Ecological fitness and interspecies interactions of food-spoilage-associated lactic acid bacteria : insights from the genome analyses and transcriptome profiles

Lactic acid bacteria (LAB) play a dual role in food manufacturing. While being indispensable for food fermentations and preservation, they are also involved in spoilage of foods and beverages, and some food-borne LAB are pathogens. Particularly, they became the main spoilage organisms in the cold-stored modified atmosphere packaged (MAP) foods. LAB species composition and their relative abundances depend on the nature of food products and preservation technology. However, two LAB species, Leuconostoc gelidum and Lactococcus piscium, have frequently been predominating at the end of shelf life in a variety of packaged and refrigerated foods of animal and plant origin. Besides the predominant species, spoilage LAB communities contain less abundant and slower growing species, such as Lactobacillus oligofermentans, the role of which in food spoilage is unclear. Taking into account the increased popularity of MAP technology combined with cold storage for preservation of minimally processed fresh foods, the need to obtain more information on the metabolism, genomics, ecology and interactions of psychrotrophic food-spoilage-associated LAB is clear. In this thesis a genomic approach was used to study these LAB. In order to characterize spoilage community members, we sequenced and annotated genomes of Lc. piscium MKFS47 and Lb. oligofermentans LMG 22743T, both isolated from broiler meat, and seven strains of Le. gelidum, isolated from vegetable-based foods. The analysis of their gene contents and their comparison with gene repertoire of other close related species allowed us to predict putative factors that might facilitate their survival in their habitats and increase competitiveness in the spoilage microbial communities. No major differences in the gene contents of the vegetable and meat Le. gelidum strains were observed that would suggest niche-specificity, therefore, indicating that the absence of strain dissemination between vegetable- and meat-processing chains is a more likely factor responsible for the reported strain segregation between vegetable and meat-based products. Lc. piscium MKFS47 was identified as an efficient producer of buttery off-odors compounds from glucose under aerobic conditions, which is in agreement with the previous inoculation studies. Time course glucose catabolism-based transcriptome profiles revealed the presence of classical carbon catabolite repression mechanism for the regulation of carbohydrate catabolism, which was relieved along with decreasing concentration of glucose. During the same time, the shift from homolactic to heterolactic fermentation mode was observed. For Lb. oligofermentans, a pentose-preferring obligate heterofermentative LAB, the induction of efficient utilization of hexoses was confirmed indicating that it has flexible carbohydrate catabolism, which can be adjusted depending on the carbohydrate sources available in the environment. Unexpectedly, transcriptome responses of Lb. oligofermentans during growth on glucose and xylose were more alike than during fermentation of ribose in the early exponential growth phase. In addition, cross induction of glucose and xylose catabolic genes by either glucose or xylose was observed. These phenomena could be governed by the CcpA transcriptional regulator, the regulation mechanism of which remains to be determined. Transcriptome-based study of interspecies interactions between three above mentioned LAB species revealed their different survival strategies to cope with competition for the common resources. Le. gelidum was shown to enhance its nutrient- (mainly carbohydrates) scavenging and growth capabilities under glucose limitation conditions when competing with the other LAB species, while the opposite was observed for Lc. piscium and Lb. oligofermentans. Such behavior might explain the competitive success and, hence, the predominance of Le. gelidum in spoilage microbial communities. Peculiarly, interspecies interactions activated expression of prophages and restriction modification systems in Lc. piscium and Lb. oligofermentans, but not in Le. gelidum. The downregulation of stress protection-related genes in all the LAB at the early growth stage was unexpected, and it requires further studies. Finally, overexpression of the numerous putative adhesins in Lb. oligofermentans during growth with other LAB could be one of the factors explaining its survival in actively growing communities in meat.Maitohappobakteerit ovat