Genomic analysis of pasteurella atlantica provides insight on its virulence factors and phylogeny and highlights the potential of reverse vaccinology in aquaculture

Pasteurellosis in farmed lumpsuckers, Cyclopterus lumpus, has emerged as a serious disease in Norwegian aquaculture in recent years. Genomic characterization of the causative agent is essential in understanding the biology of the bacteria involved and in devising an efficient preventive strategy. The genomes of two clinical Pasteurella atlantica isolates were sequenced (≈2.3 Mbp), and phylogenetic analysis confirmed their position as a novel species within the Pasteurellaceae. In silico analyses revealed 11 genomic islands and 5 prophages, highlighting the potential of mobile elements as driving forces in the evolution of this species. The previously documented pathogenicity of P. atlantica is strongly supported by the current study, and 17 target genes were recognized as putative primary drivers of pathogenicity. The expression level of a predicted vaccine target, an uncharacterized adhesin protein, was significantly increased in both broth culture and following the exposure of P. atlantica to lumpsucker head kidney leucocytes. Based on in silico and functional analyses, the strongest gene target candidates will be prioritized in future vaccine development efforts to prevent future pasteurellosis outbreaks.publishedVersio

Draft genome sequence of the bacteriocinogenic strain Enterococcus faecalis DBH18, isolated from mallard ducks (Anas platyrhynchos)

Here, we report the draft genome sequence of Enterococcus faecalis DBH18, a bacteriocinogenic lactic acid bacterium (LAB) isolated from mallard ducks (Anas platyrhynchos). The assembly contains 2,836,724 bp, with a G+C content of 37.6%. The genome is predicted to contain 2,654 coding DNA sequences (CDSs) and 50 RNAs

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

The CWPS Rubik's cube: Linking diversity of cell wall polysaccharide structures with the encoded biosynthetic machinery of selected Lactococcus lactis strains

The biosynthetic machinery for cell wall polysaccharide (CWPS) production in lactococci is encoded by a large gene cluster, designatedcwps. This locus displays considerable variation among lactococcal genomes, previously prompting a classification into three distinct genotypes (A-C). In the present study, thecwpsloci of 107 lactococcal strains were compared, revealing the presence of a fourthcwpsgenotype (type D). Lactococcal CWPSs are comprised of two saccharidic structures: a peptidoglycan-embedded rhamnan backbone polymer to which a surface-exposed, poly/oligosaccharidic side-chain is covalently linked. Chemical structures of the side-chain of seven lactococcal strains were elucidated, highlighting their diverse and strain-specific nature. Furthermore, a link betweencwpsgenotype and chemical structure was derived based on the number of glycosyltransferase-encoding genes in thecwpscluster and the presence of conserved genes encoding the presumed priming glycosyltransferase. This facilitates predictions of several structural features of lactococcal CWPSs including (a) whether the CWPS possesses short oligo/polysaccharide side-chains, (b) the number of component monosaccharides in a given CWPS structure, (c) the order of monosaccharide incorporation into the repeating units of the side-chain (for C-type strains), (d) the presence of Galfand phosphodiester bonds in the side-chain, and (e) the presence of glycerol phosphate substituents in the side-chain

