Halomonas citrativorans sp. nov., Halomonas casei sp. nov. and Halomonas colorata sp. nov., isolated from French cheese rinds.

Eight Gram-stain-negative bacterial strains were isolated from cheese rinds sampled in France. On the basis of 16S rRNA gene sequence analysis, all isolates were assigned to the genus Halomonas. Phylogenetic investigations, including 16S rRNA gene studies, multilocus sequence analysis, reconstruction of a pan-genome phylogenetic tree with the concatenated core-genome content and average nucleotide identity (ANI) calculations, revealed that they constituted three novel and well-supported clusters. The closest relative species, determined using the whole-genome sequences of the strains, were Halomonas zhanjiangensis for two groups of cheese strains, sharing 82.4 and 93.1 % ANI, and another cluster sharing 92.2 % ANI with the Halomonas profundi type strain. The strains isolated herein differed from the previously described species by ANI values <95 % and several biochemical, enzymatic and colony characteristics. The results of phenotypic, phylogenetic and chemotaxonomic analyses indicated that the isolates belonged to three novel Halomonas species, for which the names Halomonas citrativorans sp. nov., Halomonas casei sp. nov. and Halomonas colorata sp. nov. are proposed, with isolates FME63T (=DSM 113315T=CIRM-BIA2430T=CIP 111880T=LMG 32013T), FME64T (=DSM 113316T=CIRM-BIA2431T=CIP 111877T=LMG 32015T) and FME66T (=DSM 113318T=CIRM-BIA2433T=CIP 111876T=LMG 32014T) as type strains, respectively.

Molecular approaches to uncover phage-lactic acid bacteria interactions in a model community simulating fermented beverages.

Food microbial diversity and fluxes during the fermentation processes are well studied whereas phages-bacteria interactions are still poorly described in the literature. This is especially true in fermented beverages, and especially in cider, which is an alcoholic fermented apple beverage. The transcriptomic and proteomic responses of the lactic acid bacterium (LAB) Liquorilactobacillus mali UCMA 16447 to a lytic infection by phage UCMA 21115, both isolated from cider, were investigated, in order to get a better understanding of phages-bacteria interactions in such fermented beverage. During phage infection, 122 and 215 genes were differentially expressed in L. mali UCMA 16447 strain at T15 and T60 respectively, when compared to the uninfected condition. The same trends were confirmed by the proteomic study, with a total of 28 differentially expressed proteins found at T60. Overall, genes encoding cellular functions, such as carbohydrate metabolism, translation, and signal transduction, were downregulated, while genes involved in nucleotide metabolism and in the control of DNA integrity were upregulated in response to phage infection. This work also highlighted that phage infection repressed many genes involved in bacterial cell motility, and affected glycolysis.

Metatranscriptomics of cheese microbial communities: Efficiency of RNA extraction from various cheese types and of mRNA enrichment.

Microbial communities from cheeses contribute to the development of typical organoleptic properties. Metatranscriptomic analyses can be used to provide a global picture of the functioning of these communities. Our objective was to evaluate the efficiency of RNA extraction from various cheese types and to evaluate mRNA enrichment procedures for metatranscriptomic analyses. For the 32 tested cheese brands, corresponding to five cheese types, the extraction yield varied from 1 µg to 363 µg RNA per gram of cheese and, overall, the yield was lower for fresh cheeses and for the core of pressed cooked cheeses than for the other cheese types. Pressed cooked cheeses also had a lower RNA integrity than the other cheese types. For total RNA extracts from four cheeses, approximately 99% of the sequencing reads corresponded to ribosomal RNA, and mRNA enrichment by RiboPOOL and FastSelect kits decreased this percentage to a range of 75 to 97% and of 53 to 76%, respectively. Comparison of RNA libraries after mRNA enrichment with libraries of undepleted total RNA showed that the FastSelect mRNA enrichment had a lower impact on the gene expression profiles of five target cheese species than the riboPOOL kit and the oligo (dT) selection method. The procedures that we describe in the present study may be useful for metatranscriptomic analysis of various cheese types.

