Strain diversity and phage resistance in complex dairy starter cultures
Spus, M. ; Alexeeva, S.V. ; Wolkers-Rooijackers, J.C.M. ; Zwietering, M.H. ; Abee, T. ; Smid, E.J. - \ 2015
Journal of Dairy Science 98 (2015)8. - ISSN 0022-0302 - p. 5173 - 5182.
streptococcus-cremoris - lactic streptococci - lactococcus-lactis - food fermentations - bacteriophages - competition - products - genomics - bacteria - plasmid
The compositional stability of the complex Gouda cheese starter culture Ur is thought to be influenced by diversity in phage resistance of highly related strains that co-exist together with bacteriophages. To analyze the role of bacteriophages in maintaining culture diversity at the level of genetic lineages, simple blends of Lactococcus lactis strains were made and subsequently propagated for 152 generations in the absence and presence of selected bacteriophages. We first screened 102 single-colony isolates (strains) from the complex cheese starter for resistance to bacteriophages isolated from this starter. The collection of isolates represents all lactococcal genetic lineages present in the culture. Large differences were found in bacteriophage resistance among strains belonging to the same genetic lineage and among strains from different lineages. The blends of strains were designed such that 3 genetic lineages were represented by strains with different levels of phage resistance. The relative abundance of the lineages in blends with phages was not stable throughout propagation, leading to continuous changes in composition up to 152 generations. The individual resistance of strains to phage predation was confirmed as one of the factors influencing starter culture diversity. Furthermore, loss of proteolytic activity of initially proteolytic strains was found. Reconstituted blends with only 4 strains with a variable degree of phage resistance showed complex behavior during prolonged propagation. Key words: starter culture; bacteriophage; diversity; proteolytic activity
Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus
Mohedano, M.L. ; Garcia-Cayuela, T. ; Perez-Ramos, A. ; Gaiser, R.A. ; Requena, T. ; Lopez, P. - \ 2015
Journal of Industrial Microbiology and Biotechnology 42 (2015)2. - ISSN 1367-5435 - p. 247 - 253.
lactic-acid bacteria - controlled gene-expression - streptococcus-pneumoniae - lactococcus-lactis - plasmid - cloning
Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene expression in Lactobacillus. This vector is also able to replicate in Streptococcus pneumoniae and Escherichia coli. The usage of pRCR as a promoter probe was validated in Lactobacillus acidophilus by characterizing the regulation of lactacin B expression. The results show that the regulation is exerted at the transcriptional level, with lbaB gene expression being specifically induced by co-culture of the L. acidophilus bacteriocin producer and the S. thermophilus STY-31 inducer bacterium.
Evolutionary engineering to enhance starter culture performance in food fermentations
Bachmann, H. ; Pronk, J.T. ; Kleerebezem, M. ; Teusink, B. - \ 2015
Current Opinion in Biotechnology 32 (2015). - ISSN 0958-1669 - p. 1 - 7.
yeast saccharomyces-cerevisiae - adaptive evolution - lactococcus-lactis - hyperosmotic conditions - selection - growth - population - strategies - phenotypes - stability
Microbial starter cultures are essential for consistent product quality and functional properties such as flavor, texture, pH or the alcohol content of various fermented foods. Strain improvement programs to achieve desired properties in starter cultures are diverse, but developments in next-generation sequencing lead to an increased interest in evolutionary engineering of desired phenotypes. We here discuss recent developments of strain selection protocols and how computational approaches can assist such experimental design. Furthermore the analysis of evolved phenotypes and possibilities with complex consortia are highlighted. Studies carried out with mainly yeast and lactic acid bacteria demonstrate the power of evolutionary engineering to deliver strains with novel phenotypes as well as insight into underlying mechanisms.