Den mikrobielle diversiteten i mesofile starterkulturer anvendt i osteproduksjon

For ages, humankind has preserved various foods by fermentation by lactic acid bacteria (LAB), and fermentation of milk to obtain cheese can be traced back to the domestication of cattle, at least seven millennia ago. An essential ingredient in contemporary production of Dutch-type cheeses are the undefined mixed mesophilic (DL) starter cultures, which contains unknown mixtures of Lactococcus lactis strains and Leuconostoc spp.. Bacteriophages infecting Lactococcus lactis, the major contributors in the acidification of milk using mesophilic starter cultures, are recognized as the major cause of fermentation failures in dairy fermentations, disrupting the acidification process and negatively affecting the quality of the final product. The undefined mixed (DL) starter cultures are considered more robust against phage attack than the defined cultures, a characteristic gained from their large number of strains with diverse phage sensitivity. Starter cultures from different manufacturers are known to give cheeses qualitatively different characteristics, and performance differences are reported for different batches of the same starter culture, which indicates dissimilar culture compositions. Information on the microbial diversity of starter cultures is not publically available and tools to quantify the strain diversity or compare compositional differences between starter cultures does not exist. The information provided by the culture manufacturer with culture purchase does not include details beyond genus for leuconostocs, or beyond subspecies for the lactococci. In this study, the diversity of bacteria and their bacteriophages in starter cultures and dairy samples collected from three major cheese plants in Norway was investigated using molecular and DNA-sequencing based approaches. Use of a milk based-medium (GMA) in addition to the traditional M17 was instrumental in capturing a larger diversity of bacteria from starter cultures, which consequently increased the capacity to isolate bacteriophages from the dairy samples. The bacteria and bacteriophages were discriminated from each other use phage typing, revealing a large number of different bacteria as well as different bacteriophages. Interestingly, many of the strains that were only able to grow in a milkbased media, demonstrated unique phage sensitivities. A large number of phenotypically different starter bacteria with dissimilar phage sensitivities were whole-genome sequenced and characterized in pan-genome analyses. Pan-genome analyses discriminated between 21 Lactococcus lactis subsp. lactis, 28 Lactococcus lactis subsp. cremoris, as well as 12 3 Leuconostoc spp. lineages. Interestingly, the analyses did not discriminate Leuconostoc mesenteroides subsp. mesenteroides from Leuconostoc mesenteroides subsp. dextranicum, and showed that genomic variation between the isolates was much greater than between the subspecies. The diversity of Lactococcus lactis of three DL starter cultures was analyzed by targeted-amplicon sequencing of 16S rDNA, the core gene purR, and the softcore gene epsD, present in over 95% of starter culture isolates, but absent in most of the reference strains. The results revealed significant differences between the three starter cultures as well as compositional shifts during cultivation in milk. Compositional analyses of the Leuconostoc population in the five DL starters by targeted-amplicon sequencing of eno, the gene encoding for enolase, also revealed significant differences between the cultures. Three of the cultures were dominated by Leuconostoc mesenteroides subsp. cremoris while Leuconostoc pseudomesenteroides dominated in the other two. Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum was found in all DL cultures, while Leuconostoc lactis, reported to be a major constituent in fermented dairy products, was only identified in one of the cultures. This work shows that starter cultures are different both with regards to both lactococci and leuconostocs, and provides tools to describe the microbial diversity of mesophilic starter cultures. The dairy industry and starter culture manufacturers can vastly improve their ability to monitor all phases of starter culture and cheese production by implementing the methods described in this work. Routine analysis of the microbial composition of starter cultures will enable quality control of starter cultures, and enable the industry to make competent decisions regarding