Carotenoids from the ripening bacterium Brevibacterium linens impart color to the rind of the French cheese, Fourme de Montbrison (PDO).

Rind color of some high-value PDO cheeses is related to the presence of carotenoids, but little is known about the structure of the pigmented compounds and their origin. Our objective was to describe the carotenoids extracted from the rind of a French cheese, Fourme de Montbrison, and to compare them with the pigments produced by a bacterial strain used as an adjunct culture in the cheese ripening process. Eleven carotenoids were detected in the cheese rinds or in the biomass of Brevibacterium linens. Most of the carotenoids from the rinds belonged to the aryl (aromatic) carotenoid family, including hydroxylated and non-hydroxylated isorenieratene. Chlorobactene, a carotenoid rarely found in food products, was also detected. Agelaxanthin A was identified in the cheese rinds as well as in the B. linens biomass. Occurrence of this compound was previously described in only one scientific publication, where it was isolated from the sponge Agela schmidtii.

Transcription Profiling Reveals Cooperative Metabolic Interactions in a Microbial Cheese-Ripening Community Composed of Debaryomyces hansenii, Brevibacterium aurantiacum, and Hafnia alvei.

Ripening cultures containing fungi and bacteria are widely used in smear-ripened cheese production processes, but little is known about the biotic interactions of typical ripening microorganisms at the surface of cheese. We developed a lab-scale mini-cheese model to investigate the biotic interactions of a synthetic community that was composed of Debaryomyces hansenii, Brevibacterium aurantiacum, and Hafnia alvei, three species that are commonly used for smear-ripened cheese production. Transcriptomic analyses of cheese samples produced with different combinations of these three species revealed potential mechanisms of biotic interactions concerning iron acquisition, proteolysis, lipolysis, sulfur metabolism, and D-galactonate catabolism. A strong mutualistic interaction was observed between H. alvei and B. aurantiacum. We propose an explanation of this positive interaction in which B. aurantiacum would benefit from siderophore production by H. alvei, and the latter would be stimulated by the energy compounds liberated from caseins and triglycerides through the action of the proteases and lipases secreted by B. aurantiacum. In the future, it would be interesting to take the iron acquisition systems of cheese-associated strains into account for the purpose of improving the selection of the ripening culture components and their association in mixed cultures.

Comparative genomic analysis of Brevibacterium strains: insights into key genetic determinants involved in adaptation to the cheese habitat.

BACKGROUND: Brevibacterium strains are widely used for the manufacturing of surface-ripened cheeses, contributing to the breakdown of lipids and proteins and producing volatile sulfur compounds and red-orange pigments. The objective of the present study was to perform comparative genomic analyses in order to better understand the mechanisms involved in their ability to grow on the cheese surface and the differences between the strains. RESULTS: The genomes of 23 Brevibacterium strains, including twelve strains isolated from cheeses, were compared for their gene repertoire involved in salt tolerance, iron acquisition, bacteriocin production and the ability to use the energy compounds present in cheeses. All or almost all the genomes encode the enzymes involved in ethanol, acetate, lactate, 4-aminobutyrate and glycerol catabolism, and in the synthesis of the osmoprotectants ectoine, glycine-betaine and trehalose. Most of the genomes contain two contiguous genes encoding extracellular proteases, one of which was previously characterized for its activity on caseins. Genes encoding a secreted triacylglycerol lipase or involved in the catabolism of galactose and D-galactonate or in the synthesis of a hydroxamate-type siderophore are present in part of the genomes. Numerous Fe3+/siderophore ABC transport components are present, part of them resulting from horizontal gene transfers. Two cheese-associated strains have also acquired catecholate-type siderophore biosynthesis gene clusters by horizontal gene transfer. Predicted bacteriocin biosynthesis genes are present in most of the strains, and one of the corresponding gene clusters is located in a probable conjugative transposon that was only found in cheese-associated strains. CONCLUSIONS: Brevibacterium strains show differences in their gene repertoire potentially involved in the ability to grow on the cheese surface. Part of these differences can be explained by different phylogenetic positions or by horizontal gene transfer events. Some of the distinguishing features concern biotic interactions with other strains such as the secretion of proteases and triacylglycerol lipases, and competition for iron or bacteriocin production. In the future, it would be interesting to take the properties deduced from genomic analyses into account in order to improve the screening and selection of Brevibacterium strains, and their association with other ripening culture components.

