RESUMO
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.
Assuntos
Proteínas de Bactérias , Regulon , Proteínas de Bactérias/metabolismo , Competência de Transformação por DNA/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Peptídeos/genética , Feromônios/genética , Feromônios/metabolismo , Regulon/genética , Transdução de Sinais/genéticaRESUMO
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.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Hidroxiácidos/química , Níquel/metabolismo , Nucleotídeos/metabolismo , Racemases e Epimerases/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Modelos Moleculares , Níquel/química , Nucleotídeos/química , Conformação Proteica , Racemases e Epimerases/químicaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Citidina Trifosfato/metabolismo , Lactobacillus plantarum/enzimologia , Níquel/metabolismo , Nucleotídeos/metabolismo , Compostos Organometálicos/metabolismo , Racemases e Epimerases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Cristalografia por Raios X , Hidrólise , Modelos Moleculares , Níquel/química , Nucleotídeos/química , Compostos Organometálicos/química , Conformação Proteica , Racemases e Epimerases/química , Racemases e Epimerases/genética , Homologia de SequênciaRESUMO
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.
Assuntos
Aminoaciltransferases/genética , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Cisteína Endopeptidases/genética , Lactobacillus plantarum/crescimento & desenvolvimento , Lactobacillus plantarum/genética , Peptidoglicano/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Manipulação de Alimentos , Microbiologia de Alimentos , Glicosiltransferases/genética , Proteínas de Membrana/metabolismo , Peptidoglicano/genéticaRESUMO
The lactate racemase enzyme (LarA) of Lactobacillus plantarum harbors a (SCS)Ni(II) pincer complex derived from nicotinic acid. Synthesis of the enzyme-bound cofactor requires LarB, LarC, and LarE, which are widely distributed in microorganisms. The functions of the accessory proteins are unknown, but the LarB C terminus resembles aminoimidazole ribonucleotide carboxylase/mutase, LarC binds Ni and could act in Ni delivery or storage, and LarE is a putative ATP-using enzyme of the pyrophosphatase-loop superfamily. Here, we show that LarB carboxylates the pyridinium ring of nicotinic acid adenine dinucleotide (NaAD) and cleaves the phosphoanhydride bond to release AMP. The resulting biscarboxylic acid intermediate is transformed into a bisthiocarboxylic acid species by two single-turnover reactions in which sacrificial desulfurization of LarE converts its conserved Cys176 into dehydroalanine. Our results identify a previously unidentified metabolic pathway from NaAD using unprecedented carboxylase and sulfur transferase reactions to form the organic component of the (SCS)Ni(II) pincer cofactor of LarA. In species where larA is absent, this pathway could be used to generate a pincer complex in other enzymes.
Assuntos
Lactobacillus plantarum/enzimologia , NAD/análogos & derivados , Níquel/metabolismo , Racemases e Epimerases/fisiologia , Enxofre/metabolismo , Biocatálise , Carboxiliases/fisiologia , Redes e Vias Metabólicas , NAD/metabolismoRESUMO
Lactic acid racemization is involved in lactate metabolism and cell wall assembly of many microorganisms. Lactate racemase (Lar) requires nickel, but the nickel-binding site and the role of three accessory proteins required for its activation remain enigmatic. We combined mass spectrometry and x-ray crystallography to show that Lar from Lactobacillus plantarum possesses an organometallic nickel-containing prosthetic group. A nicotinic acid mononucleotide derivative is tethered to Lys(184) and forms a tridentate pincer complex that coordinates nickel through one metal-carbon and two metal-sulfur bonds, with His(200) as another ligand. Although similar complexes have been previously synthesized, there was no prior evidence for the existence of pincer cofactors in enzymes. The wide distribution of the accessory proteins without Lar suggests that it may play a role in other enzymes.