starter culture rotations in the event of phage attack. I årtusener har mennesker utnyttet melkesyrebakterier (LAB) til å konservere mat via fermentering. Produksjon av ost via fermentering av melk kan spores minst 7.000 år tilbake til domestiseringen av storfe. En essensiell ingrediens i moderne produksjon av gulost er starterkulturene, som oftest såkalt udefinerte mesofile blandingskulturer (DL) som inneholder et ukjent antall forskjellige Lactococcus lactis stammer og Leuconostoc spp.. Kjent som den hyppigste årsaken til fermenteringsfeil, er bakteriofager som angriper Lactococcus lactis, den viktigste bidragsyteren i forsuringen av melk ved bruk av mesofile starterkulturer. Bakteriofagangrep kan forstyrre forsuringsprosessen og redusere kvaliteten på sluttproduktet. Fordi de inneholder et stort antall stammer med ulik følsomhet for bakteriofager, anses de udefinerte blandingskulturene som mer robuste mot bakteriofagangrep enn definerte kulturer. Det er kjent at starterkulturer fra ulike produsenter gir ostene forskjellige kvalitetsmessige karakteristikker, en indikasjon på ulikheter i kulturkomposisjonen. Informasjon om den mikrobielle diversiteten i starterkulturene er ikke offentlig tilgjengelig og verktøy for kvantifisering av stammediversiteten eller for å sammenligne kulturkomposisjonen mellom kulturene eksisterer ikke. Kulturprodusentene oppgir ikke detaljer utover genus for Leuconostoc, eller utover underart for Lactococcus lactis. I denne studien har bakterie- og bakteriofag-diversiteten i starterkulturer og meieriprøver fra tre ulike store norske ysterier blitt undersøkt ved hjelp av molekylære og DNA-sekvenseringsbaserte metoder. Bruk av et melkebasert vekstmedium (GMA) i tillegg til det tradisjonelle vekstmediet M17 var avgjørende for å øke kapasiteten til å isolere en større diversitet av bakterier fra starterkulturene, som igjen førte til et større potensial for å isolere bakteriofager fra meieriprøvene. Ved hjelp av fagtyping ble et stort antall forskjellige bakterier og bakteriofager diskriminert fra hverandre. Et interessant funn var at mange av stammene som kun vokste på det melkebaserte mediet var følsomme for bakteriofag som M17-stammene ikke var følsomme for. Et stort antall fenotypisk forskjellige starter bakterier med ulik fagfølsomhet ble hel-genom sekvensert og karakterisert ved hjelp av pangenomiske analyser. Pan-genom analysene skilte bakteriene inn i 21 Lactococcus lactis subart lactis, 28 Lactococcus lactis subart cremoris, og 12 Leuconostoc spp. linjer. Analysen diskriminerte ikke Leuconostoc mesenteroides subart mesenteriodes fra 5 Leuconostoc mesenteroides subart dextranicum, og viste at den genomiske variasjonen mellom isolatene var mye større enn mellom subartene. Diversiteten av Lactococcus lactis ble undersøkt i tre DL starterkulturer ved «amplicon» sekvensering av 16S rDNA, «core»- genet purR, og «softcore»-genet epsD som var tilstede i over 95% av starterkultur isolatene, men var fraværende i flesteparten av referansestammene. Resultatene avslørte betydelige forskjeller mellom de tre starterkulturene og endringer i kulturkomposisjonen under kultivering i melk. Komposisjonsanalysen av Leuconostoc i fem DL starterkulturer ved «amplicon» sekvensering av eno, genet som koder for Enolase, et essensielt enzym i glykolysen avslørte også signifikante forskjeller mellom starterkulturene. Tre av kulturene var dominerte av Leuconostoc mesenteroides subsp. cremoris mens de to resterende kulturene var dominerte av Leuconostoc pseudomesenteroides. Et lavt antall av Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum ble identifisert i alle DL starterkulturene, mens Leuconostoc lactis, beskrevet i litteraturen som høyst relevant, ble kun identifisert i lave antall i en av kulturene. Dette arbeidet viser at mesofile starterkulturer er forskjellige, både med hensyn til laktokokker og leukonostokkene og inkluderer verktøy for å beskrive den mikrobielle diversiteten i mesofile starterkulturer. Ved å implementere metodene beskrevet i dette arbeidet kan meierinæringen og starterkulturprodusentene oppnå en betraktelig bedre evne til å overvåke alle faser av starterkultur og osteproduksjonen. Kvalitetskontroll av meieriproduksjonen ved å regelmessig analysere den mikrobielle komposisjonen i starterkulturene kan bidra til å redusere svinn, effektivisere produksjonen, og styrke evnen til å avgjøre hvilke kulturer som benyttes i produksjonen, samt hvilke kulturer som inkluderes i et rotasjonssystem, skulle produksjonen være utsatt for bakteriofagangrep