merkittäviä elintarvikkeiden valmistukselle kahdella eri tavalla. Ne ovat korvaamattoman tärkeitä ruuan hapattamiselle ja säilömiselle, mutta toisaalta ne myös aiheuttavat ruokien ja juomien pilaantumista, ja eräät ruokaperäiset maitohappobakteerit ovat patogeenejä. Maitohappobakteerit ovat tärkeimpiä pilaajaorganismeja kylmäsäilytettävissä suojakaasuun pakatuissa ruuissa. Niiden lajivalikoima ja eri lajien suhteelliset osuudet riippuvat ruokatuotteen luonteesta sekä säilöntäteknologiasta. Tästä huolimatta kaksi maitohappobakteerilajia, Leuconostoc gelidum ja Lactococcus piscium, tyypillisesti vallitsevat yhteisöä säilyvyysajan loppupuolella monissa erilaisissa pakatuissa ja kylmäsäilytettävissä, eläin- ja kasviperäisissä ruokatuotteissa. Näiden lajien lisäksi pilaajamaitohappobakteeriyhteisöt sisältävät harvinaisempia ja hitaammin kasvavia lajeja kuten Lactobacillus oligofermentans, jonka rooli ruuan pilaantumisessa on epäselvä. Suojakaasuun pakkaamisen suosio minimaalisesti prosessoitujen tuoreiden ruokien kylmäsäilytyksessä kasvaa jatkuvasti, joten on selvää, että ruuan pilaantumiseen liittyvien psykrotrofisten maitohappobakteerien metaboliasta, genomiikasta, ekologiasta ja vuorovaikutuksista tarvitaan lisää tietoa. Tässä väitöskirjassa näitä maitohappobakteereja tutkittiin genomiikan työkalujen avulla. Pilaajayhteisön jäsenten määrittämistä varten sekvensoimme ja annotoimme useita bakteerigenomeja: broilerinlihasta eristetyt kannat Lc. piscium MKFS47 ja Lb. oligofermentans LMG 22743T sekä seitsemän kasvipohjaisista ruuista eristettyä Le. gelidum -kantaa. Analysoimalla bakteerikantojen geenivalikoimaa sekä vertaamalla sitä lähisukuisten lajien kanssa pystyimme ennustamaan tekijöitä, jotka saattavat auttaa näitä bakteereja selviämään elinympäristöissään ja lisätä niiden kilpailukykyä pilaajabakteeriyhteisöissä. Kasviksista ja lihasta peräisin olevien Le. gelidum -kantojen geenivalikoimissa ei ollut havaittavissa suuria eroja, jotka kertoisivat elinympäristöön erikoistumisesta, joten vaikuttaa siltä, että erot kasvis- ja lihaperäisissä tuotteissa esiintyvien kantojen välillä johtuvat ennemminkin siitä, etteivät kannat leviä tuotantoketjusta toiseen. Lc. piscium MKFS47 -kannan havaittiin aerobisissa olosuhteissa tuottavan tehokkaasti glukoosista voimaisia virhehajuja aiheuttavia yhdisteitä, mikä vastaa aiempien siirrostuskokeiden tuloksia. Aikasarjana tehdyt glukoosin kataboliaan perustuvat transkriptomiprofiilit paljastivat klassisen hiili-kataboliittirepressiomekanismin, jolla bakteeri säätelee hiilihydraattien hajotusta. Sen vaikutus väheni glukoosikonsentraation laskiessa. Samaan aikaan bakteereiden havaittiin siirtyvän homolaktisesta heterolaktiseen fermentaatioon. Lb. oligofermentans on pentoosia suosiva obligaatti heterofermentatiivinen maitohappobakteeri. Kokeissa vahvistettiin, että se voidaan indusoida käyttämään tehokkaasti heksooseja. Bakteerilla siis on joustava hiilihydraattikatabolia, jota se voi säädellä saatavilla olevien hiilihydraattien mukaan. Yllättävää oli, että Lb. oligofermentansin transkriptomivasteet varhaisessa eksponentiaalisen kasvun vaiheessa olivat enemmän toistensa kaltaisia glukoosilla ja ksyloosilla kasvatettaessa kuin bakteerin fermentoidessa riboosia. Lisäksi glukoosi- ja ksyloosikataboliaan liittyvät geenit olivat indusoitavissa sekä glukoosilla että ksyloosilla. Näitä ilmiöitä saattaa hallinnoida CcpA-säätelytekijä, jonka säätelymekanismi on toistaiseksi selvittämättä. Transkriptomeihin perustuva tutkimus kolmen yllämainitun maitohappobakteerilajin vuorovaikutuksista paljasti niiden erilaiset selviytymisstrategiat yhteisistä resursseista kilpailtaessa. Le. gelidumin osoitettiin tehostavan kykyjään hankkia ravinteita (lähinnä hiilihydraatteja) sekä kasvaa glukoosirajoitetuissa olosuhteissa, kun se kilpaili muiden maitohappobakteerilajien kanssa. Lc. piscium ja Lb. oligofermentans puolestaan toimivat päinvastoin. Tämä käytös saattaa selittää Le. gelidumin kilpailumenestystä, ja siten sen vallitsevuutta pilaajabakteeriyhteisöissä. Yllättävää kyllä lajienväliset vuorovaikutukset aktivoivat profaagien ja restriktiomodifikaatiosysteemien ekspression Lc. pisciumilla ja Lb. oligofermentansilla, mutta ei Le. gelidumilla. Lisätutkimusta vaatisi myös se odottamaton havainto, että kaikilla maitohappobakteereilla esiintyi stressiltä suojautumiseen liittyvien geenien vaimentumista varhaisessa kasvuvaiheessa. Lopuksi, lukuisien ennustettujen adhesiinien yliekspressio Lb. oligofermentansilla muiden maitohappobakteerien kanssa kasvatettaessa saattaa olla yksi selitys sille, miten bakteeri selviää aktiivisesti kasvavissa yhteisöissä lihassa

