglycan d-Ala–d-Ala ends, which confirmed that MGΔpepV has an abnormal PG composition. A mutant of MGΔpepV was obtained in which growth inhibition and cell shape defects were alleviated. Genome sequencing showed that this mutant has a single point mutation in the codY gene, resulting in an arginine residue at position 218 in the DNA-binding motif of CodY being replaced by a cysteine residue. Thus, this strain was named MGΔpepVcodYR218C. Transcriptome sequencing (RNA-seq) data revealed a dramatic derepression in peptide uptake and amino acid utilization in MGΔpepVcodYR218C. A model of the connections among PepV activity, CodY regulation, and PG synthesis of L. lactis is proposed. IMPORTANCE Precise control of peptidoglycan synthesis is essential in Gram-positive bacteria for maintaining cell shape and integrity as well as resisting stresses. Although neither the dipeptidase PepV nor alanine is essential for L. lactis MG1363, adequate availability of either ensures proper cell wall synthesis. We broaden the knowledge about the dipeptidase PepV, which acts as a linker between nitrogen metabolism and cell wall synthesis in L. lactis.

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The KupA and KupB Proteins of Lactococcus lactis IL1403 Are Novel c-di-AMP Receptor Proteins Responsible for Potassium Uptake

Journal of Bacteriology ◽

10.1128/jb.00028-19 ◽

2019 ◽

Vol 201 (10) ◽

Cited By ~ 14

Author(s):

Ingrid M. Quintana ◽

Johannes Gibhardt ◽

Asan Turdiev ◽

Elke Hammer ◽

Fabian M. Commichau ◽

...

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacterium ◽

Lactococcus Lactis ◽

Second Messenger ◽

Cell Factory ◽

Content Type ◽

Potassium Homeostasis ◽

Rna Molecules ◽

High Affinity ◽

Potassium Transporters

ABSTRACT Cyclic di-AMP (c-di-AMP) is a second messenger involved in diverse metabolic processes, including osmolyte uptake, cell wall homeostasis, and antibiotic and heat resistance. In Lactococcus lactis, a lactic acid bacterium which is used in the dairy industry and as a cell factory in biotechnological processes, the only reported interaction partners of c-di-AMP are the pyruvate carboxylase and BusR, the transcription regulator of the busAB operon for glycine betaine uptake. However, recent studies uncovered a major role of c-di-AMP in the control of potassium homeostasis, and potassium is the signal that triggers c-di-AMP synthesis. In this study, we have identified KupA and KupB, which belong to the Kup/HAK/KT family, as novel c-di-AMP binding proteins. Both proteins are high-affinity potassium transporters, and their transport activities are inhibited by binding of c-di-AMP. Thus, in addition to the well-studied Ktr/Trk potassium channels, KupA and KupB represent a second class of potassium transporters that are subject to inhibition by c-di-AMP. IMPORTANCE Potassium is an essential ion in every living cell. Even though potassium is the most abundant cation in cells, its accumulation can be toxic. Therefore, the level of potassium has to be tightly controlled. In many Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in the control of potassium homeostasis by binding to potassium transporters and regulatory proteins and RNA molecules. In the lactic acid bacterium Lactococcus lactis, none of these conserved c-di-AMP-responsive molecules are present. In this study, we demonstrate that the KupA and KupB proteins of L. lactis IL1403 are high-affinity potassium transporters and that their transport activity is inhibited by the second messenger c-di-AMP.

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Genome-Wide Transcriptional Responses to Carbon Starvation in Nongrowing Lactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.03748-14 ◽

2015 ◽

Vol 81 (7) ◽

pp. 2554-2561 ◽

Cited By ~ 15

Author(s):

Onur Ercan ◽

Michiel Wels ◽

Eddy J. Smid ◽

Michiel Kleerebezem

Keyword(s):