Unprecedented diversity of lactococcal group 936 bacteriophages revealed by amplicon sequencing of the portal protein gene

Lactococcus lactis is one of the most important bacteria in dairy fermentations, being used in the production of cheese and buttermilk. The processes are vulnerable to phage attacks, and undefined mixtures of lactococcal strains are often used to reduce the risk of bacteriophage caused fermentation failure. Other preventive measures include culture rotation to prevent phage build-up and phage monitoring. Phage diversity, rather than quantity, is the largest threat to fermentations using undefined mixed starter cultures. We have developed a method for culture independent diversity analysis of lytic bacteriophages of the 936 group, the phages most commonly found in dairies. Using, as a target, a highly variable region of the portal protein gene, we demonstrate an unprecedented diversity and the presence of new 936 phages in samples taken from cheese production. The method should be useful to the dairy industry and starter culture manufacturers in their efforts to reduce phage problems.publishedVersio

Unprecedented diversity of lactococcal group 936 bacteriophages revealed by amplicon sequencing of the portal protein gene

Lactococcus lactis is one of the most important bacteria in dairy fermentations, being used in the production of cheese and buttermilk. The processes are vulnerable to phage attacks, and undefined mixtures of lactococcal strains are often used to reduce the risk of bacteriophage caused fermentation failure. Other preventive measures include culture rotation to prevent phage build-up and phage monitoring. Phage diversity, rather than quantity, is the largest threat to fermentations using undefined mixed starter cultures. We have developed a method for culture independent diversity analysis of lytic bacteriophages of the 936 group, the phages most commonly found in dairies. Using, as a target, a highly variable region of the portal protein gene, we demonstrate an unprecedented diversity and the presence of new 936 phages in samples taken from cheese production. The method should be useful to the dairy industry and starter culture manufacturers in their efforts to reduce phage problems.publishedVersio

En studie av effektene til aminosyrer og arginine deiminase (arcA) på vekst, metabolisme og genekspresjon i Enterococcus faecalis V583

In this study the roles of amino acids and arginine deiminase (arcA) in Enterococcus faecalis V583‟s growth, metabolism and gene expression were investigated. Literature describes only one pathway of arginine catabolism for Enterococcus faecalis V583 in the ADI-pathway where arginine deiminase catalyzes the deamination of L-arginine, producing L-citrulline and ammonia. A deletion of arcA was constructed in Enterococcus faecalis V583 (ΔarcA), but surprisingly L-arginine was still broken down in a glucose-limited continuous culture, suggesting an alternative arginine deiminase or alternative pathway for arginine catabolism exists in the E. faecalis V583 genome. A significant effect of the arcA deletion was observed on growth and metabolism both in batch culture with no nutritional limitation, and in glucoselimited continuous culture. Growth studies in batch culture using a defined medium with amino acid leave-out compositions showed that amino acid availability played a significant role in both E. faecalis V583 wild type and mutant growth. A significant reduction in growth rate and a more pronounced death phase was observed for the ΔarcA mutant in comparison to wild type. Growth in glucose-limited continuous culture revealed a pH-dependency on the shift between homolactic and mixed