Eric Guédon

Pleiotropic transcriptional repressor CodY senses the intracellular pool of branched-chain amino acids in Lactococcus lactis

Proteolysis is essential for supplying Lactococcus lactis with amino acids during growth in milk. Expression of the major components of the L. lactis proteolytic system, including the cell wall proteinase (PrtP), the oligopeptide transport system (Opp) and at least four intracellular peptidases (PepO1, PepN, PepC, PepDA2), was shown previously to be controlled negatively by a rich nitrogen source. The transcription of prtP, opp–pepO1, pepN and pepC genes is regulated by dipeptides in the medium. Random insertion mutants derepressed for nitrogen control in the expression of the oligopeptide transport system were isolated using an opp–lacZ fusion. A third of the mutants were targeted in the same locus. The product of the inactivated gene shared 48% identity with CodY from Bacillus subtilis, a pleiotropic repressor of the dipeptide permease operon (dpp) and several genes including genes involved in amino acid degradation and competence induction. The signal controlling CodY‐dependent repression was searched for by analysing the response of the opp–lux fusion to the addition of 67 dipeptides with different amino acid compositions. Full correlation was found between the dipeptide content in branched‐chain amino acids (BCAA; isoleucine, leucine or valine) and their ability to mediate the repression of opp–pepO1 expression. The repressive effect resulting from specific regulatory dipeptides was abolished in L. lactis mutants affected in terms of their transport or degradation into amino acids, showing that the signal was dependent on the BCAA pool in the cell. Lastly, the repression of opp–pepO1 expression was stronger in a mutant unable to degrade BCAAs, underlining the central role of BCAAs as a signal for CodY activity. This pattern of regulation suggests that, in L. lactis and possibly other Gram‐positive bacteria, CodY is a pleiotropic repressor sensing nutritional supply as a function of the BCAA pool in the cell.

Transcriptional Pattern of Genes Coding for the Proteolytic System of Lactococcus lactis and Evidence for Coordinated Regulation of Key Enzymes by Peptide Supply

The transcription of 16 genes encoding 12 peptidases (pepC, pepN, pepX, pepP, pepA, pepF2, pepDA1, pepDA2, pepQ, pepT, pepM, and pepO1), P(I) and P(III) proteinases (prtP1 and prtP3), and three transport systems (dtpT, dtpP, and opp-pepO1) of Lactococcus lactis MG1363 was analyzed in response to different environmental factors. Promoter fusions with luciferase reporter genes and/or mRNA analysis were used to study the effects of sugar sources, growth at 37 degrees C, and peptide supply on the transcription of these genes. Only transcription of the pepP gene is modulated by the source of sugar. The presence of potential catabolite-responsive element (CRE) boxes in its promoter region suggests that expression of this gene is directly controlled by catabolic repression. Elevated temperature had no significant effect on the level of transcription of these genes. prtP1, prtP3, pepC, pepN, pepX, and the opp-pepO1 operon are the most highly expressed genes in chemically defined medium, and their expression is repressed 5- to 150-fold by addition of peptide sources such as Casitone in the medium. Moreover, the transcription of prtP1, prtP3, pepC, pepN, and the opp-pepO1 operon is repressed two- to eight-fold by the dipeptides leucylproline and prolylleucine. The transcription of pepDA2 might also be repressed by the peptide sources, but this effect is not observed on the regulation of dtpT, pepP, pepA, pepF2, pepDA1, pepQ, pepT, pepM, and the dtpP operon. The significance of these results with respect to the functions of different components of the proteolytic system in L. lactis are discussed.

Intracellular effectors regulating the activity of the Lactococcus lactis CodY pleiotropic transcription regulator

CodY is a pleiotropic transcriptional regulator conserved in low‐G+C Gram‐positive bacteria. Two distinct signals have been shown independently to influence the activity of this regulator: the level of intracellular GTP in Bacillus subtilis and the level of intracellular branched‐chain amino acids (BCAA) isoleucine, leucine and valine in Lactococcus lactis. Measurement of BCAA and GTP levels in several environmental conditions showed that L. lactis CodY responded to the intracellular BCAA concentrations but not to physiological fluctuations in intracellular GTP. In addition, we demonstrated that CodY from L. lactis did not respond to intracellular GTP even when complementing CodY activity in B. subtilis. However, L. lactis CodY activity could still be modulated in B. subtilis by adding a rich nitrogen source to the growth media. This finding suggests that only BCAA are sensed by L. lactis CodY, whereas both GTP and BCAA signals may be integrated by B. subtilis CodY. The difference in the function of CodY from B. subtilis and L. lactis seems to reflect the difference in the physiology of these two bacteria.