Complete genome sequence of Leuconostoc gelidum subsp. gasicomitatum KG16-1, isolated from vacuum-packaged vegetable sausages

Leuconostoc gelidum subsp. gasicomitatum is a predominant lactic acid bacterium (LAB) in spoilage microbial communities of different kinds of modified-atmosphere packaged (MAP) food products. So far, only one genome sequence of a poultry-originating type strain of this bacterium (LMG 18811T) has been available. In the current study, we present the completely sequenced and functionally annotated genome of strain KG16-1 isolated from a vegetable-based product. In addition, six other vegetable-associated strains were sequenced to study possible “niche” specificity suggested by recent multilocus sequence typing. The genome of strain KG16-1 consisted of one circular chromosome and three plasmids, which together contained 2,035 CDSs. The chromosome carried at least three prophage regions and one of the plasmids encoded a galactan degradation cluster, which might provide a survival advantage in plant-related environments. The genome comparison with LMG 18811T and six other vegetable strains suggests no major differences between the meat- and vegetable-associated strains that would explain their “niche” specificity. Finally, the comparison with the genomes of other leuconostocs highlights the distribution of functionally interesting genes across the L. gelidum strains and the genus Leuconostoc.Peer reviewe

Complete genome sequence of Leuconostoc gelidum subsp. gasicomitatum KG16-1, isolated from vacuum-packaged vegetable sausages

Leuconostoc gelidum subsp. gasicomitatum is a predominant lactic acid bacterium (LAB) in spoilage microbial communities of different kinds of modified-atmosphere packaged (MAP) food products. So far, only one genome sequence of a poultry-originating type strain of this bacterium (LMG 18811T) has been available. In the current study, we present the completely sequenced and functionally annotated genome of strain KG16-1 isolated from a vegetable-based product. In addition, six other vegetable-associated strains were sequenced to study possible “niche” specificity suggested by recent multilocus sequence typing. The genome of strain KG16-1 consisted of one circular chromosome and three plasmids, which together contained 2,035 CDSs. The chromosome carried at least three prophage regions and one of the plasmids encoded a galactan degradation cluster, which might provide a survival advantage in plant-related environments. The genome comparison with LMG 18811T and six other vegetable strains suggests no major differences between the meat- and vegetable-associated strains that would explain their “niche” specificity. Finally, the comparison with the genomes of other leuconostocs highlights the distribution of functionally interesting genes across the L. gelidum strains and the genus Leuconostoc.Peer reviewe

Complete genome sequence of Leuconostoc gelidum subsp. gasicomitatum KG16-1, isolated from vacuum-packaged vegetable sausages