Development of the recombinase-based in vivo expression technology in Streptococcus thermophilus and validation using the lactose operon promoter
Junjua, M. ; Galia, W. ; Gaci, N. ; Uriot, O. ; Genay, M. ; Bachmann, H. ; Kleerebezem, M. ; Dary, A. ; Roussel, Y. - \ 2014
Journal of Applied Microbiology 116 (2014)3. - ISSN 1364-5072 - p. 620 - 631.
lactic-acid bacteria - gene-expression - lactococcus-lactis - human gut - yogurt - system - mice - identification - transformation - proteinase
To construct and validate the recombinase-based in vivo expression technology (R-IVET) tool in Streptococcus thermophilus (ST).
Methods and Results
The R-IVET system we constructed in the LMD-9 strain includes the plasmid pULNcreB allowing transcriptional fusion with the gene of the site-specific recombinase Cre and the chromosomal cassette containing a spectinomycin resistance gene flanked by two loxP sites. When tested in M17 medium, promoters of the genes encoding the protease PrtS, the heat-shock protein Hsp16 and of the lactose operon triggered deletion of the cassette, indicating promoter activity in these conditions. The lactose operon promoter was also found to be activated during the transit in the murine gastrointestinal tract.
The R-IVET system developed in ST is relatively stable, functional, very sensitive and can be used to assay activity of promoters, which are specifically active in in vivo conditions.
Significance and Impact of the Study
This first adaptation of R-IVET to ST provides a highly valuable tool allowing an exploration of the physiological state of ST in the GIT of mammals, fermentation processes or dairy products.
Functional implications of the microbial community structure of undefined mesophilic starter cultures
Smid, E.J. ; Erkus, O. ; Spus, M. ; Wolkers-Rooijackers, J.C.M. ; Alexeeva, S.V. ; Kleerebezem, M. - \ 2014
Microbial Cell Factories 13 (2014)suppl. 1. - ISSN 1475-2859
lactic-acid bacteria - lactococcus-lactis - subsp lactis - listeria-monocytogenes - biovar diacetylactis - metabolic models - cheddar cheese - diversity - cremoris - dairy
This review describes the recent advances made in the studies of the microbial community of complex and undefined cheese starter cultures. We report on work related to the composition of the cultures at the level of genetic lineages, on the presence and activity of bacteriophages and on the population dynamics during cheese making and during starter culture propagation. Furthermore, the link between starter composition and starter functionality will be discussed. Finally, recent advances in predictive metabolic modelling of the multi-strain cultures will be discussed in the context of microbe-microbe interactions.
Functional genomics of lactic acid bacteria: from food to health
Douillard, F.P. ; Vos, W.M. de - \ 2014
Microbial Cell Factories 13 (2014)suppl.1. - ISSN 1475-2859
lactobacillus-rhamnosus gg - human gastrointestinal-tract - sortase-dependent proteins - johnsonii strain ncc533 - gram-positive bacteria - subsp cremoris mg1363 - lactococcus-lactis - in-vitro - streptococcus-thermophilus - enterococcus-faecalis
Genome analysis using next generation sequencing technologies has revolutionized the characterization of lactic acid bacteria and complete genomes of all major groups are now available. Comparative genomics has provided new insights into the natural and laboratory evolution of lactic acid bacteria and their environmental interactions. Moreover, functional genomics approaches have been used to understand the response of lactic acid bacteria to their environment. The results have been instrumental in understanding the adaptation of lactic acid bacteria in artisanal and industrial food fermentations as well as their interactions with the human host. Collectively, this has led to a detailed analysis of genes involved in colonization, persistence, interaction and signaling towards to the human host and its health. Finally, massive parallel genome re-sequencing has provided new opportunities in applied genomics, specifically in the characterization of novel non-GMO strains that have potential to be used in the food industry. Here, we provide an overview of the state of the art of these functional genomics approaches and their impact in understanding, applying and designing lactic acid bacteria for food and health.
Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis
Douillard, F.P. ; Rasinkangas, P. ; Ossowski, I. von; Reunanen, J. ; Palva, A. ; Vos, W.M. de - \ 2014
Journal of Biological Chemistry 289 (2014)22. - ISSN 0021-9258 - p. 15764 - 15775.
complete genome sequence - gram-positive bacteria - enterococcus-faecium isolate - group-b streptococcus - lactic-acid bacteria - lactococcus-lactis - corynebacterium-diphtheriae - bacillus-anthracis - pilin subunit - reveals pili
In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.
Pathway transfer in fungi: Transporters are the key to success
Straat, L. van der; Graaff, L.H. de - \ 2014
Bioengineered 5 (2014)5. - ISSN 2165-5979 - p. 335 - 339.
itaconic acid production - saccharomyces-cerevisiae - lactococcus-lactis - aspergillus-niger - cephalosporin production - penicillium-chrysogenum - functional expression - membrane-proteins - ceft gene - overproduction
Itaconic acid is an important building block for the chemical industry. Currently, Aspergillus terreus is the main organism used for itaconic acid production. Due to the enormous citric acid production capacity of Aspergillus niger, this host is investigated as a potential itaconic acid production host. Several strategies have been tried so far: fermentation optimization, expression of cis-aconitate decarboxylase (cadA) alone and in combination with aconitase targeted to the same compartment, chassis optimization, and the heterologous expression of two transporters flanking the cadA gene. We showed that the heterologous expression of these two transporters were key to improving itaconic acid production in an A. niger strain that was unable to produce oxalic acid and gluconic acid. The expression of transporters has increased the production levels of other industrially relevant processes as well, such as ß-lactam antibiotics and bioethanol. Thus far, the role of transporters in production process optimization is a bit overlooked.
Production of aroma compounds in lactic fermentations
Smid, E.J. ; Kleerebezem, M. - \ 2014
Annual Review of Food Science and Technology 5 (2014). - ISSN 1941-1413 - p. 313 - 326.
cystathionine beta-lyase - citrate transporter citp - lactococcus-lactis - acid bacteria - flavor formation - lactobacillus-plantarum - starter cultures - streptococcus-thermophilus - proteolytic systems - food fermentations
This review describes recent scientific research on the production of aroma compounds by lactic acid bacteria (LAB) in fermented food products. We discuss the various precursor molecules for the formation of aroma compounds in connection with the metabolic pathways involved. The roles of nonmetabolic properties such as cell lysis are also described in relation to aroma formation. Finally, we provide an overview of the literature on methods to steer and control aroma formation by LAB in mixed culture fermentations. We demonstrate that the technological progress made recently in high-throughput analysis methods has been driving the development of new approaches to understand, control, and steer aroma formation in (dairy) fermentation processes. This currently entails proposing new rules for designing stable, high-performance mixed cultures constituting a selection of strains, which in concert and on the basis of their individual predicted gene contents deliver the required functionalities.
Multifactorial diversity sustains microbial community stability
Erkus, O. ; Jager, V.C.L. de; Spus, M. ; Alen-Boerrigter, I.J. van; Rijswijck, I.M.H. van; Hazelwood, L. ; Janssen, P.W. ; Hijum, S.A.F.T. van; Kleerebezem, M. ; Smid, E.J. - \ 2013
ISME Journal 7 (2013)11. - ISSN 1751-7362 - p. 2126 - 2136.