Assuntos
Proteínas de Bactérias/química , Ácido Láctico/biossíntese , Lactobacillus plantarum/enzimologia , Metaloproteínas/química , Niacina/química , Níquel/química , Racemases e Epimerases/química , Proteínas de Bactérias/genética , Sítios de Ligação , Carbono/química , Catálise , Cristalografia por Raios X , Histidina/química , Holoenzimas/química , Ácido Láctico/química , Lactobacillus plantarum/genética , Ligantes , Lisina/química , Metaloproteínas/genética , Mononucleotídeo de Nicotinamida/análogos & derivados , Mononucleotídeo de Nicotinamida/química , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Racemases e Epimerases/genética , Espectrometria de Massas por Ionização por Electrospray , EnxofreRESUMO
Competence for natural DNA transformation is a tightly controlled developmental process in streptococci. In mutans and salivarius species, the abundance of the central competence regulator σ(X) is regulated at two levels: transcriptional, by the ComRS signaling system via the σ(X)/ComX/SigX-inducing peptide (XIP), and posttranscriptional, by the adaptor protein MecA and its associated Clp ATPase, ClpC. In this study, we further investigated the mechanism and function of the MecA-ClpC control system in the salivarius species Streptococcus thermophilus. Using in vitro approaches, we showed that MecA specifically interacts with both σ(X) and ClpC, suggesting the formation of a ternary σ(X)-MecA-ClpC complex. Moreover, we demonstrated that MecA ultimately targets σ(X) for its degradation by the ClpCP protease in an ATP-dependent manner. We also identify a short sequence (18 amino acids) in the N-terminal domain of σ(X) as essential for the interaction with MecA and subsequent σ(X) degradation. Finally, increased transformability of a MecA-deficient strain in the presence of subinducing XIP concentrations suggests that the MecA-ClpCP proteolytic complex acts as an additional locking device to prevent competence under inappropriate conditions. A model of the interplay between ComRS and MecA-ClpCP in the control of σ(X) activity is proposed.
Assuntos
Proteínas de Bactérias/genética , Competência de Transformação por DNA/genética , Regulação Bacteriana da Expressão Gênica , Streptococcus thermophilus/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos , Estrutura Terciária de Proteína , Proteólise , Fator sigma/genética , Fator sigma/metabolismo , Streptococcus thermophilus/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transformação BacterianaRESUMO
Racemases catalyse the inversion of stereochemistry in biological molecules, giving the organism the ability to use both isomers. Among them, lactate racemase remains unexplored due to its intrinsic instability and lack of molecular characterization. Here we determine the genetic basis of lactate racemization in Lactobacillus plantarum. We show that, unexpectedly, the racemase is a nickel-dependent enzyme with a novel α/ß fold. In addition, we decipher the process leading to an active enzyme, which involves the activation of the apo-enzyme by a single nickel-containing maturation protein that requires preactivation by two other accessory proteins. Genomic investigations reveal the wide distribution of the lactate racemase system among prokaryotes, showing the high significance of both lactate enantiomers in carbon metabolism. The even broader distribution of the nickel-based maturation system suggests a function beyond activation of the lactate racemase and possibly linked with other undiscovered nickel-dependent enzymes.
