The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.
two component systems; Streptococcus pneumoniae; fatty acid biosynthesis; essential response regulator; transcriptional regulation
Lactobacillus collinoides CUPV237 is a strain isolated from a Basque cider. Lactobacillus collinoides is one of the most frequent species found in cider from Spain, France, or England. A notable feature of the L. collinoides CUPV237 strain is its ability to produce exopolysaccharides.
Midline laparotomy closure carries a significant risk of incisional hernia. This study examines the behavior of two new suture materials, an elastic material, polyurethane (PUe), and a barbed polydioxanone (PDXb) suture thread in a rabbit model of midline incision closure.
Three 2-cm midline incisions were made in 68 New Zealand White rabbits. The incisions were closed by running suture using four 3/0 threads: polypropylene (PP) (Surgipro®, Covidien), PUe (Assuplus®, Assut Europe), PDX (Assufil®, Assut Europe) or PDXb (Filbloc®, Assut Europe). Animals in each suture group were euthanized 3 weeks and 6 months after surgery. Histological sections of the tissue-embedded sutures were subjected to morphological, collagen expression, macrophage response and uniaxial tensiometry studies.
No signs of wound dehiscence or complications were observed. At 3 weeks, all sutures were surrounded by connective tissue composed mainly of collagen III. PUe showed greater collagen I expression than the other sutures. All sutures elicited a macrophage response that diminished from 3 weeks to 6 months (p < 0.001). This response was similar for the non-reabsorbable sutures (PP and PUe) yet PDXb showed a significantly greater response than the other reabsorbable suture (PDX) at 3 weeks (p < 0.01). At this early time point, the tensile strength of PUe was similar to that of control intact tissue (p > 0.05).
Three weeks after surgery, PUe revealed more collagen I deposition than the remaining materials and this translated to a similar biomechanical behavior to linea alba, that could avoid the appearance of short term dehiscences and thus reduce the incidence of incisional hernia. PDXb provides no additional advantages in their behavior regarding PDX suture.
Polypropylene; Polyurethane; Polydioxanone; Abdominal wall closure; Midline closure; Laparotomy closure; Barbed sutures; Elastic sutures
Lactococcus lactis is widely used as a dairy starter and has been extensively studied. Based on the acquired knowledge on its physiology and metabolism, new applications have been envisaged and there is an increasing interest of using L. lactis as a cell factory. Plasmids constitute the main toolbox for L. lactis genetic engineering and most rely on antibiotic resistant markers for plasmid selection and maintenance. In this work, we have assessed the ability of the bacteriocin Lactococcin 972 (Lcn972) gene cluster to behave as a food-grade post-segregational killing system to stabilize recombinant plasmids in L. lactis in the absence of antibiotics. Lcn972 is a non-lantibiotic bacteriocin encoded by the 11-kbp plasmid pBL1 with a potent antimicrobial activity against Lactococcus.
Attempts to clone the full lcn972 operon with its own promoter (P972), the structural gene lcn972 and the immunity genes orf2-orf3 in the unstable plasmid pIL252 failed and only plasmids with a mutated promoter were recovered. Alternatively, cloning under other constitutive promoters was approached and achieved, but bacteriocin production levels were lower than those provided by pBL1. Segregational stability studies revealed that the recombinant plasmids that yielded high bacteriocin titers were maintained for at least 200 generations without antibiotic selection. In the case of expression vectors such as pTRL1, the Lcn972 gene cluster also contributed to plasmid maintenance without compromising the production of the fluorescent mCherry protein. Furthermore, unstable Lcn972 recombinant plasmids became integrated into the chromosome through the activity of insertion sequences, supporting the notion that Lcn972 does apply a strong selective pressure against susceptible cells. Despite of it, the Lcn972 gene cluster was not enough to avoid the use of antibiotics to select plasmid-bearing cells right after transformation.
Inserting the Lcn972 cluster into segregational unstable plasmids prevents their lost by segregation and probable could be applied as an alternative to the use of antibiotics to support safer and more sustainable biotechnological applications of genetically engineered L. lactis.
Plasmid segregation; Post-segregational killing; Bacteriocins; Insertion sequences; Lactococcus lactis
Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.
Oenococcus oeni; proteome; two-dimensional electrophoresis
Ingestion of fermented foods containing high levels of biogenic amines (BA) can be deleterious to human health. Less obvious is the threat posed by BA producing organisms contained within the food which, in principle, could form BA after ingestion even if the food product itself does not initially contain high BA levels. In this work we have investigated the production of tyramine and putrescine by Lactobacillus brevis IOEB 9809, of wine origin, under simulated gastrointestinal tract (GIT) conditions.
