Global transcriptome response in Lactobacillus sakei during growth on ribose

<p>Abstract</p> <p>Background</p> <p><it>Lactobacillus sakei </it>is valuable in the fermentation of meat products and exhibits properties that allow for better preservation of meat and fish. On these substrates, glucose and ribose are the main carbon sources available for growth. We used a whole-genome microarray based on the genome sequence of <it>L. sakei </it>strain 23K to investigate the global transcriptome response of three <it>L. sakei </it>strains when grown on ribose compared with glucose.</p> <p>Results</p> <p>The function of the common regulated genes was mostly related to carbohydrate metabolism and transport. Decreased transcription of genes encoding enzymes involved in glucose metabolism and the L-lactate dehydrogenase was observed, but most of the genes showing differential expression were up-regulated. Especially transcription of genes directly involved in ribose catabolism, the phosphoketolase pathway, and in alternative fates of pyruvate increased. Interestingly, the methylglyoxal synthase gene, which encodes an enzyme unique for <it>L. sakei </it>among lactobacilli, was up-regulated. Ribose catabolism seems closely linked with catabolism of nucleosides. The deoxyribonucleoside synthesis operon transcriptional regulator gene was strongly up-regulated, as well as two gene clusters involved in nucleoside catabolism. One of the clusters included a ribokinase gene. Moreover, <it>hprK </it>encoding the HPr kinase/phosphatase, which plays a major role in the regulation of carbon metabolism and sugar transport, was up-regulated, as were genes encoding the general PTS enzyme I and the mannose-specific enzyme II complex (EII^man). Putative catabolite-responsive element (<it>cre</it>) sites were found in proximity to the promoter of several genes and operons affected by the change of carbon source. This could indicate regulation by a catabolite control protein A (CcpA)-mediated carbon catabolite repression (CCR) mechanism, possibly with the EII^manbeing indirectly involved.</p> <p>Conclusions</p> <p>Our data shows that the ribose uptake and catabolic machinery in <it>L. sakei </it>is highly regulated at the transcription level. A global regulation mechanism seems to permit a fine tuning of the expression of enzymes that control efficient exploitation of available carbon sources.</p

Primary metabolism in Lactobacillus sakei food isolates by proteomic analysis

<p>Abstract</p> <p>Background</p> <p><it>Lactobacillus sakei </it>is an important food-associated lactic acid bacterium commonly used as starter culture for industrial meat fermentation, and with great potential as a biopreservative in meat and fish products. Understanding the metabolic mechanisms underlying the growth performance of a strain to be used for food fermentations is important for obtaining high-quality and safe products. Proteomic analysis was used to study the primary metabolism in ten food isolates after growth on glucose and ribose, the main sugars available for <it>L. sakei </it>in meat and fish.</p> <p>Results</p> <p>Proteins, the expression of which varied depending on the carbon source were identified, such as a ribokinase and a D-ribose pyranase directly involved in ribose catabolism, and enzymes involved in the phosphoketolase and glycolytic pathways. Expression of enzymes involved in pyruvate and glycerol/glycerolipid metabolism were also affected by the change of carbon source. Interestingly, a commercial starter culture and a protective culture strain down-regulated the glycolytic pathway more efficiently than the rest of the strains when grown on ribose. The overall two-dimensional gel electrophoresis (2-DE) protein expression pattern was similar for the different strains, though distinct differences were seen between the two subspecies (<it>sakei </it>and <it>carnosus</it>), and a variation of about 20% in the number of spots in the 2-DE gels was observed between strains. A strain isolated from fermented fish showed a higher expression of stress related proteins growing on both carbon sources.</p> <p>Conclusions</p> <p>It is obvious from the data obtained in this study that the proteomic approach efficiently identifies differentially expressed proteins caused by the change of carbon source. Despite the basic similarity in the strains metabolic routes when they ferment glucose and ribose, there were also interesting differences. From the application point of view, an understanding of regulatory mechanisms, actions of catabolic enzymes and proteins, and preference of carbon source is of great importance.</p

Survival of Five Strains of Shiga Toxigenic Escherichia coli

The ability of foodborne pathogens to exhibit adaptive responses to stressful conditions in foods may enhance their survival when passing through the gastrointestinal system. We aimed to determine whether Escherichia coli surviving stresses encountered during a model dry-fermented sausage (DFS) production process exhibit enhanced tolerance and survival in an in vitro gastrointestinal model. Salami sausage batters spiked with five E. coli isolates, including enterohaemorrhagic E. coli strains isolated from different DFS outbreaks, were fermented in a model DFS process (20°C, 21 days). Control batters spiked with the same strains were stored at 4°C for the same period. Samples from matured model sausages and controls were thereafter exposed to an in vitro digestion challenge. Gastric exposure (pH 3) resulted in considerably reduced survival of the E. coli strains that had undergone the model DFS process. This reduction continued after entering intestinal challenge (pH 8), but growth resumed after 120 min. When subjected to gastric challenge for 120 min, E. coli that had undergone the DFS process showed about 2.3 log10⁡​ lower survival compared with those kept in sausage batter at 4°C. Our results indicated that E. coli strains surviving a model DFS process exhibited reduced tolerance to subsequent gastric challenge at low pH