Investigation of the Activity of the Microorganisms in a Reblochon-Style Cheese by Metatranscriptomic Analysis.

The microbial communities in cheeses are composed of varying bacteria, yeasts, and molds, which contribute to the development of their typical sensory properties. In situ studies are needed to better understand their growth and activity during cheese ripening. Our objective was to investigate the activity of the microorganisms used for manufacturing a surface-ripened cheese by means of metatranscriptomic analysis. The cheeses were produced using two lactic acid bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), one ripening bacterium (Brevibacterium aurantiacum), and two yeasts (Debaryomyces hansenii and Geotrichum candidum). RNA was extracted from the cheese rinds and, after depletion of most ribosomal RNA, sequencing was performed using a short-read sequencing technology that generated ~75 million reads per sample. Except for B. aurantiacum, which failed to grow in the cheeses, a large number of CDS reads were generated for the inoculated species, making it possible to investigate their individual transcriptome over time. From day 5 to 35, G. candidum accounted for the largest proportion of CDS reads, suggesting that this species was the most active. Only minor changes occurred in the transcriptomes of the lactic acid bacteria. For the two yeasts, we compared the expression of genes involved in the catabolism of lactose, galactose, lactate, amino acids, and free fatty acids. During ripening, genes involved in ammonia assimilation and galactose catabolism were down-regulated in the two species. Genes involved in amino acid catabolism were up-regulated in G. candidum from day 14 to day 35, whereas in D. hansenii, they were up-regulated mainly at day 35, suggesting that this species catabolized the cheese amino acids later. In addition, after 35 days of ripening, there was a down-regulation of genes involved in the electron transport chain, suggesting a lower cellular activity. The present study has exemplified how metatranscriptomic analyses provide insight into the activity of cheese microbial communities for which reference genome sequences are available. In the future, such studies will be facilitated by the progress in DNA sequencing technologies and by the greater availability of the genome sequences of cheese microorganisms.

Overview of a surface-ripened cheese community functioning by meta-omics analyses.

Cheese ripening is a complex biochemical process driven by microbial communities composed of both eukaryotes and prokaryotes. Surface-ripened cheeses are widely consumed all over the world and are appreciated for their characteristic flavor. Microbial community composition has been studied for a long time on surface-ripened cheeses, but only limited knowledge has been acquired about its in situ metabolic activities. We applied metagenomic, metatranscriptomic and biochemical analyses to an experimental surface-ripened cheese composed of nine microbial species during four weeks of ripening. By combining all of the data, we were able to obtain an overview of the cheese maturation process and to better understand the metabolic activities of the different community members and their possible interactions. Furthermore, differential expression analysis was used to select a set of biomarker genes, providing a valuable tool that can be used to monitor the cheese-making process.

Growth and adaptation of microorganisms on the cheese surface.

Microbial communities living on cheese surfaces are composed of various bacteria, yeasts and molds that interact together, thus generating the typical sensory properties of a cheese. Physiological and genomic investigations have revealed important functions involved in the ability of microorganisms to establish themselves at the cheese surface. These functions include the ability to use the cheese's main energy sources, to acquire iron, to tolerate low pH at the beginning of ripening and to adapt to high salt concentrations and moisture levels. Horizontal gene transfer events involved in the adaptation to the cheese habitat have been described, both for bacteria and fungi. In the future, in situ microbial gene expression profiling and identification of genes that contribute to strain fitness by massive sequencing of transposon libraries will help us to better understand how cheese surface communities function.

Investigation of Geotrichum candidum gene expression during the ripening of Reblochon-type cheese by reverse transcription-quantitative PCR.

