Ecological studies of the Lactobacillus biota in the human digestive tract and adaptation of intestinal lactobacilli to the sourdough ecosystem

Laktobazillen haben unter den Bakterien, die den menschlichen Darm bewohnen, eine ansehnliche Beachtung aufgrund ihres positiven Einflusses auf das menschliche Wohlbefinden erlangt. Die Kultivierung dieser Bakterien gilt als zuverlässig, und so wurden zahlreiche Studien unter Anwendung von Kultivierungstechniken mit selektiven Medien durchgeführt, um die Laktobazillen in intestinalen Ökosystemen zu untersuchen (Tannock, 1995; Reuter, 2001). Vor kurzem führte die Anwendung der PCR-DGGE in Kombination mit Milchsäurebakterien (MSB)-spezifischen Primern zum Nachweis von Spezies, die nicht zu den klassischen intestinalen MSB gehören, sondern vielmehr Lebensmittel-assoziiert sind, z.B. Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides and Pediococcus pentosaceus (Walter et al., 2001; Heilig et al., 2002). Interessanterweise konnten diese Spezies nicht durch Kultivierung auf Rogosa SL Agar erhalten werden (Walter et al., 2001). Das Kapitel III beschreibt die Anwendung unterschiedlicher Kultivierungsmedien und neuer Inkubationsbedingungen, um diese Schwierigkeiten zu überwinden. Menschliche Stuhlproben wurden auf selektive und nicht-selektive Agarplatten ausplattiert, und die Platten wurden unter den klassischen Bedingungen (37°C, anaerob) für intestinalen MSB sowie unter alternativen Bedingungen (30°C, 2% O2) inkubiert. Die Analyse von bakterieller Zellmasse, die von Agarplatten abgeschwemmt wurde, mittels PCR-DGGE brachte hervor, dass die Zusammensetzung der MSB-Spezies stärker von den angewandten Inkubationsbedingungen als von den Medien beeinflusst wurde. Es konnte beobachtet werden, dass Lebensmittel-assoziierte MSB wie L. sakei und Lc. mesenteroides, die bisher nicht als intestinale Bewohner beschrieben worden waren, leichter durch Einsatz der alternativen Inkubationsbedingungen kultiviert werden können. Eine Identifizierung zufällig ausgewählter Kolonien, die unter den alternativen Bedingungen auf Rogosa SL Agar gewachsen waren, zeigte, dass L. sakei einer der dominierenden Lebensmittel-assoziierten MSB in menschlichen Fäzesproben ist und dort in Keimzahlen von bis zu 106 KbE pro Gramm vorkommen kann. Ein Vergleich der kulturtechnischen Ergebnisse mit denen der PCR-DGGE-Analyse von Bakterienmassen auf Agarplatten zeigte außerdem, dass die Untersuchung der Bakterienmassen eine schnelle und zuverlässige Methode darstellt, um einen Einblick in die Spezieszusammensetzung der kultivierbaren MSB in Fäzes zu erhalten. Die Untersuchungen der intestinalen Laktobazillenpopulation in menschlichen Stuhlproben über einen längeren Zeitraum zeigte eine hohe Variabilität in der Komplexität und Stabilität der Spezieszusammensetzung (Vanhoutte et al., 2004; Walter et al., 2001). Ökologische Studien brachten hervor, dass die meisten Lactobacillus-Spezies im menschlichen Gastrointestinaltrakt (GIT) wahrscheinlich transient (allochthon) sind und entweder von Lebensmitteln oder aus der Mundhöhle stammen (Biblioni et al., 2004). Um zu untersuchen, inwieweit die oralen Laktobazillen einen Teil der fäkalen Laktobazillen ausmachen, wurde die Lactobacillus-Biota sowohl im Speichel als auch in Stuhlproben von drei Probanden untersucht und an zwei Zeitpunkten mit dreimonatigem Abstand verglichen (Kapitel IV). Die Zusammensetzung der Lactobacillus-Spezies im menschlichen Speichel und Fäzes war Individuum-spezifisch und fluktuierte in einem gewisse Maße, dennoch konnten die Spezies Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus rhamnosus und Lactobacillus vaginalis an beiden Zeitpunkten sowohl im Speichel als auch Fäzes der Probanden nachgewiesen werden. Durch RAPD-PCR-Analyse konnte gezeigt werden, dass mehrere Stämme dieser Spezies im Speichel und in Fäzes desselben Probanden vorhanden waren. Stämme von L. gasseri und L. vaginalis mit identischen RAPD-Mustern konnten aus beiden Speichelproben isoliert werden, was darauf hinweist, dass diese Spezies in der Mundhöhle autochthon sind. Die Ergebnisse dieses Kapitels gemeinsam mit kürzlich publizierten Daten stellen ein starkes Indiz dafür dar, dass einige Laktobazillen, die aus Stuhlproben isoliert werden können, aus der Mundhöhle stammen und somit im Intestinum allochthon sind. Laktobazillen sind in unterschiedlichen Ökosystemen gefunden worden und aufgrund ihrer kommerziellen Verwendung in der