lactic-acid bacteria - complete genome sequence - lactococcus-lactis - dairy environment - subsp lactis - raw-milk - cremoris - plasmids - cheese - identification
Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty
Comparative Genomics Analysis of Streptococcus Isolates from the Human Small Intestine Reveals their Adaptation to a Highly Dynamic Ecosystem
Bogert, B. van den; Boekhorst, J. te; Herrmann, R. ; Smid, E.J. ; Zoetendal, E.G. ; Kleerebezem, M. - \ 2013
PLoS ONE 8 (2013)12. - ISSN 1932-6203
human gastrointestinal-tract - group-b streptococcus - prokaryotic genomes - lactococcus-lactis - bacterial samples - transport-system - microbiota - pneumoniae - competence - sequences
The human small-intestinal microbiota is characterised by relatively large and dynamic Streptococcus populations. In this study, genome sequences of small-intestinal streptococci from S. mitis, S. bovis, and S. salivarius species-groups were determined and compared with those from 58 Streptococcus strains in public databases. The Streptococcus pangenome consists of 12,403 orthologous groups of which 574 are shared among all sequenced streptococci and are defined as the Streptococcus core genome. Genome mining of the small-intestinal streptococci focused on functions playing an important role in the interaction of these streptococci in the small-intestinal ecosystem, including natural competence and nutrient-transport and metabolism. Analysis of the small-intestinal Streptococcus genomes predicts a high capacity to synthesize amino acids and various vitamins as well as substantial divergence in their carbohydrate transport and metabolic capacities, which is in agreement with observed physiological differences between these Streptococcus strains. Gene-specific PCR-strategies enabled evaluation of conservation of Streptococcus populations in intestinal samples from different human individuals, revealing that the S. salivarius strains were frequently detected in the small-intestine microbiota, supporting the representative value of the genomes provided in this study. Finally, the Streptococcus genomes allow prediction of the effect of dietary substances on Streptococcus population dynamics in the human small-intestine
Oxygen relieves the CO2 and acetate dependency of Lactobacillus johnsonii NCC 533
Hertzberger, R.Y. ; Pridmore, R.D. ; Gysler, C. ; Kleerebezem, M. ; Teixeira de Mattos, M.J. - \ 2013
PLoS ONE 8 (2013)2. - ISSN 1932-6203 - 8 p.
population heterogeneity - transcriptome analysis - pyruvate oxidase - streptococcus-thermophilus - carbamoyl-phosphate - lactococcus-lactis - aerobic growth - plantarum - acid - bacteria
Oxygen relieves the CO2 and acetate dependency of Lactobacillus johnsonii NCC 533. The probiotic Lactobacillus johnsonii NCC 533 is relatively sensitive to oxidative stress; the presence of oxygen causes a lower biomass yield due to early growth stagnation. We show however that oxygen can also be beneficial to this organism as it relieves the requirement for acetate and CO2 during growth. Both on agar- and liquid-media, anaerobic growth of L. johnsonii NCC 533 requires CO2 supplementation of the gas phase. Switching off the CO2 supply induces growth arrest and cell death. The presence of molecular oxygen overcomes the CO2 dependency. Analogously, L. johnsonii NCC 533 strictly requires media with acetate to sustain anaerobic growth, although supplementation at a level that is 100-fold lower (120 microM) than the concentration in regular growth medium for lactobacilli already suffices for normal growth. Analogous to the CO2 requirement, oxygen supply relieves this acetate-dependency for growth. The L. johnsonii NCC 533 genome indicates that this organism lacks genes coding for pyruvate formate lyase (PFL) and pyruvate dehydrogenase (PDH), both CO2 and acetyl-CoA producing systems. Therefore, C1- and C2- compound production is predicted to largely depend on pyruvate oxidase activity (POX). This proposed role of POX in C2/C1-generation is corroborated by the observation that in a POX deficient mutant of L. johnsonii NCC 533, oxygen is not able to overcome acetate dependency nor does it relieve the CO2 dependency.
O-glycosylation as a novel control mechanism of peptidoglycan hydrolase activity
Rolain, T. ; Bernard, E. ; Beaussart, A. ; Degand, H. ; Courtin, P. ; Egge-Jacobsen, W. ; Bron, P.A. ; Morsomme, P. ; Kleerebezem, M. ; Chapot-Chartier, M.P. ; Dufrêne, Y.F. ; Hols, P. - \ 2013
Journal of Biological Chemistry 288 (2013)31. - ISSN 0021-9258 - p. 22233 - 22247.