Lactococcus Lactis ◽

Natural Transformation ◽

Recovery Period ◽

Transcriptional Responses ◽

Content Type ◽

Gene Set ◽

Expression Of Genes ◽

Genome Wide ◽

Elevated Expression ◽

Branched Chain

ABSTRACTThis paper describes the transcriptional adaptations of nongrowing, retentostat cultures ofLactococcus lactisto starvation. Near-zero-growth cultures (μ = 0.0001 h−1) obtained by extended retentostat cultivation were exposed to starvation by termination of the medium supply for 24 h, followed by a recovery period of another 24 h by reinitiating the medium supply to the retentostat culture. During starvation, the viability of the culture was largely retained, and the expression of genes involved in transcription and translational machineries, cell division, and cell membrane energy metabolism was strongly repressed. Expression of these genes was largely recovered following the reinitiation of the medium supply. Starvation triggered the elevated expression of genes associated with synthesis of branched-chain amino acids, histidine, purine, and riboflavin. The expression of these biosynthesis genes was found to remain at an elevated level after reinitiation of the medium supply. In addition, starvation induced the complete gene set predicted to be involved in natural competence inL. lactisKF147, and the elevated expression of these genes was sustained during the subsequent recovery period, but our attempts to experimentally demonstrate natural transformation in these cells failed. Mining the starvation response gene set identified a conservedcis-acting element that resembles the lactococcal CodY motif in the upstream regions of genes associated with transcription and translational machineries, purine biosynthesis, and natural transformation inL. lactis, suggesting a role for CodY in the observed transcriptome adaptations to starvation in nongrowing cells.

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Reducing the Level of Undecaprenyl Pyrophosphate Synthase Has Complex Effects on Susceptibility to Cell Wall Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00794-13 ◽

2013 ◽

Vol 57 (9) ◽

pp. 4267-4275 ◽

Cited By ~ 16

Author(s):

Yong Heon Lee ◽

John D. Helmann

Keyword(s):

Cell Wall ◽

Single Point ◽

Single Point Mutation ◽

Wild Type ◽

Content Type ◽

Ribosome Binding ◽

Peptidoglycan Biosynthesis ◽

Elevated Expression ◽

Antibacterial Target ◽

Bacterial Peptidoglycan

ABSTRACTUndecaprenyl pyrophosphate synthase (UppS) catalyzes the formation of the C55lipid carrier (UPP) that is essential for bacterial peptidoglycan biosynthesis. We selected here a vancomycin (VAN)-resistant derivative ofBacillus subtilisW168 that contains a single-point mutation in the ribosome-binding site of theuppSgene designateduppS1. Genetic reconstruction experiments demonstrate that theuppS1allele is sufficient to confer low-level VAN resistance and causes reduced UppS translation. The decreased level of UppS rendersB. subtilisslightly more susceptible to many late-acting cell wall antibiotics, including β-lactams, but significantly more resistant to fosfomycin andd-cycloserine, antibiotics that interfere with the very early steps of cell wall synthesis. We further show that theuppS1allele leads to slightly elevated expression of the σMregulon, possibly helping to compensate for the stress caused by a decrease in UPP levels. Notably, theuppS1mutation increases resistance to VAN, fosfomycin, andd-cycloserine in wild-type cells, but this effect is greatly reduced or eliminated in asigMmutant background. Our findings suggest that, although UppS is an attractive antibacterial target, incomplete inhibition of UppS function may lead to increased resistance to some cell wall-active antibiotics.

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Task Distribution between Acetate and Acetoin Pathways To Prolong Growth inLactococcus lactisunder Respiration Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.01005-18 ◽

2018 ◽

Vol 84 (18) ◽

Cited By ~ 3

Author(s):

Bénédicte Cesselin ◽

Christel Garrigues ◽

Martin B. Pedersen ◽

Célia Roussel ◽

Alexandra Gruss ◽

...

Keyword(s):