acid fermentation. Transcriptional analysis by real-time PCR also showed pH-dependency of pflA transcription, revealing a down-regulation of pflA in pH 6.5 culture compared to pH 7.5 culture. A reduction in free phosphate concentrations in the mutant cultures, together with provisional genomic data of a polyphosphate kinase (ppnK) gene at EF2670 indicates E. faecalis V583 might be able to produce polyphosphates in stressful environments. A significant reduction in biomass for the ΔarcA mutant in comparison to wild type also suggests a less efficient metabolism for the ΔarcA mutant. Complementation of the E. faecalis V583ΔarcA mutant was performed, providing an intact arcA gene in trans, and construction of a double deletion mutant E. faecalis V583ΔglnAΔarcA was also performed. A preliminary growth study in batch culture showed no growth difference between E. faecalis V583ΔarcA and the complemented E. faecalis V583ΔarcA, suggesting that providing an intact arcA gene is not sufficient to restore ADI-pathway function. A significant difference between E. faecalis V583ΔglnA and V583ΔglnAΔarcA was observed, with V583ΔglnAΔarcA growing significantly slower, but reaching a higher OD600 before initiating stationary phase. I dette arbeidet har effekten av ulike aminosyrer og arginine deiminase (arcA) på vekst, metabolisme og genekspresjon i Enterococcus faecalis V583 blitt undersøkt. Kun en vei for nedbrytning av L-arginine har så langt blitt beskrevet for E. faecalis V583. Her deamineres Larginine i en reaksjon katalysert av arginine deiminase, og L-citrulline og ammoniakk blir dannet. En mutant med delesjon av arcA (ΔarcA) ble konstruert i E. faecalis V583. Data fra glukose-begrenset kontinuerlig kultur viste imidlertid at L- arginine fortsatt ble brutt ned i mutanten, noe som indikerer at en alternativ arginine deiminase, eller en alternativ katabolsk vei eksisterer for E. faecalis V583. En signifikant effekt av arcA delesjonen på vekst og metabolisme ble observert i kultur både med og uten glukose-begrensning. Vekstforsøk i batchkulturer i et definert medium, der enkelte aminosyrer ble utelatt, viste at tilgjengeligheten av aminosyrer har en signifikant rolle i veksten til både ΔarcA mutanten og villtypen av E. faecalis V583. Videre ble en signifikant reduksjon av veksthastigheten og en mer utpreget dødsfase observert hos ΔarcA mutanten sammenlignet med villtypen. Vekst i glukose-begrenset kontinuerlig kultur viste et pH-avhengig skifte mellom homolaktisk og blandet syrefermentering. Transkripsjonsanalyser, gjort med sanntids-PCR, viste også en effekt av pH på transkripsjonsnivå av pflA, med en nedregulering av pflA i pH 6.5 kulturen kontra pH 7.5 kulturen. En reduksjon i mengden fritt fosfat i mutanten, sammen med et gen med homologi til polyfosfat kinase (ppnK) i EF2670, foreslår at E. faecalis V583 kan være i stand til å danne polyfosfater under stress. En signifikant reduksjon av biomassen for ΔarcA mutanten, i forhold til villtypen, indikerer en mindre effektiv metabolisme hos mutanten kontra villtypen. Komplementering av ΔarcA mutanten der et intakt arcAble tilført in trans, ,og konstruksjon av en dobbel delesjonsmutant E. faecalis V583ΔglnAΔarcA ble gjort. Et innledende vekstforsøki batchkultur viste ingen forskjellig mellom E. faecalis V583ΔarcA og E. faecalis V583ΔarcA komplementert. Denne observasjonen indikerer at et intakt arcA gen ikke er tilstrekkelig for å gjenopprette arginine deiminase-funksjonen i cellen. En signifikant forskjell av vekst mellom E. faecalis V583ΔglnA and V583ΔglnAΔarcA ble også observert. V583ΔglnAΔarcA viste en signifikant reduksjon i veksthastighet, men vokser til en signifikant høyere OD600 før stasjonærfase innledes