An in vitro model that simulates the normal physiological conditions in the human digestive tract, as well as Caco-2 epithelial human cell lines, was used to challenge L. brevis IOEB 9809, which produced both tyramine and putrescine under all conditions tested. In the presence of BA precursors and under mild gastric stress, a correlation between enhancement of bacterial survival and a synchronous transcriptional activation of the tyramine and putrescine biosynthetic pathways was detected. High levels of both BA were observed after exposure of the bacterium to Caco-2 cells.
L. brevis IOEB 9809 can produce tyramine and putrescine under simulated human digestive tract conditions. The results indicate that BA production may be a mechanism that increases bacterial survival under gastric stress.
Biogenic amines; Lactic acid bacteria; Putrescine; Tyramine; Food safety; Food toxicity
Probiotics, prebiotics and synbiotics are frequently-used components for the elaboration of functional food. Currently, most of the commercialized probiotics are limited to a few strains of the genera Bifidobacteria, Lactobacillus and Streptococcus, most of which produce exopolysaccharides (EPS). This suggests that the beneficial properties of these microorganisms may be related to the biological activities of these biopolymers. In this work we report that a 2-substituted-(1,3)-β-d-glucan of non-dairy bacterial origin has a prebiotic effect on three probiotic strains. Moreover, the presence of this β-d-glucan potentiates in vitro adhesion of the probiotic Lactobacillus plantarum WCFS1 to human intestinal epithelial cells.
β-glucans; probiotics; prebiotics; Lactobacillus plantarum
Among Gram-positive bacteria, CtsR (Class Three Stress gene Repressor) mainly regulates the expression of genes encoding the Clp ATPases and the ClpP protease. To gain a better understanding of the biological significance of the CtsR regulon in response to heat-shock conditions, we performed a global proteomic analysis of Lactobacillus plantarum WCFS1 and ΔctsR mutant strains under optimal or heat stress temperatures. Total protein extracts from bacterial cells were analyzed by two-dimensional gel fractionation. By comparing maps from different culture conditions and different L. plantarum strains, image analysis revealed 23 spots with altered levels of expression. The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes. Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the ΔctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1. Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control. Our results could add new outlooks in order to determine the complex biological role of CtsR-mediated stress response in lactic acid bacteria.
ClpE; CtsR; heat; Lactobacillus plantarum; stress
Biogenic amines in food constitute a human health risk. Here we report that tyramine-producing Enterococcus durans strain IPLA655 (from cheese) was able to produce tyramine under conditions simulating transit through the gastrointestinal tract. Activation of the tyramine biosynthetic pathway contributed to binding and immunomodulation of enterocytes.
Exopolysaccharides have prebiotic potential and contribute to the rheology and texture of fermented foods. Here we have analyzed the in vitro bioavailability and immunomodulatory properties of the 2-substituted (1,3)-β-d-glucan-producing bacterium Pediococcus parvulus 2.6. It resists gastrointestinal stress, adheres to Caco-2 cells, and induces the production of inflammation-related cytokines by polarized macrophages.
Exopolysaccharides play an important role in the rheology and texture of fermented foods, and among these β-glucans have immunomodulating properties. We show that the overproduction of the Pediococcus parvulus GTF glycosyltransferase in an uncapsulated Lactococcus lactis strain results in synthesis and secretion (300 mg liter−1) of a position 2-substituted (1→3)-β-d-glucan that has potential use as a food additive.
Lactococcus lactis subsp. lactis bv. diacetylactis strains are aroma-producing organisms used in starter cultures for the elaboration of dairy products. This species is essentially a fermentative microorganism, which cometabolizes glucose and citrate to yield aroma compounds through the diacetyl/acetoin biosynthetic pathway. Our previous results have shown that under acidic growth Lactococcus bv. diacetylactis CRL264 expresses coordinately the genes responsible for citrate transport and its conversion into pyruvate. In the present work the impact of acidic growth on glucose, citrate, and pyruvate metabolism of Lactococcus bv. diacetylactis CRL264 has been investigated by proteomic analysis. The results indicated that acid growth triggers the conversion of citrate, but not glucose, into α-acetolactate via pyruvate. Moreover, they showed that low pH has no influence on levels of lactate dehydrogenase and pyruvate dehydrogenase. Therefore, the influence of external pH on regulation of the diacetyl/acetoin biosynthetic pathway in Lactococcus bv. diacetylactis CRL264 has been analyzed at the transcriptional level. Expression of the als, aldB, aldC, and butBA genes encoding the enzymes involved in conversion of pyruvate into aroma compounds has been investigated by primer extension, reverse transcription-PCR analysis, and transcriptional fusions. The results support that this biosynthetic pathway is induced at the transcriptional level by acidic growth conditions, presumably contributing to lactococcal pH homeostasis by synthesis of neutral compounds and by decreasing levels of pyruvate.