Lebensmittelindustrie Gegenstand umfassender Forschung gewesen (Hammes und Hertel, 2003). Einige Lactobacillus-Spezies lassen sich häufig sowohl in fermentierten Lebensmitteln als auch im menschlichen GIT nachweisen, jedoch ist der genetische Hintergrund für diese ökologische Vielseitigkeit noch weitgehend unbekannt. Lactobacillus reuteri ist ein dominantes Mitglied in der Mikrobiota von Typ II Sauerteigfermentationen (Meroth et al., 2003) und gilt als einer der echten autochthonen Lactobacillus-Spezies bei Menschen (Reuter, 2001). In Kapitel V und VI wurde die von Walter et al. (2001) entwickelte "in vivo expression technology (IVET)" angewandt, um bei dem Sauerteigisolat L. reuteri LTH5531 Gene (sogenannte in vivo induzierte (ivi)-Gene) zu identifizieren, die während des Wachstums in einer Typ II Sauerteigfermentation (Kapitel V) bzw. während der Passage durch den GIT einer Maus (Kapitel VI) eine erhöhte Expression zeigen. Während der Sauerteigfermentation wurden 38 induzierte und stark exprimierte Genfusionen gefunden (Kapitel V), die auf der Basis der verfügbaren Sequenzen eine Identifizierung von 29 Genen erlaubten. Vier Gene kodierten für Stress-verwandte Funktionen (z.B. Säure- und allgemeine Stressantwort) und spiegeln somit die harschen Bedingungen in der Sauerteigfermentation wider. Weitere 8 Gene kodierten für Proteine, die in Transport und Synthese von Aminosäuren und Nukleotiden involviert sind, was eine limitierte Verfügbarkeit beider Komponenten während der Sauerteigfermentation anzeigte. Die restlichen Gene waren entweder Teil von Stoffwechselwegen, die in keiner Korrelation zum Ökosystem standen, oder kodierten für hypothetische Proteine. Die Identifizierung einer putativen Proteinase und einer Komponente des Argininedeiminase-Stoffwechsels ist von technologischer Bedeutung, da beide dahinter stehenden Systeme potenziell an der Bildung von Aromavorläufern beteiligt sein können. Bemerkenswerterweise wurden bei Anwendung der IVET mit der Genombibliothek, die bereits erfolgreich bei der Sauerteigstudie eingesetzt wurde (Kapitel V), keine ivi-Promotoren während der Passage von L. reuteri LTH5531 durch den GIT einer Maus identifiziert (Kapitel VI). Mit Hilfe der IVET werden durch die Expression eines "essentiellen Wachstumsfaktors" (in unserem System die Erythromycinresistenz vermittelt durch ErmGT) aktive Promotoren selektioniert, da diese Wachstum und/oder Kolonisierung des Organismus im Ökosystem erlauben (Rainey, 1999; Walter et al., 2001). Deshalb muss die Expression eines ivi-Promotors im Ökosystem permanent erfolgen und hoch genug sein, um ein vergleichbares Wachstum von ivi-Klonen und Klonen mit konstitutiven Promoter zu erhalten, insbesondere im GIT, wo langsam wachsende Bakterien sonst ausgewaschen werden. Die Ergebnisse aus Kapitel V und VI deuten darauf hin, dass L. reuteri LTH5531 keine stark exprimierten und "GIT induzierbaren" Gene besitzt, obwohl der Stamm 38 im Sauerteig spezifisch induzierbare Gene besitzt. Ivi-Gene sind wahrscheinlich eher für die Wettbewerbsfähigkeit bzw. das ökologische Verhalten eines Organismus in einem spezifischen Ökosystem verantwortlich, als Gene, die in unterschiedlichen Ökosystemen gleich stark exprimiert werden (Rainey, 1999; Gal et al., 2003; Walter et al., 2005). Somit sind Eigenschaften, die von ivi-Genen kodiert werden, eher für die Adaption verantwortlich und das Ausmaß ihrer Expression würde durch natürliche Selektion in der Art gestaltet werden, dass die ökologische Fitness verbessert wird. Die Identifizierung von 38 Sauerteig-spezifischen ivi-Genfusionen in L. reuteri LTH5531 spiegelt die lange Adaptation von LTH5531 an das Ökosystem Sauerteig wider, ebenso wie die ivi-Gene von L. reuteri 100-23, der aus Ratten isoliert wurde, die Adaption dieses Keim an den GIT von Ratten widerspiegelt (Walter et al., 2003). In der Tat wurde der Stamm LTH5531 aus einem Sauerteig isoliert, der mit einem industriellen Starter inokuliert wurde. Dieser industrielle Starter wurde über mehrere Jahre propagiert, so dass die enthaltenen Keime genug Zeit hatten sich zu adaptieren. In Übereinstimmung damit stehen die Ergebnisse von Meroth et al. (2003), die unter Anwendung der RAPD-PCR zeigten, dass der Stamm LTH5531 bereits in einem kommerziellen Typ II Sauerteigstarter enthalten war, der 10 Jahre vor der Isolierung des Stammes Gegenstand von Untersuchungen war. Das deutet darauf hin, dass sich der Stamm LTH5531 an das Ökosystem Sauerteig seit