The microbial diversity of mesophilic starter cultures used in cheese production

For ages, humankind has preserved various foods by fermentation by lactic acid bacteria (LAB), and fermentation of milk to obtain cheese can be traced back to the domestication of cattle, at least seven millennia ago. An essential ingredient in contemporary production of Dutch-type cheeses are the undefined mixed mesophilic (DL) starter cultures, which contains unknown mixtures of Lactococcus lactis strains and Leuconostoc spp.. Bacteriophages infecting Lactococcus lactis, the major contributors in the acidification of milk using mesophilic starter cultures, are recognized as the major cause of fermentation failures in dairy fermentations, disrupting the acidification process and negatively affecting the quality of the final product. The undefined mixed (DL) starter cultures are considered more robust against phage attack than the defined cultures, a characteristic gained from their large number of strains with diverse phage sensitivity. Starter cultures from different manufacturers are known to give cheeses qualitatively different characteristics, and performance differences are reported for different batches of the same starter culture, which indicates dissimilar culture compositions. Information on the microbial diversity of starter cultures is not publically available and tools to quantify the strain diversity or compare compositional differences between starter cultures does not exist. The information provided by the culture manufacturer with culture purchase does not include details beyond genus for leuconostocs, or beyond subspecies for the lactococci. In this study, the diversity of bacteria and their bacteriophages in starter cultures and dairy samples collected from three major cheese plants in Norway was investigated using molecular and DNA-sequencing based approaches. Use of a milk based-medium (GMA) in addition to the traditional M17 was instrumental in capturing a larger diversity of bacteria from starter cultures, which consequently increased the capacity to isolate bacteriophages from the dairy samples. The bacteria and bacteriophages were discriminated from each other use phage typing, revealing a large number of different bacteria as well as different bacteriophages. Interestingly, many of the strains that were only able to grow in a milkbased media, demonstrated unique phage sensitivities. A large number of phenotypically different starter bacteria with dissimilar phage sensitivities were whole-genome sequenced and characterized in pan-genome analyses. Pan-genome analyses discriminated between 21 Lactococcus lactis subsp. lactis, 28 Lactococcus lactis subsp. cremoris, as well as 12 3 Leuconostoc spp. lineages. Interestingly, the analyses did not discriminate Leuconostoc mesenteroides subsp. mesenteroides from Leuconostoc mesenteroides subsp. dextranicum, and showed that genomic variation between the isolates was much greater than between the subspecies. The diversity of Lactococcus lactis of three DL starter cultures was analyzed by targeted-amplicon sequencing of 16S rDNA, the core gene purR, and the softcore gene epsD, present in over 95% of starter culture isolates, but absent in most of the reference strains. The results revealed significant differences between the three starter cultures as well as compositional shifts during cultivation in milk. Compositional analyses of the Leuconostoc population in the five DL starters by targeted-amplicon sequencing of eno, the gene encoding for enolase, also revealed significant differences between the cultures. Three of the cultures were dominated by Leuconostoc mesenteroides subsp. cremoris while Leuconostoc pseudomesenteroides dominated in the other two. Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum was found in all DL cultures, while Leuconostoc lactis, reported to be a major constituent in fermented dairy products, was only identified in one of the cultures. This work shows that starter cultures are different both with regards to both lactococci and leuconostocs, and provides tools to describe the microbial diversity of mesophilic starter cultures. The dairy industry and starter culture manufacturers can vastly improve their ability to monitor all phases of starter culture and cheese production by implementing the methods described in this work. Routine analysis of the microbial composition of starter cultures will enable quality control of starter cultures, and enable the industry to make competent decisions regarding