Assuntos
Ácido Láctico/metabolismo , Lactobacillus plantarum/enzimologia , Níquel , Racemases e Epimerases/metabolismo , Lactobacillus plantarum/genética , Dobramento de Proteína , Racemases e Epimerases/genética , EstereoisomerismoRESUMO
Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/genética , Proteoma/genética , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Células CACO-2 , Cromatografia Líquida , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/ultraestrutura , Humanos , Intestinos/citologia , Intestinos/microbiologia , Lactococcus lactis/metabolismo , Lactococcus lactis/ultraestrutura , Microscopia Eletrônica , Anotação de Sequência Molecular , Dados de Sequência Molecular , Família Multigênica , Fragmentos de Peptídeos/análise , Plasmídeos , Probióticos/química , Proteólise , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Tripsina/químicaRESUMO
Peptidoglycan (PG) is the major component of Gram positive bacteria cell wall and is essential for bacterial integrity and shape. Bacteria synthesize PG hydrolases (PGHs) which are able to cleave bonds in their own PG and play major roles in PG remodelling required for bacterial growth and division. Our aim was to identify the main PGHs in Lactobacillus casei BL23, a lactic acid bacterium with probiotic properties.The PGH complement was first identified in silico by amino acid sequence similarity searches of the BL23 genome sequence. Thirteen PGHs were detected with different predicted hydrolytic specificities. Transcription of the genes was confirmed by RT-PCR. A proteomic analysis combining the use of SDS-PAGE and LC-MS/MS revealed the main seven PGHs synthesized during growth of L. casei BL23. Among these PGHs, LCABL_02770 (renamed Lc-p75) was identified as the major one. This protein is the homolog of p75 (Msp1) major secreted protein of Lactobacillus rhamnosus GG, which was shown to promote survival and growth of intestinal epithelial cells. We identified its hydrolytic specificity on PG and showed that it is a γ-D-glutamyl-L-lysyl-endopeptidase. It has a marked specificity towards PG tetrapeptide chains versus tripeptide chains and for oligomers rather than monomers. Immunofluorescence experiments demonstrated that Lc-p75 localizes at cell septa in agreement with its role in daughter cell separation. It is also secreted under an active form as detected in zymogram. Comparison of the muropeptide profiles of wild type and Lc-p75-negative mutant revealed a decrease of the amount of disaccharide-dipeptide in the mutant PG in agreement with Lc-p75 activity. As a conclusion, Lc-p75 is the major L. casei BL23 PGH with endopeptidase specificity and a key role in daughter cell separation. Further studies will aim at investigating the role of Lc-p75 in the anti-inflammatory potential of L. casei BL23.
Assuntos
Endopeptidases/biossíntese , Lacticaseibacillus casei/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Serina Endopeptidases/química , Cromatografia Líquida/métodos , Proteínas do Sistema Complemento , Biologia Computacional/métodos , Eletroforese em Gel de Poliacrilamida , Endopeptidases/genética , Teste de Complementação Genética , Genoma Bacteriano , Hidrólise , Modelos Biológicos , Mutação , Peptídeos/química , Fenótipo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Transcrição GênicaRESUMO
In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.
Assuntos
Cápsulas Bacterianas/química , Parede Celular/química , Lactococcus lactis/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologia , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Carboidratos , Células Cultivadas , Cromossomos Bacterianos , Macrófagos/microbiologia , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Polissacarídeos Bacterianos/ultraestruturaRESUMO
In streptococcal species, the key step of competence development is the transcriptional induction of comX, which encodes the alternative sigma factor sigma(X), which positively regulates genes necessary for DNA transformation. In Streptococcus species belonging to the mitis and mutans groups, induction of comX relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In Streptococcus thermophilus, a species belonging to the salivarius group, the oligopeptide transporter Ami is essential for comX expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of comX induction in S. thermophilus LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for comX induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored comX induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in S. thermophilus and Streptococcus salivarius strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for comX induction in species from the salivarius group, which differs from the classical phosphorelay three-component system identified previously in streptococci.
Assuntos
Regulação Bacteriana da Expressão Gênica , Feromônios/metabolismo , Percepção de Quorum , Transdução de Sinais , Streptococcus thermophilus/fisiologia , Transformação Genética , Proteínas de Bactérias/biossíntese , Perfilação da Expressão Gênica , Óperon , Peptídeos/genética , Peptídeos/metabolismo , Feromônios/genética , Deleção de Sequência , Fatores de Transcrição/biossínteseRESUMO
In this report, we describe the amino acid metabolism and amino acid dependency of the dairy bacterium Streptococcus thermophilus LMG18311 and compare them with those of two other characterized lactic acid bacteria, Lactococcus lactis and Lactobacillus plantarum. Through the construction of a genome-scale metabolic model of S. thermophilus, the metabolic differences between the three bacteria were visualized by direct projection on a metabolic map. The comparative analysis revealed the minimal amino acid auxotrophy (only histidine and methionine or cysteine) of S. thermophilus LMG18311 and the broad variety of volatiles produced from amino acids compared to the other two bacteria. It also revealed the limited number of pyruvate branches, forcing this strain to use the homofermentative metabolism for growth optimization. In addition, some industrially relevant features could be identified in S. thermophilus, such as the unique pathway for acetaldehyde (yogurt flavor) production and the absence of a complete pentose phosphate pathway.