Lactococcus lactis subsp. lactis biovar diacetylactis CRL264 is a natural strain isolated from cheese (F. Sesma, D. Gardiol, A. P. de Ruiz Holgado, and D. de Mendoza, Appl. Environ. Microbiol. 56:2099-2103, 1990). The effect of citrate on the growth parameters at a very acidic pH value was studied with this strain and with derivatives whose citrate uptake capacity was genetically manipulated. The culture pH was maintained at 4.5 to prevent alkalinization of the medium, a well-known effect of citrate metabolism. In the presence of citrate, the maximum specific growth rate and the specific glucose consumption rate were stimulated. Moreover, a more efficient energy metabolism was revealed by analysis of the biomass yields relative to glucose consumption or ATP production. Thus, it was shown that the beneficial effect of citrate on growth under acid stress conditions is not primarily due to the concomitant alkalinization of the medium but stems from less expenditure of ATP, derived from glucose catabolism, to achieve pH homeostasis. After citrate depletion, a deleterious effect on the final biomass was apparent due to organic acid accumulation, particularly acetic acid. On the other hand, citrate metabolism endowed cells with extra ability to counteract lactic and acetic acid toxicity. In vivo 13C nuclear magnetic resonance provided strong evidence for the operation of a citrate/lactate exchanger. Interestingly, the greater capacity for citrate transport correlated positively with the final biomass and growth rates of the citrate-utilizing strains. We propose that increasing the citrate transport capacity of CRL264 could be a useful strategy to improve further the ability of this strain to cope with strongly acidic conditions.
Enterocin AS-48 production and immunity characters are encoded by 10 genes (as-48ABCC1DD1EFGH) of the pMB2 plasmid from the Enterococcus faecalis S-48 strain. Among these, as-48A, encoding the AS-48 peptide, and the as-48BC genes constitute a cluster required for AS-48 biogenesis and full immunity. In this study, the levels of expression of this cluster have been altered by insertion and site-directed mutagenesis as well as by expression coupled to trans complementation. Phenotypic studies of the mutants have indicated cotranscription of the three genes and revealed that the inactivation of as-48B prevents the production of AS-48, thus confirming its essentiality in AS-48 biogenesis. These studies have also supported the involvement of as-48C in enterocin immunity. In addition, they established that the intergenic region between the as-48A and as-48B genes is decisive for AS-48 expression, since a 3-bp substitution, which should disrupt a potential 47-nucleotide complex secondary structure, resulted in a hypoproducing phenotype. Transcriptional analyses of the E. faecalis wild-type and mutant strains supports the possibility that the as-48ABC genes are transcribed from the PA promoter located upstream of as-48A. Moreover, analysis and bioinformatic predictions of RNA folding indicate that as-48ABC mRNA is processed at the secondary structure located between as-48A and as-48B. Thus, synthesis of the AS-48 peptide appears to be controlled at the posttranscriptional level and is uncoupled from as-48BC translation. This mechanism of genetic regulation has not been previously described for the regulation of bacteriocin expression in enterococci.
A large variety of lactic acid bacteria (LAB) can utilize citrate under fermentative conditions. Although much information concerning the metabolic pathways leading to citrate utilization by LAB has been gathered, the mechanisms regulating these pathways are obscure. In Weissella paramesenteroides (formerly called Leuconostoc paramesenteroides), transcription of the citMDEFCGRP citrate operon and the upstream divergent gene citI is induced by the presence of citrate in the medium. Although genetic experiments have suggested that CitI is a transcriptional activator whose activity can be modulated in response to citrate availability, specific details of the interaction between CitI and DNA remained unknown. In this study, we show that CitI recognizes two A+T-rich operator sites located between citI and citM and that the DNA-binding affinity of CitI is increased by citrate. Subsequently, this citrate signal propagation leads to the activation of the cit operon through an enhanced recruitment of RNA polymerase to its promoters. Our results indicate that the control of CitI by the cellular pools of citrate provides a mechanism for sensing the availability of citrate and adjusting the expression of the cit operon accordingly. In addition, this is the first reported example of a transcription factor directly functioning as a citrate-activated switch allowing the cell to optimize the generation of metabolic energy.
