From meadows to milk to mucosa – adaptation of Streptococcus and Lactococcus species to their nutritional environments

Lactic acid bacteria (LAB) are indigenous to food-related habitats as well as associated with the mucosal surfaces of animals. The LAB family Streptococcaceae consists of the genera Lactococcus and Streptococcus. Members of the family include the industrially important species Lactococcus lactis, which has a long history safe use in the fermentative food industry, and the disease-causing streptococci Streptococcus pneumoniae and Streptococcus pyogenes. The central metabolic pathways of the Streptococcaceae family have been extensively studied because of their relevance in the industrial use of some species, as well as their influence on virulence of others. Recent developments in high-throughput proteomic and DNA-microarray techniques, in in vivo NMR studies, and importantly in whole-genome sequencing have resulted in new insights into the metabolism of the Streptococcaceae family. The development of cost-effective high-throughput sequencing has resulted in the publication of numerous whole-genome sequences of lactococcal and streptococcal species. Comparative genomic analysis of these closely related but environmentally diverse species provides insight into the evolution of this family of LAB and shows that the relatively small genomes of members of the Streptococcaceae family have been largely shaped by the nutritionally rich environments they inhabit.

From meadows to milk to mucosa – adaptation of Streptococcus and Lactococcus species to their nutritional environments

Lactic acid bacteria (LAB) are indigenous to food-related habitats as well as associated with the mucosal surfaces of animals. The LAB family Streptococcaceae consists of the genera Lactococcus and Streptococcus. Members of the family include the industrially important species Lactococcus lactis, which has a long history safe use in the fermentative food industry, and the disease-causing streptococci Streptococcus pneumoniae and Streptococcus pyogenes. The central metabolic pathways of the Streptococcaceae family have been extensively studied because of their relevance in the industrial use of some species, as well as their influence on virulence of others. Recent developments in high-throughput proteomic and DNA-microarray techniques, in in vivo NMR studies, and importantly in whole-genome sequencing have resulted in new insights into the metabolism of the Streptococcaceae family. The development of cost-effective high-throughput sequencing has resulted in the publication of numerous whole-genome sequences of lactococcal and streptococcal species. Comparative genomic analysis of these closely related but environmentally diverse species provides insight into the evolution of this family of LAB and shows that the relatively small genomes of members of the Streptococcaceae family have been largely shaped by the nutritionally rich environments they inhabit.

Branched chain aldehydes: production and breakdown pathways and relevance for flavour in foods

Branched aldehydes, such as 2-methyl propanal and 2- and 3-methyl butanal, are important flavour compounds in many food products, both fermented and non-fermented (heat-treated) products. The production and degradation of these aldehydes from amino acids is described and reviewed extensively in literature. This paper reviews aspects influencing the formation of these aldehydes at the level of metabolic conversions, microbial and food composition. Special emphasis was on 3-methyl butanal and its presence in various food products. Knowledge gained about the generation pathways of these flavour compounds is essential for being able to control the formation of desired levels of these aldehydes

ClaR—a novel key regulator of cellobiose and lactose metabolism in Lactococcus lactis IL1403

In a number of previous studies, our group has discovered an alternative pathway for lactose utilization in Lactococcus lactis that, in addition to a sugar-hydrolyzing enzyme with both P-β-glucosidase and P-β-galactosidase activity (BglS), engages chromosomally encoded components of cellobiose-specific PTS (PTSCel-Lac), including PtcA, PtcB, and CelB. In this report, we show that this system undergoes regulation via ClaR, a novel activator protein from the RpiR family of transcriptional regulators. Although RpiR proteins are widely distributed among lactic acid bacteria, their roles have yet to be confirmed by functional assays. Here, we show that ClaR activity depends on intracellular cellobiose-6-phosphate availability, while other sugars such as glucose or galactose have no influence on it. We also show that ClaR is crucial for activation of the bglS and celB expression in the presence of cellobiose, with some limited effects on ptcA and ptcB activation. Among 190 of carbon sources tested, the deletion of claR reduces L. lactis growth only in lactose- and/or cellobiose-containing media, suggesting a narrow specificity of this regulator within the context of sugar metabolism

Overview on sugar metabolism and its control in Lactococcus lactis - The input from in vivo NMR