mindestens 10 Jahren angepasst hat. Die Ergebnisse aus Kapitel V zeigen deutlich, dass die IVET eine geeignete Methode ist, die Erkenntnisse über die Genexpression und metabolische Aktivität von Bakterien während Lebensmittelfermentationen zu erweitern. Die gesammelten Ergebnisse stellen eine wichtige molekulare Grundlage dar, auf deren Basis verbesserte Starterorganismen für die Nutzung in der Lebensmittelindustrie entwickelt werden können. Darüber hinaus zeigen die Ergebnisse von Kapitel VI die Notwendigkeit in ökologischen Studien hoch-adaptierte, autochthone Stämme zu verwenden, um Kenntnisse über adaptive Wechselwirkungen zu erlangen, die für den ökologischen Erfolg dieser Bakterien in ihrem natürlichen Ökosystem sowie während der Lebensmittelfermentation verantwortlich sind.Among the bacteria inhabiting the human gut, lactobacilli have received considerable attention, due to their putative health promoting effects (Reid, 1999; Vaughan et al., 1999). Cultivation of lactobacilli is considered to be reliable and numerous studies using plating on selective media have been performed to investigate these bacteria in intestinal ecosystems (Tannock, 1995; Reuter, 2001). Recently, the application of PCR-DGGE in combination with primers specific for lactic acid bacteria (LAB) detected species which are not considered to be intestinal inhabitants but food-associated, such as Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides and Pediococcus pentosaceus (Walter et al., 2001; Heilig et al., 2002). Remarkably, these species could not be recovered by traditional bacteriological culture on Rogosa SL agar (Walter et al., 2001). In Chapter III, different cultivation media, as well as new incubation conditions were applied to overcome these difficulties. Human faecal samples were plated on selective and non-selective media and incubated under standard condition (37°C, anaerobiosis) for faecal LAB as well as alternative condition (30°C, 2% O2). PCR-DGGE analyses of resuspended bacterial biomass (RBB) obtained from agar plates revealed that the species composition of the recovered LAB was affected stronger by the incubation condition than by the used medium. It was observed that food-associated LAB such as L. sakei and Lc. mesenteroides, hitherto not described as intestinal inhabitants, are more easily selected when the alternative incubation condition is used. Identification of randomly picked colonies grown under the alternative condition on Rogosa SL agar showed that L. sakei is one of the predominant food-associated LAB species in faecal samples, reaching counts of up to 106 CFU per gram faeces. Comparison of the results of bacteriological culture with those obtained by PCR-DGGE analysis of the RBB showed that investigation of RBB is a fast and reliable method to gain insight into the species composition of culturable LAB in faeces. Examination of the faecal Lactobacillus populations over longer periods has revealed marked variation in the complexity and stability of these populations among human subjects (Vanhoutte et al., 2004, Walter et al., 2001). Ecological studies indicate that most Lactobacillus species found in the human gastrointestinal tract (GIT) are likely to be transient (allochthonous), originating from either the oral cavity or food (reviewed in Bibiloni et al., 2004). In order to investigate if oral lactobacilli constitute a part of the faecal Lactobacillus biota, the Lactobacillus biota of saliva and faeces of three human subjects were investigated and compared at two time-points in a three months interval (Chapter IV). The species composition of the Lactobacillus biota of human saliva and faeces was found to be subject-specific and fluctuated to some degree, but the species Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus vaginalis were detected at both time-points in saliva and faecal samples of individual subjects. RAPD-PCR analysis indicated that several strains of these species were present both in the oral cavity and in the faecal samples of the same subject. Oral isolates of the species L. gasseri and L. vaginalis showing identical RAPD types were found to persist over time, suggesting that these species are autochthonous to the oral cavity. The results of Chapter IV, together with recently published data (reviewed in Bibiloni et al., 2004), give strong evidence that some lactobacilli found in human faeces are allochthonous to the intestine and originate