Postgenomic Analysis of Streptococcus thermophilus Cocultivated in Milk with Lactobacillus delbrueckii subsp. bulgaricus: Involvement of Nitrogen, Purine, and Iron Metabolism

ABSTRACT 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 H2O2 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.

Sulfur Amino Acid Metabolism and Its Control in Lactococcus lactis IL1403

Cysteine and methionine availability influences many processes in the cell. In bacteria, transcription of the specific genes involved in the synthesis of these two amino acids is usually regulated by different mechanisms or regulators. Pathways for the synthesis of cysteine and methionine and their interconversion were experimentally determined for Lactococcus lactis, a lactic acid bacterium commonly found in food. A new gene, yhcE, was shown to be involved in methionine recycling to cysteine. Surprisingly, 18 genes, representing almost all genes of these pathways, are under the control of a LysR-type activator, FhuR, also named CmbR. DNA microarray experiments showed that FhuR targets are restricted to this set of 18 genes clustered in seven transcriptional units, while cysteine starvation modifies the transcription level of several other genes potentially involved in oxidoreduction processes. Purified FhuR binds a 13-bp box centered 46 to 53 bp upstream of the transcriptional starts from the seven regulated promoters, while a second box with the same consensus is present upstream of the first binding box, separated by 8 to 10 bp. O-Acetyl serine increases FhuR binding affinity to its binding boxes. The overall view of sulfur amino acid metabolism and its regulation in L. lactis indicates that CysE could be a master enzyme controlling the activity of FhuR by providing its effector, while other controls at the enzymatic level appear to be necessary to compensate the absence of differential regulation of the genes involved in the interconversion of methionine and cysteine and other biosynthesis genes.

Fructose Utilization in Lactococcus lactis as a Model for Low-GC Gram-Positive Bacteria: Its Regulator, Signal, and DNA-Binding Site

In addition to its role as carbon and energy source, fructose metabolism was reported to affect other cellular processes, such as biofilm formation by streptococci and bacterial pathogenicity in plants. Fructose genes encoding a 1-phosphofructokinase and a phosphotransferase system (PTS) fructose-specific enzyme IIABC component reside commonly in a gene cluster with a DeoR family regulator in various gram-positive bacteria. We present a comprehensive study of fructose metabolism in Lactococcus lactis, including a systematic study of fru mutants, global messenger analysis, and a molecular characterization of its regulation. The fru operon is regulated at the transcriptional level by both FruR and CcpA and at the metabolic level by inducer exclusion. The FruR effector is fructose-1-phosphate (F1P), as shown by combined analysis of transcription and measurements of the intracellular F1P pools in mutants either unable to produce this metabolite or accumulating it. The regulation of the fru operon by FruR requires four adjacent 10-bp direct repeats. The well-conserved organization of the fru promoter region in various low-GC gram-positive bacteria, including CRE boxes as well as the newly defined FruR motif, suggests that the regulation scheme defined in L. lactis could be applied to these bacteria. Transcriptome profiling of fruR and fruC mutants revealed that the effect of F1P and FruR regulation is limited to the fru operon in L. lactis. This result is enforced by the fact that no other targets for FruR were found in the available low-GC gram-positive bacteria genomes, suggesting that additional phenotypical effects due to fructose metabolism do not rely directly on FruR control, but rather on metabolism.