The wide application of lactic acid bacteria in the production of fermented foods depends to a great extent on the unique features of sugar metabolism in these organisms. The relative metabolic simplicity and the availability of genetic tools made Lactococcus lactis the organism of choice to gain insight into metabolic and regulatory networks. In vivo nuclear magnetic resonance has proven a very useful technique to monitor non-invasively the dynamics of intracellular metabolite and co-factor pools following a glucose pulse. Examples of the application of this methodology to identify metabolic bottlenecks and regulatory sites are presented. The use of this information to direct metabolic engineering strategies is illustrated. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved

Investigation of the adaptation of Lactococcus lactis to isoleucine starvation integrating dynamic transcriptome and proteome information

Background: Amino acid assimilation is crucial for bacteria and this is particularly true for Lactic Acid Bacteria (LAB) that are generally auxotroph for amino acids. The global response of the Lmodel Lactococcus lactis ssp. lactis was characterized during progressive isoleucine starvation in batch culture using a chemically defined medium in which isoleucine concentration was fixed so as to become the sole limiting nutriment. Dynamic analyses were performed using transcriptomic and proteomic approaches and the results were analysed conjointly with fermentation kinetic data. Results: The response was first deduced from transcriptomic analysis and corroborated by proteomic results. It occurred progressively and could be divided into three major mechanisms: (i) a global down-regulation of processes linked to bacterial growth and catabolism (transcription, translation, carbon metabolism and transport, pyrimidine and fatty acid metabolism), (ii) a specific positive response related to the limiting nutrient (activation of pathways of carbon or nitrogen metabolism and leading to isoleucine supply) and (iii) an unexpected oxidative stress response (positive regulation of aerobic metabolism, electron transport, thioredoxin metabolism and pyruvate dehydrogenase). The involvement of various regulatory mechanisms during this adaptation was analysed on the basis of transcriptomic data comparisons. The global regulator CodY seemed specifically dedicated to the regulation of isoleucine supply. Other regulations were massively related to growth rate and stringent response. Conclusion: This integrative biology approach provided an overview of the metabolic pathways involved during isoleucine starvation and their regulations. It has extended significantly the physiological understanding of the metabolism of L. lactis ssp. lactis. The approach can be generalised to other conditions and will contribute significantly to the identification of the biological processes involved in complex regulatory networks of micro-organisms

Respiratory Physiology of Lactococcus lactis in Chemostat Cultures and Its Effect on Cellular Robustness in Frozen and Freeze-Dried Starter Cultures

In this study, we used chemostat cultures to analyze the quantitative effects of the specific growth rate and respiration on the metabolism in Lactococcus lactis CHCC2862 and on the downstream robustness of cells after freezing or freeze-drying. Under anaerobic conditions, metabolism remained homofermentative, although biomass yields varied with the dilution rate (D). In contrast, metabolism shifted with the dilution rate under respiration-permissive conditions. At D = 0.1 h-1, no lactate was produced, while lactate formation increased with higher dilution rates. Thus, a clear metabolic shift was observed, from flavor-forming respiratory metabolism at low specific growth rates to mixed-acid respiro-fermentative metabolism at higher specific growth rates. Quantitative analysis of the respiratory activity, lactose uptake rate, and metabolite production rates showed that aerobic acetoin formation provided most of the NADH consumed in respiration. Moreover, the maintenance-associated lactose consumption under respiration-permissive conditions was only 10% of the anaerobic value, either due to higher respiratory yield of ATP on consumed lactose or due to lower maintenance-related ATP demand. The cultivation conditions also affected the quality of the starter cultures produced. Cells harvested under respiration-permissive conditions at D = 0.1 h-1 were less robust after freeze-drying and had lower acidification activity for subsequent milk acidification, whereas respiration-permissive conditions at the higher dilution rates led to robust cells that performed equally well or better than anaerobic cells.IMPORTANCELactococcus lactis is used in large quantities by the food and biotechnology industries. L. lactis can use oxygen for respiration if heme is supplied in the growth medium. This has been extensively studied in batch cultures using various mutants, but quantitative studies of how the cell growth affects respiratory metabolism, energetics, and cell quality are surprisingly scarce. Our results demonstrate that the respiratory metabolism of L. lactis is remarkably flexible and can be modulated by controlling the specific growth rate. We also link the physiological state of cells during cultivation to the quality of frozen or freeze-dried cells, which is relevant to the industry that may lack understanding of such relationships. This study extends our knowledge of respiratory metabolism in L. lactis and its impact on frozen and freeze-dried starter culture products, and it illustrates the influence of cultivation conditions and microbial physiology on the quality of starter cultures