starter culture rotations in the event of phage attack. I årtusener har mennesker utnyttet melkesyrebakterier (LAB) til å konservere mat via fermentering. Produksjon av ost via fermentering av melk kan spores minst 7.000 år tilbake til domestiseringen av storfe. En essensiell ingrediens i moderne produksjon av gulost er starterkulturene, som oftest såkalt udefinerte mesofile blandingskulturer (DL) som inneholder et ukjent antall forskjellige Lactococcus lactis stammer og Leuconostoc spp.. Kjent som den hyppigste årsaken til fermenteringsfeil, er bakteriofager som angriper Lactococcus lactis, den viktigste bidragsyteren i forsuringen av melk ved bruk av mesofile starterkulturer. Bakteriofagangrep kan forstyrre forsuringsprosessen og redusere kvaliteten på sluttproduktet. Fordi de inneholder et stort antall stammer med ulik følsomhet for bakteriofager, anses de udefinerte blandingskulturene som mer robuste mot bakteriofagangrep enn definerte kulturer. Det er kjent at starterkulturer fra ulike produsenter gir ostene forskjellige kvalitetsmessige karakteristikker, en indikasjon på ulikheter i kulturkomposisjonen. Informasjon om den mikrobielle diversiteten i starterkulturene er ikke offentlig tilgjengelig og verktøy for kvantifisering av stammediversiteten eller for å sammenligne kulturkomposisjonen mellom kulturene eksisterer ikke. Kulturprodusentene oppgir ikke detaljer utover genus for Leuconostoc, eller utover underart for Lactococcus lactis. I denne studien har bakterie- og bakteriofag-diversiteten i starterkulturer og meieriprøver fra tre ulike store norske ysterier blitt undersøkt ved hjelp av molekylære og DNA-sekvenseringsbaserte metoder. Bruk av et melkebasert vekstmedium (GMA) i tillegg til det tradisjonelle vekstmediet M17 var avgjørende for å øke kapasiteten til å isolere en større diversitet av bakterier fra starterkulturene, som igjen førte til et større potensial for å isolere bakteriofager fra meieriprøvene. Ved hjelp av fagtyping ble et stort antall forskjellige bakterier og bakteriofager diskriminert fra hverandre. Et interessant funn var at mange av stammene som kun vokste på det melkebaserte mediet var følsomme for bakteriofag som M17-stammene ikke var følsomme for. Et stort antall fenotypisk forskjellige starter bakterier med ulik fagfølsomhet ble hel-genom sekvensert og karakterisert ved hjelp av pangenomiske analyser. Pan-genom analysene skilte bakteriene inn i 21 Lactococcus lactis subart lactis, 28 Lactococcus lactis subart cremoris, og 12 Leuconostoc spp. linjer. Analysen diskriminerte ikke Leuconostoc mesenteroides subart mesenteriodes fra 5 Leuconostoc mesenteroides subart dextranicum, og viste at den genomiske variasjonen mellom isolatene var mye større enn mellom subartene. Diversiteten av Lactococcus lactis ble undersøkt i tre DL starterkulturer ved «amplicon» sekvensering av 16S rDNA, «core»- genet purR, og «softcore»-genet epsD som var tilstede i over 95% av starterkultur isolatene, men var fraværende i flesteparten av referansestammene. Resultatene avslørte betydelige forskjeller mellom de tre starterkulturene og endringer i kulturkomposisjonen under kultivering i melk. Komposisjonsanalysen av Leuconostoc i fem DL starterkulturer ved «amplicon» sekvensering av eno, genet som koder for Enolase, et essensielt enzym i glykolysen avslørte også signifikante forskjeller mellom starterkulturene. Tre av kulturene var dominerte av Leuconostoc mesenteroides subsp. cremoris mens de to resterende kulturene var dominerte av Leuconostoc pseudomesenteroides. Et lavt antall av Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum ble identifisert i alle DL starterkulturene, mens Leuconostoc lactis, beskrevet i litteraturen som høyst relevant, ble kun identifisert i lave antall i en av kulturene. Dette arbeidet viser at mesofile starterkulturer er forskjellige, både med hensyn til laktokokker og leukonostokkene og inkluderer verktøy for å beskrive den mikrobielle diversiteten i mesofile starterkulturer. Ved å implementere metodene beskrevet i dette arbeidet kan meierinæringen og starterkulturprodusentene oppnå en betraktelig bedre evne til å overvåke alle faser av starterkultur og osteproduksjonen. Kvalitetskontroll av meieriproduksjonen ved å regelmessig analysere den mikrobielle komposisjonen i starterkulturene kan bidra til å redusere svinn, effektivisere produksjonen, og styrke evnen til å avgjøre hvilke kulturer som benyttes i produksjonen, samt hvilke kulturer som inkluderes i et rotasjonssystem, skulle produksjonen være utsatt for bakteriofagangrep