Assuntos
Aminoácidos/metabolismo , Lactobacillus plantarum/metabolismo , Lactococcus lactis/metabolismo , Redes e Vias Metabólicas/genética , Streptococcus thermophilus/metabolismo , Acetaldeído/metabolismo , Metaboloma , Modelos Biológicos , Piruvatos/metabolismo , Streptococcus thermophilus/genéticaRESUMO
FtsH proteins have dual chaperone-protease activities and are involved in protein quality control under stress conditions. Although the functional role of FtsH proteins has been clearly established, the regulatory mechanisms controlling ftsH expression in gram-positive bacteria remain largely unknown. Here we show that ftsH of Lactobacillus plantarum WCFS1 is transiently induced at the transcriptional level upon a temperature upshift. In addition, disruption of ftsH negatively affected the growth of L. plantarum at high temperatures. Sequence analysis and mapping of the ftsH transcriptional start site revealed a potential operator sequence for the CtsR repressor, partially overlapping the -35 sequence of the ftsH promoter. In order to verify whether CtsR is able to recognize and bind the ftsH promoter, CtsR proteins of Bacillus subtilis and L. plantarum were overproduced, purified, and used in DNA binding assays. CtsR from both species bound specifically to the ftsH promoter, generating a single protein-DNA complex, suggesting that CtsR may control the expression of L. plantarum ftsH. In order to confirm this hypothesis, a DeltactsR mutant strain of L. plantarum was generated. Expression of ftsH in the DeltactsR mutant strain was strongly upregulated, indicating that ftsH of L. plantarum is negatively controlled by CtsR. This is the first example of an ftsH gene controlled by the CtsR repressor, and the first of the low-G+C gram-positive bacteria where the regulatory mechanism has been identified.
Assuntos
Proteases Dependentes de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico , Lactobacillus plantarum/crescimento & desenvolvimento , Proteínas Repressoras/metabolismo , Proteases Dependentes de ATP/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Deleção de Genes , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Lactobacillus plantarum/fisiologia , Mutação , Regiões Promotoras Genéticas , Regulon , Proteínas Repressoras/genéticaRESUMO
Streptococcus thermophilus is one of the most widely used lactic acid bacteria in the dairy industry, in particular in yoghurt manufacture, where it is associated with Lactobacillus delbrueckii subsp. bulgaricus. This bacterial association, known as a proto-cooperation, is poorly documented at the molecular and regulatory levels. We thus investigate the kinetics of the transcriptomic and proteomic modifications of S. thermophilus LMG 18311 in response to the presence of L. delbrueckii subsp. bulgaricus ATCC 11842 during growth in milk at two growth stages. Seventy-seven different genes or proteins (4.1% of total coding sequences), implicated mainly in the metabolism of nitrogen (24%), nucleotide base (21%), and iron (20%), varied specifically in coculture. One of the most unpredicted results was a significant decrease of most of the transcripts and enzymes involved in purine biosynthesis. Interestingly, the expression of nearly all genes potentially encoding iron transporters of S. thermophilus decreased, whereas that of iron-chelating dpr as well as that of the fur (perR) regulator genes increased, suggesting a reduction in the intracellular iron concentration, probably in response to H(2)O(2) production by L. bulgaricus. The present study reveals undocumented nutritional exchanges and regulatory relationships between the two yoghurt bacteria, which provide new molecular clues for the understanding of their associative behavior.