from the oral cavity. Lactobacilli have been detected in diverse environments and have been the subject of considerable research due to their commercial use in the food industry (reviewed in Hammes and Hertel, 2003). Several Lactobacillus species are commonly detected in both fermented food and the human GIT, but the genetic background for this ecological versatility is poorly understood. Lactobacillus reuteri is a dominant member of the microbiota of type II sourdough fermentations (Meroth et al., 2003) and is considered one of the truly autochthonous Lactobacillus species in humans (Reuter, 2001). The in vivo expression technology (IVET) developed by Walter et al. (2003) was used to identify genes (so-called ivi genes) of the sourdough isolate L. reuteri LTH5531 that show elevated levels of expression during growth of this organism in a type II sourdough fermentation (Chapter V) and during passage through the GIT of mice (Chapter VI). Thirty-eight induced fusions were found to be highly expressed during the sourdough fermentation (Chapter V), and 29 genes could be identified on the basis of the available sequence information. Four genes encoded stress-related functions (e.g. acid and general stress response) reflecting the harsh conditions prevailing during sourdough fermentation. Further eight genes were involved in acquisition and synthesis of amino acids and nucleotides, indicating their limited availability in sourdough. The remaining genes were either part of functionally unrelated pathways or encoded hypothetical proteins. The identification of a putative proteinase and a component of the arginine deiminase pathway are of technological interest, as they are potentially involved in the formation of aroma precursors. Remarkably, IVET with the genomic library that was successfully used in the sourdough study (Chapter V) did not detect ivi promoters when LTH5531 inhabited the GIT of mice (Chapter VI). With IVET, active promoters are selected by expression of an "essential growth factor" (in our system the erythromycin resistance mediated by ErmGT) that allows the organism to colonize and/ or grow in the ecosystem (Rainey, 1999, Walter et al., 2003). Expression of ivi promoters in particular ecosystems must therefore be permanent and strong in order to allow comparable growth rates of ivi clones and clones bearing constitutive promoters, especially in the GIT, where inactive bacteria are washed out. The findings of Chapter V and VI indicate that L. reuteri LTH5531 does not possess strongly expressed "GIT inducible" genes, while possessing 38 ones specifically induced in sourdough. Ivi genes are more likely to contribute to the ecological performance of an organism in a specific environment than genes expressed equally in a broad range of habitats (Rainey, 1999, Gal et al, 2003, Walter et al., 2005). Therefore, traits encoded by ivi genes are likely to be adaptive and the extent of their expression would be shaped by natural selection to improve ecological fitness. The presence of thirty-eight "sourdough specific" ivi fusions in L. reuteri LTH5531 probably reflects the long term adaptation of LTH5531 to the sourdough environment, just as ivi genes detected in strain 100-23 reflect adaptation of this GIT isolate to the rodent GIT (Walter et al., 2003). Indeed, LTH5531 was isolated from an experimental sourdough that had been inoculated with an industrial starter. This industrial starter has been propagated over several years, giving the organisms present sufficient time to adapt. In accordance with this, by using RAPD-PCR, Meroth et al. (2003) showed that strain LTH5531 was present in a commercial type II sourdough starter collected 10 years prior isolation of LTH5531, thus indicating that this strain has adapted to the sourdough environment for at least 10 years. The results of Chapter V clearly demonstrated that knowledge of gene expression and metabolic activities of bacteria during food fermentations can be obtained by applying IVET. The results collected provide an important molecular basis on which improved starter strains can be developed for industrial exploitation. Moreover, the results of Chapter VI show the importance of working with highly adapted, autochthonous strains in studies of microbial ecology in order to reveal the adaptive interactions responsible for the ecological success of these bacteria in their natural environment or during food fermentations