Lactobacillus sakei metabolisme og diversitet

Lactic acid bacteria are associated with food fermentation, acidification and preservation. Lactobacillus sakei is an industrially important species mainly due to its ability to ferment and preserve meat. It is used as starter culture for industrial meat fermentation and has potential as a biopreservative to extend storage life and ensure microbial safety of meat and fish products. The work in this thesis aims at increasing the understanding of the primary metabolism of various L. sakei food isolates, and at defining the diversity existing among these. Growth characteristics on various media, carbohydrate-fermentation abilities and acidification properties tested in a meat model, were demonstrated to vary between strains. By genetic fingerprint techniques, a distinction between two genetic groups consistent with the two L. sakei subspecies, sakei and carnosus, was observed, with the majority of strains belonging to the latter. Microarray-based comparative genome hybridization using an array mainly based on the sequenced L. sakei strain 23K was introduced for clustering the strains. The same division into two genetic groups was observed, and a detailed view of the gene content between various test strains compared to the 23K strain was obtained. By pulsed field gel electrophoresis genome sizes were estimated to vary from 1.880 to 2.175 Mb, and the 23K genome was among the smallest. Consequently, a large part of the 23K genome belongs to a common gene pool of the species. The majority of genes important for adaption to meat products, the ability to utilize meat components, and robustness during meat processing and storage were conserved, indicative of the role these genes play in niche specialization within the species. Proteomic analysis was used to study the primary metabolism in different strains when grown on ribose compared with glucose, the main sugars available for L. sakei in meat and fish. Increased expression was observed for proteins directly involved in ribose catabolism and the phosphoketolase pathway, as well as pyruvate and glycerol/glycerolipid metabolism. Simultanously, enzymes involved in the glycolytic pathway were less expressed. These findings were confirmed at the level of gene expression using microarrays, and it was also obvious that ribose catabolism is tightly linked with catabolism of nucleosides. Moreover, enzymes important in the regulation of carbon metabolism and in sugar transport were induced. A global regulation mechanism seems to permit a fine tuning of the expression of enzymes that control efficient exploitation of available carbon sources.Melkesyrebakterier er forbundet med fermentering, syrning og konservering av mat. Lactobacillus sakei er en industrielt viktig art hovedsaklig på grunn av evnen den har til å fermentere og konservere kjøtt. Den brukes som starterkultur for industriell kjøttfermentering og har potensiale for å forlenge holdbarhet og ivareta mikrobiell trygghet for kjøtt- og fiskeprodukter. Målet for arbeidet i denne avhandlingen var å øke forståelsen omkring primærmetabolismen til forskjellige L. sakei stammer isolert fra mat, og i tillegg studere mangfoldet som eksisterer blant disse. Vekstegenskaper i forskjellige medier, karbohydrat-fermenteringsevner og evne til syreproduksjon testet i en kjøttmodell ble vist å variere mellom stammene. Ved å bruke genetiske fingerprintteknikker kunne to genetiske grupper skjelnes fra hverandre. Grupperingen oppnådd i dette arbeidet var forenlig med de to L. sakei underartene, sakei og carnosus, med flesteparten av stammene tilhørende den sistnevnte. Mikroarray-basert komparativ genom hybridisering ved bruk av et array hovedsaklig basert på den sekvenserte stammen L. sakei 23K, ble også benyttet for å gruppere stammene. Dette gav den samme inndelingen som ved bruk av genetisk fingerprintmetodikk, og gav også et detaljert bilde av geninnholdet mellom teststammene sammenliknet med 23K stammen. Ved pulsfelt gelelektroforese ble genomstørrelsene beregnet til å variere fra 1.880 til 2.175 Mb, og 23K genomet var blant de minste. Følgelig hører en stor del av 23K genomet til en felles genpool for arten. De fleste av genene som er viktige for tilpasning til et liv i kjøttprodukter, som evnen til å utnytte komponenter fra kjøtt og til å overleve kjøttprosessering og lagring var bevart, noe som antyder betydningen disse genene har i nisjespesialisering. Proteomanalyse ble benyttet for å studere primærmetabolismen i de forskjellige stammene etter vekst på ribose sammenliknet med glukose, hovedsukrene som er tilgjengelig for L. sakei i kjøtt og fisk. Økt produksjon ble observert for proteiner som er direkte involvert i ribosenedbrytning og fosfoketolaseveien, og i pyruvat- and glycerol/glycerolipid-metabolisme. Samtidig var enzymer involvert i den glykolytiske veien mindre uttrykt. Disse funnene ble bekreftet på geneksperesjonsnivå ved å bruke mikroarray. Transkripsjonsstudiene viste også at riboseutnyttelse er tett knyttet opp mot utnyttelse av nukleosider. Dessuten var enzymer som er viktige innen regulering av karbonmetabolisme og i sukkertransport indusert. Resultatene tyder på tilstedeværelse av en global regulatorisk mekanisme som finjusterer uttrykket av gener som koder for enzymer som kontrollerer effektiv utnyttelse av tilgjengelige karbonkilder.Nofim