Lactic Acid ◽

Stationary Phase ◽

Lactococcus Lactis ◽

Time Course ◽

Biomass Yield ◽

Fine Tuning ◽

Ph Homeostasis ◽

Content Type ◽

Growth And Survival ◽

Bacterial Physiology

ABSTRACTLactococcus lactisis the main bacterium used for food fermentation and is a candidate for probiotic development. In addition to fermentation growth, supplementation with heme under aerobic conditions activates a cytochrome oxidase, which promotes respiration metabolism. In contrast to fermentation, in which cells consume energy to produce mainly lactic acid, respiration metabolism dramatically changes energy metabolism, such that massive amounts of acetic acid and acetoin are produced at the expense of lactic acid. Our goal was to investigate the metabolic changes that correlate with significantly improved growth and survival during respiration growth. Using transcriptional time course analyses, mutational analyses, and promoter-reporter fusions, we uncover two main pathways that can explain the robust growth and stability of respiration cultures. First, the acetate pathway contributes to biomass yield in respiration without affecting medium pH. Second, the acetoin pathway allows cells to cope with internal acidification, which directly affects cell density and survival in stationary phase. Our results suggest that manipulation of these pathways will lead to fine-tuning respiration growth, with improved yield and stability.IMPORTANCELactococcus lactisis used in food and biotechnology industries for its capacity to produce lactic acid, aroma, and proteins. This species grows by fermentation or by an aerobic respiration metabolism when heme is added. Whereas fermentation leads mostly to lactic acid production, respiration produces acetate and acetoin. Respiration growth leads to greatly improved bacterial growth and survival. Our study aims at deciphering mechanisms of respiration metabolism that have a major impact on bacterial physiology. Our results showed that two metabolic pathways (acetate and acetoin) are key elements of respiration. The acetate pathway contributes to biomass yield. The acetoin pathway is needed for pH homeostasis, which affects metabolic activities and bacterial viability in stationary phase. This study clarifies key metabolic elements that are required to maintain the growth advantage conferred by respiration metabolism and has potential uses in strain optimization for industrial and biomedical applications.

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Contribution of Lactococcus lactis Reducing Properties to the Downregulation of a Major Virulence Regulator in Staphylococcus aureus, theagrSystem

Applied and Environmental Microbiology ◽

10.1128/aem.02287-14 ◽

2014 ◽

Vol 80 (22) ◽

pp. 7028-7035 ◽

Cited By ~ 16

Author(s):

Sébastien Nouaille ◽

Lucie Rault ◽

Sophie Jeanson ◽

Pascal Loubière ◽

Yves Le Loir ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Lactic Acid ◽

Lactococcus Lactis ◽

Response Regulator ◽

Food Poisoning ◽

Accessory Gene ◽

Content Type ◽

Accessory Gene Regulator ◽

Virulence Regulator ◽

Reducing Properties

ABSTRACTStaphylococcus aureusis a major cause of food poisoning outbreaks associated with dairy products, because of the ingestion of preformed enterotoxins. The biocontrol ofS. aureususing lactic acid bacteria (LAB) offers a promising opportunity to fight this pathogen while respecting the product ecosystem. We had previously established the ability ofLactococcus lactis, a lactic acid bacterium widely used in the dairy industry, to downregulate a major staphylococcal virulence regulator, the accessory gene regulator (agr) system, and, as a consequence,agr-controlled enterotoxins. In the present paper, we have shown that the oxygen-independent reducing properties ofL. lactiscontribute toagrdownregulation. Neutralizing lactococcal reduction by adding potassium ferricyanide or maintaining the oxygen pressure constant at 50% releasedagrdownregulation in the presence ofL. lactis. This downregulation still occurred in anS. aureus srrAmutant, indicating that the staphylococcal respiratory response regulator SrrAB was not the only component in the signaling pathway. Therefore, this study clearly demonstrates the ability ofL. lactisreducing properties to interfere with the expression ofS. aureusvirulence, thus highlighting this general property of LAB as a lever to control the virulence expression of this major pathogen in a food context and beyond.

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The Proline Variant of the W[F/L/M][T/S]R Cyclic Di-GMP Binding Motif Suppresses Dependence on Signal Association for Regulator Function

Journal of Bacteriology ◽

10.1128/jb.00344-17 ◽

2017 ◽

Vol 199 (19) ◽

Cited By ~ 8

Author(s):

Daniel M. Chodur ◽

Linda Guo ◽

Meng Pu ◽

Eric Bruger ◽

Nico Fernandez ◽

...

Keyword(s):