lactobacillus-plantarum wcfs1 - diamino acid endopeptidase - lactococcus-lactis - teichoic-acids - staphylococcus-aureus - cell-separation - bacterial-peptidoglycan - n-acetylglucosaminidase - listeria-monocytogenes - protein glycosylation
Acm2, the major autolysin of Lactobacillus plantarum, is a tripartite protein. Its catalytic domain is surrounded by an O-glycosylated N-terminal region rich in Ala, Ser, and Thr (AST domain), which is of low complexity and unknown function, and a C-terminal region composed of five SH3b peptidoglycan (PG) binding domains. Here, we investigate the contribution of these two accessory domains and of O-glycosylation to Acm2 functionality. We demonstrate that Acm2 is an N-acetylglucosaminidase and identify the pattern of O-glycosylation (21 mono-N-acetylglucosamines) of its AST domain. The O-glycosylation process is species-specific as Acm2 purified from Lactococcus lactis is not glycosylated. We therefore explored the functional role of O-glycosylation by purifying different truncated versions of Acm2 that were either glycosylated or non-glycosylated. We show that SH3b domains are able to bind PG and are responsible for Acm2 targeting to the septum of dividing cells, whereas the AST domain and its O-glycosylation are not involved in this process. Notably, our data reveal that the lack of O-glycosylation of the AST domain significantly increases Acm2 enzymatic activity, whereas removal of SH3b PG binding domains dramatically reduces this activity. Based on this antagonistic role, we propose a model in which access of the Acm2 catalytic domain to its substrate may be hindered by the AST domain where O-glycosylation changes its conformation and/or mediates interdomain interactions. To the best of our knowledge, this is the first time that O-glycosylation is shown to control the activity of a bacterial enzyme.
Effects of dietary arabinoxylan-oligosaccharides (AXOS) and endogenous probiotics on the growth performance, non-specific immunity and gut micrbiota of juvenile Siberian sturgeon (Acipenser baerii)
Geraylou, Z. ; Souffreau, C. ; Rurangwa, E. ; Meester, L. de; Courtin, C.M. ; Delcour, J.A. ; Buyse, J. ; Ollevier, F. - \ 2013
Fish and Shellfish Immunology 35 (2013)3. - ISSN 1050-4648 - p. 766 - 775.
lactic-acid bacteria - bacillus-subtilis - lactococcus-lactis - disease resistance - intestinal microbiota - scophthalmus-maximus - labeo-rohita - in-vitro - fish - supplementation
We investigated the effects of administration of putative endogenous probiotics Lactococcus lactis spp. lactis or Bacillus circulans, alone and in combination with arabinoxylan-oligosaccharides (AXOS), a new class of candidate prebiotics, in juvenile Siberian sturgeon (Acipenser baerii). Eight experimental diets were tested: basal diet (Diet 1), basal diet supplemented with 2% AXOS (Diet 2), or L. lactis ST G81 (Diet 3), L. lactis ST G45 (Diet 4), B. circulans ST M53 (Diet 5), L. lactis ST G81 + 2% AXOS (Diet 6), L. lactis ST G45 + 2% AXOS (Diet 7), B. circulans ST M53 + 2% AXOS (Diet 8). After four weeks, growth performance and feed conversion ratio significantly improved in fish fed diet 7. Innate immune responses of fish were boosted with both AXOS and probiotic diets, however synergistic effects of AXOS and probiotic diets were only observed for phagocytic and alternative complement activity. Phagocytic and respiratory burst activity of fish macrophage increased in fish fed diet 2 and 7, while humoral immune responses only increased in fish fed diet 7. Pyrosequencing analysis (16S rDNA) of the hindgut microbiota demonstrated that AXOS improved the colonization or/and growth capacity of L. lactis, as a higher relative abundance of L. lactis was observed in fish receiving diet 7. However, no observable colonization of B. circulans was found in the hindgut of fish fed diet 5 or 8, containing this bacterium. The dietary L. lactis ST G45 + 2% AXOS caused significant alterations in the intestinal microbiota by significantly decreasing in bacterial diversity, demonstrated by the fall in richness and Shannon diversity, and improved growth performance and boosted immune responses of Siberian sturgeon.