ELECTROMYOGRAPHIC INTER-LIMB ASYMMETRY IN BENCH PRESS EXERCISE IN ELITE PARALYMPICS WEIGHTLIFTERS

The purpose of this study was to describe inter-limb asymmetry in three muscle groups in a sample of Paralympic weightlifters during an 80% RM bench press. The sample was composed of 7 subjects belonging to the Chilean elite powerlifting. Surface electromyography was assessed in major pectoral, deltoid anterior and triceps brachii. The magnitude of the response was calculated through root mean square (RMS). Symmetry Index was calculated for an interlimb differences measure. Only the pectoralis major muscle showed significant differences between limbs (right 84.7 ± 41.3; left 66.1 ± 19.3 RMS) (p=0.05) and the SI median greatest value (19.74 ± 24.59%). Anterior deltoid showed high individual differences in two athletes with upper 80% SI values. More studies should assess asymmetry with the objective to decrease this injuries risk factor

Genomic Characterization of Dairy Associated Leuconostoc Species and Diversity of Leuconostocs in Undefined Mixed Mesophilic Starter Cultures

Undefined mesophilic mixed (DL-type) starter cultures are composed of predominantly Lactococcus lactis subspecies and 1–10% Leuconostoc spp. The composition of the Leuconostoc population in the starter culture ultimately affects the characteristics and the quality of the final product. The scientific basis for the taxonomy of dairy relevant leuconostocs can be traced back 50 years, and no documentation on the genomic diversity of leuconostocs in starter cultures exists. We present data on the Leuconostoc population in five DL-type starter cultures commonly used by the dairy industry. The analyses were performed using traditional cultivation methods, and further augmented by next-generation DNA sequencing methods. Bacterial counts for starter cultures cultivated on two different media, MRS and MPCA, revealed large differences in the relative abundance of leuconostocs. Most of the leuconostocs in two of the starter cultures were unable to grow on MRS, emphasizing the limitations of culture-based methods and the importance of careful media selection or use of culture independent methods. Pan-genomic analysis of 59 Leuconostoc genomes enabled differentiation into twelve robust lineages. The genomic analyses show that the dairy-associated leuconostocs are highly adapted to their environment, characterized by the acquisition of genotype traits, such as the ability to metabolize citrate. In particular, Leuconostoc mesenteroides subsp. cremoris display telltale signs of a degenerative evolution, likely resulting from a long period of growth in milk in association with lactococci. Great differences in the metabolic potential between Leuconostoc species and subspecies were revealed. Using targeted amplicon sequencing, the composition of the Leuconostoc population in the five commercial starter cultures was shown to be significantly different. Three of the cultures were dominated by Ln. mesenteroides subspecies cremoris. Leuconostoc pseudomesenteroides dominated in two of the cultures while Leuconostoc lactis, reported to be a major constituent in fermented dairy products, was only present in low amounts in one of the cultures. This is the first in-depth study of Leuconostoc genomics and diversity in dairy starter cultures. The results and the techniques presented may be of great value for the dairy industry