Vibrio Vulnificus ◽

Single Point ◽

Extracellular Polysaccharide ◽

Single Point Mutation ◽

Binding Motif ◽

Content Type ◽

Bacterial Signaling ◽

Direct Binding ◽

Opportunistic Human Pathogen ◽

The Impact

ABSTRACT Vibrio vulnificus is an estuarine bacterium and potent opportunistic human pathogen. It enters the food chain by asymptomatically colonizing a variety of marine organisms, most notably oysters. Expression of the brp-encoded extracellular polysaccharide, which enhances cell-surface adherence, is regulated by cyclic di-GMP (c-di-GMP) and the activator BrpT. The Vibrio cholerae and Vibrio parahaemolyticus homologs VpsT and CpsQ, directly bind c-di-GMP via a novel W[F/L/M][T/S]R motif, and c-di-GMP binding is absolutely required for activity. Notably, BrpT belongs to a distinct subclass of VpsT-like regulators that harbor a proline in the third position of the c-di-GMP binding motif (WLPR), and the impact of this change on activity is unknown. We show that the brp locus is organized as two linked operons with BrpT specifically binding to promoters upstream of brpA and brpH. Expression data and structural modeling suggested that BrpT might be less dependent on c-di-GMP binding for activity than VpsT or CpsQ. We show that the affinity of BrpT for c-di-GMP is low and that signal binding is not a requisite for BrpT function. Furthermore, a BrpT mutant engineered to carry a canonical WLTR motif (BrpTP124T) bound c-di-GMP with high affinity and its activity was now c-di-GMP dependent. Conversely, introduction of the WLPR motif into VpsT suppressed its dependence on c-di-GMP for activity. This is the first demonstration of reduced dependence on signal association for regulator function within this motif family. Thus, BrpT defines a new class of VpsT-like transcriptional regulators, and the WLPR motif variant may similarly liberate the activity of other subclass members. IMPORTANCE A Vibrio genome may encode nearly 100 proteins that make, break, and bind c-di-GMP, underscoring its central role in the physiology of these bacteria. The activity of the biofilm regulators VpsT of V. cholerae and CpsQ of V. parahaemolyticus is regulated by the direct binding of c-di-GMP via a novel W[F/L/M][T/S]R motif. The V. vulnificus homolog, BrpT, bears an unusual WLPR variant and remains active at low intracellular c-di-GMP levels. This suggests that the WLPR motif may also liberate the activity of other members of this subclass. A single point mutation at the 3rd position of the motif was sufficient to moderate dependence on c-di-GMP binding for activator function, highlighting the simplicity with which complex bacterial signaling networks can be rewired.

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Enhanced Transmission of Antibiotic Resistance in Campylobacter jejuni Biofilms by Natural Transformation

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04066-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7573-7575 ◽

Cited By ~ 37

Author(s):

Junghee Bae ◽

Euna Oh ◽

Byeonghwa Jeon

Keyword(s):

Antibiotic Resistance ◽

Campylobacter Jejuni ◽

Natural Transformation ◽

Antibiotic Resistance Genes ◽

Dna Transformation ◽

Planktonic Cells ◽

Content Type ◽

Enhanced Transmission ◽

Food Borne ◽

Foreign Dna

ABSTRACTCampylobacter jejuniis a leading food-borne pathogen, and its antibiotic resistance is of serious concern to public health worldwide.C. jejuniis naturally competent for DNA transformation and freely takes up foreign DNA harboring genetic information responsible for antibiotic resistance. In this study, we demonstrate thatC. jejunitransfers antibiotic resistance genes more frequently in biofilms than in planktonic cells by natural transformation.

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Production of isomelezitose from sucrose by engineered glucansucrases

Amylase ◽

10.1515/amylase-2017-0008 ◽

2017 ◽

Vol 1 (1) ◽

Cited By ~ 3

Author(s):

Gregory L. Côté ◽

Christopher A. Dunlap ◽

Karl E. Vermillion ◽

Christopher D. Skory

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Biomedical Applications ◽

Large Scale ◽

Single Point ◽

Single Point Mutation ◽

Scale Production ◽

Acceptor Substrate ◽

Large Scale Production ◽

High Yields

AbstractCertain lactic acid bacteria produce glycosyltransferases known as glucansucrases, which synthesize α-D-glucans via glucosyl transfer from sucrose. We recently reported on the formation of the unusual trisaccharide isomelezitose in low yields by a variety of glucansucrases. Isomelezitose is a rare non-reducing trisaccharide, with the structure α-d-glucopyranosyl- (1→6)-β-d-fructofuranosyl-(2↔1)-α-d-glucopyranoside. In this work, we describe the synthesis of isomelezitose in high yields by variants of glucansucrases engineered to contain a single point mutation at a key leucine residue involved in acceptor substrate binding. Some variants produce isomelezitose in yields up to 57%. This method is amenable to large-scale production of isomelezitose for food, industrial and biomedical applications.

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