Modeling Lactococcus lactis using a genome-scale flux model

BACKGROUND: Genome-scale flux models are useful tools to represent and analyze microbial metabolism. In this work we reconstructed the metabolic network of the lactic acid bacteria Lactococcus lactis and developed a genome-scale flux model able to simulate and analyze network capabilities and whole-cell function under aerobic and anaerobic continuous cultures. Flux balance analysis (FBA) and minimization of metabolic adjustment (MOMA) were used as modeling frameworks. RESULTS: The metabolic network was reconstructed using the annotated genome sequence from L. lactis ssp. lactis IL1403 together with physiological and biochemical information. The established network comprised a total of 621 reactions and 509 metabolites, representing the overall metabolism of L. lactis. Experimental data reported in the literature was used to fit the model to phenotypic observations. Regulatory constraints had to be included to simulate certain metabolic features, such as the shift from homo to heterolactic fermentation. A minimal medium for in silico growth was identified, indicating the requirement of four amino acids in addition to a sugar. Remarkably, de novo biosynthesis of four other amino acids was observed even when all amino acids were supplied, which is in good agreement with experimental observations. Additionally, enhanced metabolic engineering strategies for improved diacetyl producing strains were designed. CONCLUSION: The L. lactis metabolic network can now be used for a better understanding of lactococcal metabolic capabilities and potential, for the design of enhanced metabolic engineering strategies and for integration with other types of 'omic' data, to assist in finding new information on cellular organization and function

Regulering av energimetabolisme i enterococcus faecalis studert med transkriptom-, proteom- og metabolomanalyser