Towards metagenome-scale models for industrial applications-the case of Lactic Acid Bacteria
Branco Dos Santos, F. ; Vos, W.M. de; Teusink, B. - \ 2013
Current Opinion in Biotechnology 24 (2013)2. - ISSN 0958-1669 - p. 200 - 206.
comparative systems biology - lactobacillus-plantarum - lactococcus-lactis - metabolic reconstruction - intestinal microbiota - escherichia-coli - gut - growth - identification - fermentation
We review the uses and limitations of modelling approaches that are in use in the field of Lactic Acid Bacteria (LAB). We describe recent developments in model construction and computational methods, starting from application of such models to monocultures. However, since most applications in food biotechnology involve complex nutrient environments and mixed cultures, we extend the scope to discuss developments in modelling such complex systems. With metagenomics and meta-functional genomics data becoming available, the developments in genome-scale community models are discussed. We conclude that exploratory tools are available and useful, but truly predictive mechanistic models will remain a major challenge in the field
Metabolic shifts: a fitness perspective for microbial cell factories
Goel, A. ; Wortel, M.T. ; Molenaar, D. ; Teusink, B. - \ 2012
Biotechnology Letters 34 (2012)12. - ISSN 0141-5492 - p. 2147 - 2160.
lactic-acid bacteria - heterologous protein secretion - escherichia-coli - saccharomyces-cerevisiae - lactococcus-lactis - bacillus-subtilis - pyruvate metabolism - overflow metabolism - aerobic glycolysis - glucose-metabolism
Performance of industrial microorganisms as cell factories is limited by the capacity to channel nutrients to desired products, of which optimal production usually requires careful manipulation of process conditions, or strain improvement. The focus in process improvement is often on understanding and manipulating the regulation of metabolism. Nonetheless, one encounters situations where organisms are remarkably resilient to further optimization or their properties become unstable. Therefore it is important to understand the origin of these apparent limitations to find whether and how they can be improved. We argue that by considering fitness effects of regulation, a more generic explanation for certain behaviour can be obtained. In this view, apparent process limitations arise from trade-offs that cells faced as they evolved to improve fitness. A deeper understanding of such trade-offs using a systems biology approach can ultimately enhance performance of cell factories.
Transcriptomes reveal genetic signatures underlying physiological variations imposed by different fermentation conditions in Lactobacillus plantarum
Bron, P.A. ; Wels, M. ; Bongers, R.S. ; Bokhorst-van de Veen, H. van; Wiersma, A. ; Overmars, L. ; Marco, M.L. ; Kleerebezem, M. - \ 2012
PLoS ONE 7 (2012)7. - ISSN 1932-6203
lactic-acid bacteria - complete genome sequence - lactococcus-lactis - ribonucleotide reductase - escherichia-coli - stationary-phase - microarray data - aerobic growth - diversity - pathways
Lactic acid bacteria (LAB) are utilized widely for the fermentation of foods. In the current post-genomic era, tools have been developed that explore genetic diversity among LAB strains aiming to link these variations to differential phenotypes observed in the strains investigated. However, these genotype-phenotype matching approaches fail to assess the role of conserved genes in the determination of physiological characteristics of cultures by environmental conditions. This manuscript describes a complementary approach in which Lactobacillus plantarum WCFS1 was fermented under a variety of conditions that differ in temperature, pH, as well as NaCl, amino acid, and O2 levels. Samples derived from these fermentations were analyzed by full-genome transcriptomics, paralleled by the assessment of physiological characteristics, e.g., maximum growth rate, yield, and organic acid profiles. A data-storage and -mining suite designated FermDB was constructed and exploited to identify correlations between fermentation conditions and industrially relevant physiological characteristics of L. plantarum, as well as the associated transcriptome signatures. Finally, integration of the specific fermentation variables with the transcriptomes enabled the reconstruction of the gene-regulatory networks involved. The fermentation-genomics platform presented here is a valuable complementary approach to earlier described genotype-phenotype matching strategies which allows the identification of transcriptome signatures underlying physiological variations imposed by different fermentation conditions.
Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli
García-Cayuela, T. ; Cadiñanos, L.P. de; Mohedano, M.L. ; Palencia, P.F. de; Boden, D. ; Wells, J. ; Peláez, C. ; López, P. ; Requena, T. - \ 2012
Applied Microbiology and Biotechnology 96 (2012)1. - ISSN 0175-7598 - p. 171 - 181.
lactococcus-lactis - streptococcus-pneumoniae - genetic tools - in-vitro - green - cremoris - red - transformation - expression - faecalis
Fluorescent reporter genes are valuable tools for real-time monitoring of gene expression in living cells. In this study we describe the construction of novel promoter-probe vectors containing a synthetic mCherry fluorescent protein gene, codon-optimized for lactic acid bacteria, divergently linked, or not, to a gene encoding the S65T and F64L variant of the green fluorescent protein. The utility of the transcriptional fusion vectors was demonstrated by the cloning of a single or two divergent promoter regions and by the quantitative evaluation of fluorescence during growth of Lactococcus lactis, Enterococcus faecalis, and Escherichia coli.
Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling
Remus, D.M. ; Kranenburg, R. van; Swam, I.I. van; Taverne, N. ; Bongers, R.S. ; Wels, M. ; Wells, J. ; Bron, P.A. ; Kleerebezem, M. - \ 2012
Microbial Cell Factories 11 (2012)1. - ISSN 1475-2859 - 10 p.
lactic-acid bacteria - gene-expression omnibus - streptococcus-pneumoniae - lactococcus-lactis - exopolysaccharide biosynthesis - microarray data - subsp cremoris - rhamnosus gg - identification - strains
Background - Bacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well described. Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action. Results - The Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production. Two of these clusters appear to encode all functions required for capsular polysaccharide formation (cps2A-J and cps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-I and cps3A-J). We constructed L. plantarum WCFS1 gene deletion mutants that lack individual (¿cps1A-I, ¿cps2A-J, ¿cps3A-J and ¿cps4A-J) or combinations of cps clusters (¿cps1A-3J and ¿cps1A-3I, ¿cps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. The cps cluster deletions influenced the expression of variable gene sets in the individual cps cluster mutants, but also considerable numbers of up- and down-regulated genes were shared between mutants in cps cluster 1 and 2, as well as between mutant in cps clusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness of cps1A-I and cps3A-J, all clusters are active and functional in L. plantarum. The ¿cps1A-I strain produced surface polysaccharides in equal amounts as compared to the wild-type strain, while the polysaccharides were characterized by a reduced molar mass and the lack of rhamnose. The mutants that lacked functional copies of cps2A-J, cps3A-J or cps4A-J produced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of the cps cluster mutations on toll-like receptor (TLR)-mediated human nuclear factor (NF)-¿B activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to a L. plantarum wild-type derivative, TLR2 activation remained unaffected by the ¿cps1A-I and ¿cps3A-J mutants but appeared slightly increased after stimulation with the ¿cps2A-J and ¿cps4A-J mutants, while the ¿cps1A-3J and ¿cps1A-3J, ¿cps4A-J mutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling. Conclusions - Our study reveals that modulation of surface glycan characteristics in L. plantarum highlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently complete cps clusters (cps2A-J and cps4A-J) contributed individually to this shielding, the removal of all cps clusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis in L. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.
Impact of respiration on resistance of Lactobacillus plantarum WCFS1 to acid stress
Watanabe, M. ; Veen, S. van der; Abee, T. - \ 2012
Applied and Environmental Microbiology 78 (2012)11. - ISSN 0099-2240 - p. 4062 - 4064.
lactococcus-lactis - oxidative stress - bacteria - bacillus
This study shows that growth under respiration conditions has a negative impact on the survival of stationary-phase cultures of Lactobacillus plantarum WCFS1 at low pHs and that viability loss at critical values is associated with the formation of radicals and loss of membrane integrity.