A study on the effects of aminoacids and arginine deiminase (arcA) on growth, metabolism and gene expression in Enterococcus faecalis V583

In this study the roles of amino acids and arginine deiminase (arcA) in Enterococcus faecalis V583‟s growth, metabolism and gene expression were investigated. Literature describes only one pathway of arginine catabolism for Enterococcus faecalis V583 in the ADI-pathway where arginine deiminase catalyzes the deamination of L-arginine, producing L-citrulline and ammonia. A deletion of arcA was constructed in Enterococcus faecalis V583 (ΔarcA), but surprisingly L-arginine was still broken down in a glucose-limited continuous culture, suggesting an alternative arginine deiminase or alternative pathway for arginine catabolism exists in the E. faecalis V583 genome. A significant effect of the arcA deletion was observed on growth and metabolism both in batch culture with no nutritional limitation, and in glucoselimited continuous culture. Growth studies in batch culture using a defined medium with amino acid leave-out compositions showed that amino acid availability played a significant role in both E. faecalis V583 wild type and mutant growth. A significant reduction in growth rate and a more pronounced death phase was observed for the ΔarcA mutant in comparison to wild type. Growth in glucose-limited continuous culture revealed a pH-dependency on the shift between homolactic and mixed acid fermentation. Transcriptional analysis by real-time PCR also showed pH-dependency of pflA transcription, revealing a down-regulation of pflA in pH 6.5 culture compared to pH 7.5 culture. A reduction in free phosphate concentrations in the mutant cultures, together with provisional genomic data of a polyphosphate kinase (ppnK) gene at EF2670 indicates E. faecalis V583 might be able to produce polyphosphates in stressful environments. A significant reduction in biomass for the ΔarcA mutant in comparison to wild type also suggests a less efficient metabolism for the ΔarcA mutant. Complementation of the E. faecalis V583ΔarcA mutant was performed, providing an intact arcA gene in trans, and construction of a double deletion mutant E. faecalis V583ΔglnAΔarcA was also performed. A preliminary growth study in batch culture showed no growth difference between E. faecalis V583ΔarcA and the complemented E. faecalis V583ΔarcA, suggesting that providing an intact arcA gene is not sufficient to restore ADI-pathway function. A significant difference between E. faecalis V583ΔglnA and V583ΔglnAΔarcA was observed, with V583ΔglnAΔarcA growing significantly slower, but reaching a higher OD600 before initiating stationary phase. I dette arbeidet har effekten av ulike aminosyrer og arginine deiminase (arcA) på vekst, metabolisme og genekspresjon i Enterococcus faecalis V583 blitt undersøkt. Kun en vei for nedbrytning av L-arginine har så langt blitt beskrevet for E. faecalis V583. Her deamineres Larginine i en reaksjon katalysert av arginine deiminase, og L-citrulline og ammoniakk blir dannet. En mutant med delesjon av arcA (ΔarcA) ble konstruert i E. faecalis V583. Data fra glukose-begrenset kontinuerlig kultur viste imidlertid at L- arginine fortsatt ble brutt ned i mutanten, noe som indikerer at en alternativ arginine deiminase, eller en alternativ katabolsk vei eksisterer for E. faecalis V583. En signifikant effekt av arcA delesjonen på vekst og metabolisme ble observert i kultur både med og uten glukose-begrensning. Vekstforsøk i batchkulturer i et definert medium, der enkelte aminosyrer ble utelatt, viste at tilgjengeligheten av aminosyrer har en signifikant rolle i veksten til både ΔarcA mutanten og villtypen av E. faecalis V583. Videre ble en signifikant reduksjon av veksthastigheten og en mer utpreget dødsfase observert hos ΔarcA mutanten sammenlignet med villtypen. Vekst i glukose-begrenset kontinuerlig kultur viste et pH-avhengig skifte mellom homolaktisk og blandet syrefermentering. Transkripsjonsanalyser, gjort med sanntids-PCR, viste også en effekt av pH på transkripsjonsnivå av pflA, med en nedregulering av pflA i pH 6.5 kulturen kontra pH 7.5 kulturen. En reduksjon i mengden fritt fosfat i mutanten, sammen med et gen med homologi til polyfosfat kinase (ppnK) i EF2670, foreslår at E. faecalis V583 kan være i stand til å danne polyfosfater under stress. En signifikant reduksjon av biomassen for ΔarcA mutanten, i forhold til villtypen, indikerer en mindre effektiv metabolisme hos mutanten kontra villtypen. Komplementering av ΔarcA mutanten der et intakt arcAble tilført in trans, ,og konstruksjon av en dobbel delesjonsmutant E. faecalis V583ΔglnAΔarcA ble gjort. Et innledende vekstforsøki batchkultur viste ingen forskjellig mellom E. faecalis V583ΔarcA og E. faecalis V583ΔarcA komplementert. Denne observasjonen indikerer at et intakt arcA gen ikke er tilstrekkelig for å gjenopprette arginine deiminase-funksjonen i cellen. En signifikant forskjell av vekst mellom E. faecalis V583ΔglnA and V583ΔglnAΔarcA ble også observert. V583ΔglnAΔarcA viste en signifikant reduksjon i veksthastighet, men vokser til en signifikant høyere OD600 før stasjonærfase innledes