The YycFG two-component system, originally identified in Bacillus subtilis, is highly conserved among gram-positive bacteria with low G+C contents. In Streptococcus pneumoniae, the YycF response regulator has been reported to be essential for cell growth, but the signal to which it responds and the gene members of the regulon remain unclear. In order to investigate the role of YycFG in S. pneumoniae, we increased the expression of yycF by using a maltose-inducible vector and analyzed the genome-wide effects on transcription and protein expression during the course of yycF expression. The induction of yycF expression increased histidine kinase yycG transcript levels, suggesting an autoregulation of the yycFG operon. Evidence from both proteomic and microarray transcriptome studies as well as analyses of membrane fatty acid composition indicated that YycFG is involved in the regulation of fatty acid biosynthesis pathways and in determining fatty acid chain lengths in membrane lipids. In agreement with recent transcriptome data on pneumococcal cells depleted of YycFG, we also identified several other potential members of the YycFG regulon that are required for virulence and cell wall biosynthesis and metabolism.
In this study we describe the expression pattern of the Leuconostoc paramesenteroides citMCDEFGRP operon in response to the addition of citrate to the growth medium. An 8.8-kb polycistronic transcript, which includes the citMCDEFGRP genes, was identified; its synthesis was dramatically induced upon addition of citrate to the growth medium. We also found that expression of the cit operon is subjected to posttranscriptional regulation, since processing sites included in four complex secondary structures (I, II, III, and IV) were identified by Northern blot analysis and mapped by primer extension. Upstream of the citMCDEFGRP operon a divergent open reading frame, whose expression was also increased by citrate, was identified by DNA sequencing and designated citI. The start and end sites of transcription of the cit operon and citI gene were mapped. The start sites are separated by a stretch of 188 bp with a very high A+T content of 77% and are preceded by transcriptional promoters. The end sites of the transcripts are located next to the 3′ end of two secondary structures characteristic of ρ-independent transcriptional terminators. The effect of the citI gene on expression of the cit operon was studied in Escherichia coli. The presence of the citI gene in cis and in trans resulted in increased activity of the cit promoter. These data provide the first evidence that citrate fermentation in Leuconostoc is regulated at the transcriptional level by a transcriptional activator rather than by a repressor.
Citrate transport in Lactococcus lactis subsp. lactis biovar diacetylactis is catalyzed by citrate permease P (CitP), which is encoded by the plasmidic citP gene. We have shown previously that citP is included in the citQRP operon, which is mainly transcribed from the P1 promoter in L. lactis subsp. lactis biovar diacetylactis. Furthermore, transcription of citQRP and citrate transport are not induced by the presence of citrate in the growth medium. In this work, we analyzed the influence of the extracellular pH on the expression of citP. The citrate transport system is induced by natural acidification of the medium during cell growth and by a shift to media buffered at acidic pHs. This inducible response to acid stress takes place at the transcriptional level and seems to be due to increased utilization of the P1 promoter. Increased transcription correlates with increased synthesis of CitP and results in higher citrate transport activity catalyzed by the cells. Finally, this acid stress response seems to provide L. lactis subsp. lactis biovar diacetylactis with a selective advantage resulting from cometabolism of glucose and citrate at low pHs.
Both bacteriophage PBS1 deoxyribonucleic acid (DNA) (in which all the thymine residues are replaced by uracil) and phage φW-14 DNA [in which half the thymine residues are replaced by 5-(aminobutylaminomethyl)uracil or 5-putrescinylthymine] exhibit comparable competing abilities for uptake of homologous DNA in a Bacillus subtilis competent system. But, whereas PBS1 DNA leads to a decrease in transformation frequencies compatible with its competing ability for DNA uptake, φW-14 DNA decreases transformation frequencies by a factor up to eightfold higher. The effect of φW-14 DNA on transformation frequencies is visible even at a concentration level that does not decrease transforming DNA uptake. No such effect was observed with heterologous DNA containing presumably ionically bound putrescine. Low concentrations of φW-14 DNA decreased the number of double (nonlinked) transformants more than single transformants. The influence on transformation was abolished when φW-14 DNA was added 20 min after addition of transforming DNA, i.e., when the recombination process was terminated. The putrescine-containing DNA also decreased retention of trichloroacetic acid-precipitable radioactivity of homologous DNA taken up. We conclude that φW-14 DNA inhibits some intracellular process(es) at the level of recombination. In addition, there is evidence that φW-14 DNA, but not heterologous DNA with ionically bound putrescine, binds also to site(s) on the cell surface other than receptors for homologous DNA.