Leuconostoc gelidum subsp. gasicomitatum is a predominant lactic acid bacterium (LAB) in spoilage microbial communities of different kinds of modified-atmosphere packaged (MAP) food products. So far, only one genome sequence of a poultry-originating type strain of this bacterium (LMG 18811T) has been available. In the current study, we present the completely sequenced and functionally annotated genome of strain KG16-1 isolated from a vegetable-based product. In addition, six other vegetable-associated strains were sequenced to study possible “niche” specificity suggested by recent multilocus sequence typing. The genome of strain KG16-1 consisted of one circular chromosome and three plasmids, which together contained 2,035 CDSs. The chromosome carried at least three prophage regions and one of the plasmids encoded a galactan degradation cluster, which might provide a survival advantage in plant-related environments. The genome comparison with LMG 18811T and six other vegetable strains suggests no major differences between the meat- and vegetable-associated strains that would explain their “niche” specificity. Finally, the comparison with the genomes of other leuconostocs highlights the distribution of functionally interesting genes across the L. gelidum strains and the genus Leuconostoc.Peer reviewe

Complete genome sequence of Leuconostoc gelidum subsp. gasicomitatum KG16-1, isolated from vacuum-packaged vegetable sausages

Abstract Leuconostoc gelidum subsp. gasicomitatum is a predominant lactic acid bacterium (LAB) in spoilage microbial communities of different kinds of modified-atmosphere packaged (MAP) food products. So far, only one genome sequence of a poultry-originating type strain of this bacterium (LMG 18811T) has been available. In the current study, we present the completely sequenced and functionally annotated genome of strain KG16-1 isolated from a vegetable-based product. In addition, six other vegetable-associated strains were sequenced to study possible “niche” specificity suggested by recent multilocus sequence typing. The genome of strain KG16-1 consisted of one circular chromosome and three plasmids, which together contained 2,035 CDSs. The chromosome carried at least three prophage regions and one of the plasmids encoded a galactan degradation cluster, which might provide a survival advantage in plant-related environments. The genome comparison with LMG 18811T and six other vegetable strains suggests no major differences between the meat- and vegetable-associated strains that would explain their “niche” specificity. Finally, the comparison with the genomes of other leuconostocs highlights the distribution of functionally interesting genes across the L. gelidum strains and the genus Leuconostoc

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Genebank identifiers of the nucleotide sequences of atpA, pheS and rpoA genes, used for the phylogenetic analyses. Table S2. NCBI Refseq/Genbank assembly accession numbers for the comparative genomic analysis of Leuconostoc species (29.09.2015). Table S3. L. gelidum subsp. gasicomitatum draft genomes statistics. Figure S1. Phylogenetic tree showing the relationship of L. citreum 1300_LCIT, L. gelidum subsp. gasicomitatum 1301_LGAS and L. inhae LMG 22919 genomes to other Leuconostoc species. (PDF 406 kb

Metagenomic and metatranscriptomic analysis of the microbial community in Swiss-type Maasdam cheese during ripening

In Swiss-type cheeses, characteristic nut-like and sweet flavor develops during the cheese ripening due to the metabolic activities of cheese microbiota. Temperature changes during warm and cold room ripening, and duration of ripening can significantly change the gene expression of the cheese microbiota, which can affect the flavor formation. In this study, a metagenomic and metatranscriptomic analysis of Swiss-type Maasdam cheese was performed on samples obtained during ripening in the warm and cold rooms. We reconstructed four different bacterial genomes (Lactococcus lactis, Lactobacillus rhamnosus, Lactobacillus helveticus, and Propionibacterium freudenreichii subsp. shermanii strain JS) from the Maasdam cheese to near completeness. Based on the DNA and RNA mean coverage, Lc. lactis strongly dominated (similar to 80-90%) within the cheese microbial community. Genome annotation showed the potential for the presence of several flavor forming pathways in these species, such as production of methanethiol, free fatty acids, acetoin, diacetyl, acetate, ethanol, and propionate. Using the metatranscriptomic data, we showed that, with the exception of Lc. lactis, the central metabolism of the microbiota was downregulated during cold room ripening suggesting that fewer flavor compounds such as acetoin and propionate were produced. In contrast, Lc. lactis genes related to the central metabolism, including the vitamin biosynthesis and homolactic fermentation, were upregulated during cold room ripening.Peer reviewe