Large Plasmid Complement Resolved: Complete Genome Sequencing of Lactobacillus plantarum MF1298, a Candidate Probiotic Strain Associated with Unfavorable Effect

Considerable attention has been given to the species Lactobacillus plantarum regarding its probiotic potential. L. plantarum strains have shown health benefits in several studies, and even nonstrain-specific claims are allowed in certain markets. L. plantarum strain MF1298 was considered a candidate probiotic, demonstrating in vitro probiotic properties and the ability to survive passage through the human intestinal tract. However, the strain showed an unfavorable effect on symptoms in subjects with irritable bowel syndrome in a clinical trial. The properties and the genome of this strain are thus of general interest. Obtaining the complete genome of strain MF1298 proved difficult due to its large plasmid complement. Here, we exploit a combination of sequencing approaches to obtain the complete chromosome and plasmid assemblies of MF1298. The Oxford Nanopore Technologies MinION long-read sequencer was particularly useful in resolving the unusually large number of plasmids in the strain, 14 in total. The complete genome sequence of 3,576,440 basepairs contains 3272 protein-encoding genes, of which 315 are located on plasmids. Few unique regions were found in comparison with other L. plantarum genomes. Notably, however, one of the plasmids contains genes related to vitamin B12 (cobalamin) turnover and genes encoding bacterial reverse transcriptases, features not previously reported for L. plantarum. The extensive plasmid information will be important for future studies with this strain.publishedVersio

Health and Safety Considerations of Fermented Sausages

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Growth Behavior of Listeria monocytogenes in a Traditional Norwegian Fermented Fish Product (Rakfisk), and Its Inhibition through Bacteriophage Addition

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Comparison of UV-C and Pulsed UV Light Treatments for Reduction of Salmonella, Listeria monocytogenes, and Enterohemorrhagic Escherichia coli on Eggs

Ten percent of all strong-evidence foodborne outbreaks in the European Union are caused by Salmonella related to eggs and egg products. UV light may be used to decontaminate egg surfaces and reduce the risk of human salmonellosis infections. The efficiency of continuous UV-C (254 nm) and pulsed UV light for reducing the viability of Salmonella Enteritidis, Listeria monocytogenes, and enterohemorrhagic Escherichia coli on eggs was thoroughly compared. Bacterial cells were exposed to UVC light at fluences from 0.05 to 3.0 J/cm2 (10 mW/cm2, for 5 to 300 s) and pulsed UV light at fluences from 1.25 to 18.0 J/cm2, resulting in reductions ranging from 1.6 to 3.8 log, depending on conditions used. Using UV-C light, it was possible to achieve higher reductions at lower fluences compared with pulsed UV light. When Salmonella was stacked on a small area or shielded in feces, the pulsed UV light seemed to have a higher penetration capacity and gave higher bacterial reductions. Microscopy imaging and attempts to contaminate the interior of the eggs with Salmonella through the eggshell demonstrated that the integrity of the eggshell was maintained after UV light treatments. Only minor sensory changes were reported by panelists when the highest UV doses were used. UV-C and pulsed UV light treatments appear to be useful decontamination technologies that can be implemented in continuous processing.submittedVersio

Large Plasmid Complement Resolved: Complete Genome Sequencing of Lactobacillus plantarum MF1298, a Candidate Probiotic Strain Associated with Unfavorable Effect

Considerable attention has been given to the species Lactobacillus plantarum regarding its probiotic potential. L. plantarum strains have shown health benefits in several studies, and even nonstrain-specific claims are allowed in certain markets. L. plantarum strain MF1298 was considered a candidate probiotic, demonstrating in vitro probiotic properties and the ability to survive passage through the human intestinal tract. However, the strain showed an unfavorable effect on symptoms in subjects with irritable bowel syndrome in a clinical trial. The properties and the genome of this strain are thus of general interest. Obtaining the complete genome of strain MF1298 proved difficult due to its large plasmid complement. Here, we exploit a combination of sequencing approaches to obtain the complete chromosome and plasmid assemblies of MF1298. The Oxford Nanopore Technologies MinION long-read sequencer was particularly useful in resolving the unusually large number of plasmids in the strain, 14 in total. The complete genome sequence of 3,576,440 basepairs contains 3272 protein-encoding genes, of which 315 are located on plasmids. Few unique regions were found in comparison with other L. plantarum genomes. Notably, however, one of the plasmids contains genes related to vitamin B12 (cobalamin) turnover and genes encoding bacterial reverse transcriptases, features not previously reported for L. plantarum. The extensive plasmid information will be important for future studies with this strain

Chicken fillets subjected to UV-C and pulsed UV light: Reduction of pathogenic and spoilage bacteria, and changes in sensory quality

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