Lactic Acid Bacteria (LAB) are widely used as starter culture in food fermentation. Among LAB also pathogenic bacteria are found particular in enterococci and streptococci. Enterococcus faecalis is a gut commensal bacterium but certain isolates have been shown to be pathogenic while others are foodgrade bacteria in LAB fermented food commodities. E. faecalis ferments sugars through different pathways, resulting in homo- or mixed acid fermentation. In homolactic bacteria glucose is converted to lactate in an ATP producing reaction. In mixed acid fermentation, in addition to lactate production, glucose is also converted to acetate, acetoin, formate, ethanol and CO2. However, there is limited information regarding to regulation of the central energy metabolism of E. faecalis. The aim of this work was to extend our knowledge with respect to the central energy metabolism of E. faecalis by employing metabolite, transcriptome and proteome approaches. High performance liquid chromatography and gas chromatography were used for metabolite measurements. DNA microarray technology and two dimensional gel electrophoresis combined with mass spectrometry analysis were used in transcription and protein expression analysis, respectively. Combining these approaches has not been performed in metabolic analysis in E. faecalis and this should give an in-depth understanding about regulation of the central energy metabolism in E. faecalis. This work showed that in absence of ldh (lactate dehydrogenase) gene, E. faecalis metabolizes glucose to ethanol, formate and acetoin. The change from homolactic to mixed acid fermentation affected expression of several genes and proteins mostly involved in energy metabolism. These genes play an important role in the regulatory network controlling energy metabolism in E. faecalis including acetoin production, and NAD+/NADH ratio. Additional studies were carried out in order to investigate the mixed acid fermentation of wild-type E. faecalis in chemostat during steady state and glucose limiting growth. Growth at three different growth rates demonstrated that the bacterium responded differently depending on the growth rate. At the highest dilution rate (D=0.4 h-1) most of the glucose was converted to lactate while at the lowest dilution rate (D=0.05 h-1) it changed towards mixed acids fermentation. Interestingly, increased growth rate induced the transcription of the ldh gene while the amount of Ldh protein was more or less unaffected. The differences in glucose energy metabolism at different growth and pHs between E. faecalis and two other LAB (Streptococcus pyogenes and Lactococcus lactis) and their LDH negative mutants were also investigated. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. The final study explored the effect of ascorbate on growth in the absence of glucose and showed that E. faecalis can grow on ascorbate. In summary, the work presented in this thesis gave new insights in regulation and strengthens our knowledge regarding the metabolic pathways of glucose fermentation through the metabolite analysis, regulation of transcription and protein expression.Melkesyrebakterier brukes som startkulturer i en rekke ulike gjæringsreaksjoner i forbindelse med produksjon av mat. Enkelte melkesyrebakterier har også evnen til å forårsake sykdom, og dette gjelder spesielt for enterokokker og streptokokker. Enterococcus faecalis er en kommensal tarmbakterie. Likevel finner man innenfor denne arten både patogene isolater såvel som stammer benyttet i fermentering av matvarer. E. faecalis bryter ned sukker gjennom flere ulike veier, med enten melkesyre (homolaktisk gjæring) eller en blanding av syrer (blandet syregjæring) som endeprodukt. Homolaktiske bakterier bryter ned glukose til melkesyre i en reaksjonskjede som produserer ATP. Ved blandet syregjæring av glukose produseres det i tillegg til melkesyre også eddiksyre, acetoin, maursyre, etanol og CO2. Det er imidlertid lite informasjon om reguleringen av energimetabolismen i E. faecalis tilgjengenlig. Målet med arbeidet bak denne avhandlingen har derfor vært å tilegne oss kunnskap om den sentrale energimetabolismen i E. faecalis ved hjelp av ulike metoder for å studere metabolitter, transkriptomet og proteomet. Væskekromatografi og gasskromatografi ble brukt til metabolittmålinger, mens DNA mikromatriseteknologi og to-dimensjonal gelelektroforese kombinert med massespektroskopi ble brukt til henholdsvis transkripsjon- og proteinanalyser. Kombinasjonen av disse metodene har ikke tidligere blitt brukt i metabolske studier av E. faecalis, og vil derfor forhåpentligvis gi en dypere forståelse av overgangen mellom homolaktisk- og blandet syregjæring. Våre studier viser at i fravær av ldh genet, som koder for laktatdehydrogenase, blir glukose brutt med til etanol, maursyre og acetoin. Denne overgangen fra homolaktisk til blandet syregjøring påvirker uttrykket av en rekke gener og proteiner involvert i energimetabolismen. Genene innehar viktige roller i det regulatoriske nettverket som kontrollerer energimetabolismen i E. faecalis, og inkluderer gener involvert i produksjon av acetoin og balansen mellom NAD+/NADH. Videre studier ble også gjort for å undersøke blandet syregjæring i villtype E. faecalis i kjemostat ved likevektstilstand og glukosebegrenset vekst. Vekst ved tre forskjellige veksthastigheter viste av bakterien responderer forskjellig avhengig av veksthastighet. Ved den høyeste fortynningshastigheten (D=0.4 h-1) ble det meste av glukosen omdannet til melkesyre, mens en endring i retning av blandet syrefermentering ble observert ved den laveste fortynningshastigheten (D=0.05 h-1). Interessant nok så førte økt veksthastighet til økt transkripsjon av ldh-genet, men mengden Ldh-protein var tilnærmet uendret. Forskjellene i nedbrytning av glukose ved forskjellige veksthastigheter og ved forskjellig pH mellom E. faecalis og to andre melkesyrebakterier (Streptococcus pyogenes and Lactococcus lactis) ble også undersøkt. Det er her verdt å merke seg at inaktivering av ldh genene hadde liten innvirkning på veksthastigheten til de ulike bakteriene i kjemisk definert medium under mikroaerofile vekstforhold. Inaktiveringen av ldh påvirket også bakterienes evne til å utnytte andre substrater enn glukose som karbonkilde. I det siste arbeidet i avhandlingen ble det vist at E. faecalis i fravær av glukose er istand til å vokse på askorbinsyre. Sett under ett har arbeidet som er presentert i denne avhandlingen, gjennom analyser av metabolitter, transkripsjonregulering og proteinuttrykk, gitt økt innsikt i reguleringen av og styrket vår kjennskap til veiene for nedbrytning av glukose.Norges Forskningrå