Analysis of temporal gene regulation of Listeria monocytogenes revealed distinct regulatory response modes after exposure to high pressure processing

Background The pathogen Listeria (L.) monocytogenes is known to survive heat, cold, high pressure, and other extreme conditions. Although the response of this pathogen to pH, osmotic, temperature, and oxidative stress has been studied extensively, its reaction to the stress produced by high pressure processing HPP (which is a preservation method in the food industry), and the activated gene regulatory network (GRN) in response to this stress is still largely unknown. Results We used RNA sequencing transcriptome data of L. monocytogenes (ScottA) treated at 400 MPa and 8(circle)C, for 8 min and combined it with current information in the literature to create a transcriptional regulation database, depicting the relationship between transcription factors (TFs) and their target genes (TGs) in L. monocytogenes. We then applied network component analysis (NCA), a matrix decomposition method, to reconstruct the activities of the TFs over time. According to our findings, L. monocytogenes responded to the stress applied during HPP by three statistically different gene regulation modes: survival mode during the first 10 min post-treatment, repair mode during 1 h post-treatment, and re-growth mode beyond 6 h after HPP. We identified the TFs and their TGs that were responsible for each of the modes. We developed a plausible model that could explain the regulatory mechanism that L. monocytogenes activated through the well-studied CIRCE operon via the regulator HrcA during the survival mode. Conclusions Our findings suggest that the timely activation of TFs associated with an immediate stress response, followed by the expression of genes for repair purposes, and then re-growth and metabolism, could be a strategy of L. monocytogenes to survive and recover extreme HPP conditions. We believe that our results give a better understanding of L. monocytogenes behavior after exposure to high pressure that may lead to the design of a specific knock-out process to target the genes or mechanisms. The results can help the food industry select appropriate HPP conditions to prevent L. monocytogenes recovery during food storage.Peer reviewe

Lactobacillus oligofermentans glucose, ribose and xylose transcriptomes show higher similarity between glucose and xylose catabolism-induced responses in the early exponential growth phase

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High-pressure processing-induced transcriptome response during recovery of Listeria monocytogenes

Background High-pressure processing (HPP) is a commonly used technique in the food industry to inactivate pathogens, including L. monocytogenes. It has been shown that L. monocytogenes is able to recover from HPP injuries and can start to grow again during long-term cold storage. To date, the gene expression profiling of L. monocytogenes during HPP damage recovery at cooling temperature has not been studied. In order identify key genes that play a role in recovery of the damage caused by HPP treatment, we performed RNA-sequencing (RNA-seq) for two L. monocytogenes strains (barotolerant RO15 and barosensitive ScottA) at nine selected time points (up to 48 h) after treatment with two pressure levels (200 and 400 MPa). Results The results showed that a general stress response was activated by SigB after HPP treatment. In addition, the phosphotransferase system (PTS; mostly fructose-, mannose-, galactitol-, cellobiose-, and ascorbate-specific PTS systems), protein folding, and cobalamin biosynthesis were the most upregulated genes during HPP damage recovery. We observed that cell-division-related genes (divIC, dicIVA, ftsE, and ftsX) were downregulated. By contrast, peptidoglycan-synthesis genes (murG, murC, and pbp2A) were upregulated. This indicates that cell-wall repair occurs as a part of HPP damage recovery. We also observed that prophage genes, including anti-CRISPR genes, were induced by HPP. Interestingly, a large amount of RNA-seq data (up to 85%) was mapped to Rli47, which is a non-coding RNA that is upregulated after HPP. Thus, we predicted that Rli47 plays a role in HPP damage recovery in L. monocytogenes. Moreover, gene-deletion experiments showed that amongst peptidoglycan biosynthesis genes, pbp2A mutants are more sensitive to HPP. Conclusions We identified several genes and mechanisms that may play a role in recovery from HPP damage of L. monocytogenes. Our study contributes to new information on pathogen inactivation by HPP.Peer reviewe