Assuntos
Ferro/metabolismo , Lactobacillus delbrueckii/crescimento & desenvolvimento , Leite/microbiologia , Nitrogênio/metabolismo , Purinas/metabolismo , Streptococcus thermophilus/química , Streptococcus thermophilus/genética , Animais , Proteínas de Bactérias/análise , Técnicas de Cocultura , Perfilação da Expressão Gênica , Proteoma/análise , Streptococcus thermophilus/crescimento & desenvolvimentoRESUMO
The blp(St) cluster of Streptococcus thermophilus LMD-9 was recently shown to contain all the genetic information required for the production of bacteriocins active against other S. thermophilus strains. In this study, we further investigated the antimicrobial activity of S. thermophilus LMD-9 by testing the susceptibility of 31 bacterial species (87 strains). We showed that LMD-9 displays an inhibitory spectrum targeted toward related gram-positive bacteria, including pathogens such as Listeria monocytogenes. Using deletion mutants, we investigated the contribution of the three putative bacteriocin-encoding operons blpD(St)-orf2, blpU(St)-orf3, and blpE(St)-blpF(St) (bac(St) operons) and of the blpG(St) gene, which encodes a putative modification protein, to the inhibitory spectrum and immunity of strain LMD-9. Our results present evidence that the blp(St) locus encodes a multipeptide bacteriocin system called thermophilin 9. Among the four class II bacteriocin-like peptides encoded within the bac(St) operons, BlpD(St) alone was sufficient to inhibit the growth of most thermophilin 9-sensitive species. The blpD(St) gene forms an operon with its associated immunity gene(s), and this functional bacteriocin/immunity module could easily be transferred to Lactococcus lactis. The remaining three Bac(St) peptides, BlpU(St), BlpE(St), and BlpF(St), confer poor antimicrobial activity but act as enhancers of the antagonistic activity of thermophilin 9 by an unknown mechanism. The blpG(St) gene was also shown to be specifically required for the antilisteria activity of thermophilin 9, since its deletion abolished the sensitivities of most Listeria species. By complementation of the motility deficiency of Escherichia coli dsbA, we showed that blpG(St) encodes a functional thiol-disulfide oxidase, suggesting an important role for disulfide bridges within thermophilin 9.
Assuntos
Bacteriocinas/genética , Família Multigênica/genética , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Streptococcus thermophilus/genética , Sequência de Aminoácidos , Bacteriocinas/farmacologia , Sequência de Bases , Primers do DNA/genética , Deleção de Genes , Vetores Genéticos/genética , Bactérias Gram-Positivas/efeitos dos fármacos , Dados de Sequência Molecular , Óperon/genética , Peptídeos/genética , Peptídeos/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/genética , Análise de Sequência de DNA , Especificidade da Espécie , Streptococcus thermophilus/patogenicidadeRESUMO
Streptococcus thermophilus PB18 can grow between 20 degrees and 52 degrees C and is resistant to various stresses such as heat, acidic or cold shock. During cold shock, a protein of 21.5 kDa was previously shown to be induced in S. thermophilus. In addition to its cold-shock induction, 2D-PAGE revealed that the 21.5-kDa protein was also expressed during the stationary phase of growth. The recent access to the genome sequence of S. thermophilus LMG18311 allowed the identification of a 173-amino acid protein displaying a strong homology between the 21.5-kDa protein and members of the Dps family of proteins. Specific staining of non-denaturing polyacrylamide gel electrophoresis (ND-PAGE) followed by two-dimensional PAGE (2D-PAGE) showed that the 21.5-kDa protein was an iron-binding protein.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Ligação ao Ferro/isolamento & purificação , Streptococcus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Temperatura Baixa , Eletroforese em Gel Bidimensional , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/genética , Ponto Isoelétrico , Dados de Sequência Molecular , Peso Molecular , Alinhamento de Sequência , Streptococcus/genética , Streptococcus/isolamento & purificaçãoRESUMO
Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.