Needle in a Whey-Stack: PhRACS as a Discovery Tool for Unknown Phage-Host Combinations

The field of metagenomics has rapidly expanded to become the go-to method for complex microbial community analyses. However, there is currently no straightforward route from metagenomics to traditional culture-based methods of strain isolation, particularly in (bacterio)phage biology, leading to an investigative bottleneck. Here, we describe a method that exploits specific phage receptor binding protein (RBP)-host cell surface receptor interaction enabling isolation of phagehost combinations from an environmental sample. The method was successfully applied to two complex sample types-a dairy-derived whey sample and an infant fecal sample, enabling retrieval of specific and culturable phage hosts.IMPORTANCE PhRACS aims to bridge the current divide between in silico genetic analyses (i.e., phageomic studies) and traditional culture-based methodology. Through the labeling of specific bacterial hosts with fluorescently tagged recombinant phage receptor binding proteins and the isolation of tagged cells using flow cytometry, PhRACS allows the full potential of phageomic data to be realized in the wet laboratory

The forgotten role of food cultures

Fermentation is one of if not the oldest food processing technique, yet it is still an emerging field when it comes to its numerous mechanisms of action and potential applications. The effect of microbial activity on the taste, bioavailability and preservation of the nutrients and the different food matrices has been deciphered by the insights of molecular microbiology. Among those roles of fermentation in the food chain, biopreservation remains the one most debated. Presumably because it has been underestimated for quite a while, and only considered - based on a food safety and technological approach - from the toxicological and chemical perspective. Biopreservation is not considered as a traditional use, where it has been by design - but forgotten - as the initial goal of fermentation. The 'modern' use of biopreservation is also slightly different from the traditional use, due mainly to changes in cooling of food and other ways of preservation, Extending shelf life is considered to be one of the properties of food additives, classifying - from our perspective - biopreservation wrongly and forgetting the role of fermentation and food cultures. The present review will summarize the current approaches of fermentation as a way to preserve and protect the food, considering the different way in which food cultures and this application could help tackle food waste as an additional control measure to ensure the safety of the food

Neutralizing antibodies to Omicron after the fourth SARS-CoV-2 mRNA vaccine dose in immunocompromised patients highlight the need of additional boosters

IntroductionImmunocompromised patients have been shown to have an impaired immune response to COVID-19 vaccines.MethodsHere we compared the B-cell, T-cell and neutralizing antibody response to WT and Omicron BA.2 SARS-CoV-2 virus after the fourth dose of mRNA COVID-19 vaccines in patients with hematological malignancies (HM, n=71), solid tumors (ST, n=39) and immune-rheumatological (IR, n=25) diseases. The humoral and T-cell responses to SARS-CoV-2 vaccination were analyzed by quantifying the anti-RBD antibodies, their neutralization activity and the IFN-γ released after spike specific stimulation.ResultsWe show that the T-cell response is similarly boosted by the fourth dose across the different subgroups, while the antibody response is improved only in patients not receiving B-cell targeted therapies, independent on the pathology. However, 9% of patients with anti-RBD antibodies did not have neutralizing antibodies to either virus variants, while an additional 5.7% did not have neutralizing antibodies to Omicron BA.2, making these patients particularly vulnerable to SARS-CoV-2 infection. The increment of neutralizing antibodies was very similar towards Omicron BA.2 and WT virus after the third or fourth dose of vaccine, suggesting that there is no preferential skewing towards either virus variant with the booster dose. The only limited step is the amount of antibodies that are elicited after vaccination, thus increasing the probability of developing neutralizing antibodies to both variants of virus.DiscussionThese data support the recommendation of additional booster doses in frail patients to enhance the development of a B-cell response directed against Omicron and/or to enhance the T-cell response in patients treated with anti-CD20