Quorum-Sensing Regulation of the Production of Blp Bacteriocins in Streptococcus thermophilus

The blp gene cluster identified in the genome sequences of Streptococcus thermophilus (blp(St)) LMG18311, CNRZ1066, and LMD-9 displays all the characteristics of a class II bacteriocin locus. In the present study, we showed that the blp(St) locus is only fully functional in strain LMD-9 and regulates the production of antimicrobial peptides that inhibit strains LMG18311 and CNRZ1066. The blp(St) cluster of LMD-9 contains 23 genes that are transcriptionally organized in six operons: blpABC(St) (peptide transporter genes and pheromone gene); blpRH(St) (two-component regulatory system genes); blpD(St)-orf1, blpU(St)-orf3, and blpE-F(St) (bacteriocin precursors and immunity genes); and blpG-X(St) (unknown function). All the operons, except the regulatory unit blpRH(St), were shown to be coregulated at the transcriptional level by a quorum-sensing mechanism involving the mature S. thermophilus pheromone BlpC* (BlpC*(St)), which was extracellularly detected as two active forms (30 and 19 amino acids). These operons are differentially transcribed depending on growth phase and pheromone concentration. They all contain a motif with two imperfect direct repeats in their mapped promoter regions that could serve as binding sites of the response regulator BlpR(St). Through the construction of deletion mutants, the blp(St) locus of strain LMD-9 was shown to encode all the essential functions associated with bacteriocin production, quorum-sensing regulation, and immunity.

A new morphogenesis pathway in bacteria : unbalanced activity of cell wall synthesis machineries leads to coccus-to-rod transition and filamentation in ovococci.

Bacteria display a variety of shapes, which have biological relevance. In most eubacteria, cell shape is maintained by the tough peptidoglycan (PG) layer of the cell wall, the sacculus. The organization of PG synthesis machineries, orchestrated by different cytoskeletal elements, determines the specific shapes of sacculi. In rod‐shaped bacteria, the actin‐like (MreB) and the tubuline‐like (FtsZ) cytoskeletons control synthesis of the sidewall (elongation) and the crosswall (septation) respectively. Much less is known concerning cell morphogenesis in cocci, which lack MreB proteins. While spherical cocci exclusively display septal growth, ovococci additionally display peripheral growth, which is responsible of the slight longitudinal expansion that generates their ovoid shape. Here, we report that the ovococcus Lactococcus lactis has the ability to become rod‐shaped. L. lactis IL1403 wild‐type cells form long aseptate filaments during both biofilm and planktonic growth in a synthetic medium. Nascent PG insertion and the division protein FtsK localize in multiple peripheral rings regularly spaced along the filaments. We show that filamentation results from septation inhibition, and that penicillin‐binding proteins PBP2x and PBP2b play a direct role in this process. We propose a model for filament formation in L. lactis, and discuss the possible biological role of such morphological differentiation.

Physiology of Streptococcus thermophilus during the late stage of milk fermentation with special regard to sulfur amino-acid metabolism

Streptococcus thermophilus is a thermophilic lactic acid bacterium widely used as starter in the manufacture of dairy products in particular in yoghurt manufacture in combination with Lactobacillus delbrueckii ssp. bulgaricus. However, in spite of its massive use, the physiological state of S. thermophilus in milk has hardly been investigated. We established the first map of the cytosolic proteome of S. thermophilus LMG18311 grown in milk. It comprises 203 identified proteins corresponding to 32% of theoretical proteome. In addition, using proteomic and transcriptomic approaches, we analyzed the physiology of LMG18311 during its late stage of growth in milk (between 2h30 and 5h30). It revealed the up‐regulation of (i) peptides and AA transporters and of specific AA biosynthetic pathways notably for sulfur AA and (ii) genes and proteins involved in the metabolism of various sugars. These two effects were also observed in LMG18311 grown in milk in coculture with L. bulgaricus although the effect on sugar metabolism was less pronounced. It suggests that the stimulatory effect of Lactobacillus on the Streptococcus growth is more complex than AA or peptides supply.

Overview of a surface-ripened cheese community functioning by meta-omics analyses

Cheese ripening is a complex biochemical process driven by microbial communities composed of both eukaryotes and prokaryotes. Surface-ripened cheeses are widely consumed all over the world and are appreciated for their characteristic flavor. Microbial community composition has been studied for a long time on surface-ripened cheeses, but only limited knowledge has been acquired about its in situ metabolic activities. We applied metagenomic, metatranscriptomic and biochemical analyses to an experimental surface-ripened cheese composed of nine microbial species during four weeks of ripening. By combining all of the data, we were able to obtain an overview of the cheese maturation process and to better understand the metabolic activities of the different community members and their possible interactions. Furthermore, differential expression analysis was used to select a set of biomarker genes, providing a valuable tool that can be used to monitor the cheese-making process.