I ricercatori che mancano alle imprese

Testimonianze di Fabio Dal Bello (scientific & QC director Sacco), Germano Scarpa (Presidente Biofarma). Confronto con Maurizio Fermeglia (rettore Università di Trieste), conduce Filiberto Zovico, fondatore ItalyPost, autore di Nuove imprese. Chi sono i champions che competono con le global companies (Egea)

Inducible Gene Expression in Lactobacillus reuteri LTH5531 during Type II Sourdough Fermentation

Lactobacillus reuteri LTH5531 is a dominant member of the microbiota of type II sourdough fermentations. To investigate the genetic background of the ecological performance of LTH5531, in vivo expression technology was used to identify promoters that show elevated levels of expression during growth of this organism in a type II sourdough fermentation. Thirty-eight sourdough-induced fusions were detected, and 29 genes could be identified on the basis of the available sequence information. Four genes encoded stress-related functions (e.g., acid and general stress response), reflecting the harsh conditions prevailing during sourdough fermentation. Further, eight genes were involved in acquisition and synthesis of amino acids and nucleotides, indicating their limited availability in sourdough. The remaining genes were either part of functionally unrelated pathways or encoded hypothetical proteins. The identification of a putative proteinase and a component of the arginine deiminase pathway is of technological interest, as they are potentially involved in the formation of aroma precursors. Our study allowed insight into the transcriptional response of Lactobacillus reuteri to the dough environment, which establishes the molecular basis to investigate bacterial properties that are likely to contribute to the ecological performance of the organism and influence the final outcome of the fermentation

The role of wave-exposure and human impacts in regulating the distribution of alternative habitats on NW Mediterranean rocky reefs

The global decline of canopy-forming macroalgae has stimulated research on the mechanism regulating shifts among alternative habitats on rocky reefs. The effects of sea urchin grazing and alterations of environmental conditions are now acknowledged as the main drivers of shifts between canopy-formers and encrusting coralline barrens and algal turfs, respectively. The conditions under which these mechanisms operate remains, however, somewhat elusive. This is mostly a consequence of the fact that our current understanding has been generated by envisioning habitat shifts as dichotomic, at odds with rocky reef landscapes being composed by mosaics of habitats and with evidence of strong interactions among the species that compose each of the alternative habitats. Using data from a long-term sampling program and path analysis, we investigated how wave-exposure and human-induced degradation of environmental conditions regulate the mechanisms maintaining algal canopies formed by Cystoseira crinita, barren habitats and algal turfs as alternative states on subtidal reefs in the NW Mediterranean. In the Tuscan Archipelago, wave-exposure had positive effects on sea urchins, which, likely due to their low mean density, had weak effects on each of the alternative habitats. Canopy-forming macroalgae resulted, instead, to exert strong negative effects on the abundance of algal turfs. Since data from the Tuscan Archipelago did not explain any of the variation in the abundance of C. crinita canopies, a further analysis was performed including data from the coast of Tuscany to assess the role of cumulative human impacts in regulating habitat shifts. This showed that degradation of environmental conditions is a direct cause of the decline of macroalgal canopies, indirectly favouring the dominance of algal turfs. Our study suggests that management of human impacts should be considered a priority for preserving subtidal canopies formed by Cystoseira in the NW Mediterranean and that conservation efforts based exclusively on the control of sea urchin populations might be doomed to failure in some areas