Cheese ripening involves the activity of various bacteria, yeasts or molds, which contribute to the development of the typical color, flavor and texture of the final product. In situ measurements of gene expression are increasingly being used to improve our understanding of the microbial flora activity in cheeses. The objective of the present study was to investigate the physiology and metabolic activity of Geotrichum candidum during the ripening of Reblochon-type cheeses by quantifying mRNA transcripts at various ripening times. The expression of 80 genes involved in various functions could be quantified with a correct level of biological repeatability using a set of three stable reference genes. As ripening progresses, a decrease in expression was observed for genes involved in cell wall organization, translation, vesicular mediated transport, and in cytoskeleton constituents and ribosomal protein genes. There was also a decrease in the expression of mitochondrial F1F0 ATP synthase and plasma membrane H(+) ATPase genes. Some genes involved in the catabolism of lactate, acetate and ethanol were expressed to a greater extent at the beginning of ripening. During the second part of ripening, there was an increased expression of genes involved in the transport and catabolism of amino acids, which could be attributed to a change in the energy source. There was also an increase in the expression of genes involved in autophagy and of genes possibly involved in lifespan determination. Quantification of mRNA transcripts may also be used to produce bioindicators relevant for cheesemaking, for example when considering genes encoding enzymes involved in the catabolism of amino acids.

Construction of a dairy microbial genome catalog opens new perspectives for the metagenomic analysis of dairy fermented products.

BACKGROUND: Microbial communities of traditional cheeses are complex and insufficiently characterized. The origin, safety and functional role in cheese making of these microbial communities are still not well understood. Metagenomic analysis of these communities by high throughput shotgun sequencing is a promising approach to characterize their genomic and functional profiles. Such analyses, however, critically depend on the availability of appropriate reference genome databases against which the sequencing reads can be aligned. RESULTS: We built a reference genome catalog suitable for short read metagenomic analysis using a low-cost sequencing strategy. We selected 142 bacteria isolated from dairy products belonging to 137 different species and 67 genera, and succeeded to reconstruct the draft genome of 117 of them at a standard or high quality level, including isolates from the genera Kluyvera, Luteococcus and Marinilactibacillus, still missing from public database. To demonstrate the potential of this catalog, we analysed the microbial composition of the surface of two smear cheeses and one blue-veined cheese, and showed that a significant part of the microbiota of these traditional cheeses was composed of microorganisms newly sequenced in our study. CONCLUSIONS: Our study provides data, which combined with publicly available genome references, represents the most expansive catalog to date of cheese-associated bacteria. Using this extended dairy catalog, we revealed the presence in traditional cheese of dominant microorganisms not deliberately inoculated, mainly Gram-negative genera such as Pseudoalteromonas haloplanktis or Psychrobacter immobilis, that may contribute to the characteristics of cheese produced through traditional methods.

Dynamics of Penicillium camemberti growth quantified by real-time PCR on Camembert-type cheeses under different conditions of temperature and relative humidity.

Penicillium camemberti plays a major role in the flavor and appearance of Camembert-type cheeses. However, little is known about its mycelium growth kinetics during ripening. We monitored the growth of P. camemberti mycelium in Camembert-type cheeses using real-time PCR in 4 ripening runs, performed at 2 temperatures (8 and 16°C) and 2 relative humidities (88 and 98%). These findings were compared with P. camemberti quantification by spore concentration. During the first phase, the mycelium grew but no spores were produced, regardless of the ripening conditions. During the second phase, which began when lactose was depleted, the concentration of spores increased, especially in the cheeses ripened at 16°C. Sporulation was associated with a large decrease in the mycelial concentration in the cheeses ripened at 16°C and 98% relative humidity. It was hypothesized that lactose is the main energy source for the growth of P. camemberti mycelium at the beginning of ripening and that its depletion would trigger stress, resulting in sporulation.

Quantification of yeast and bacterial gene transcripts in retail cheeses by reverse transcription-quantitative PCR.

The cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts of Corynebacterium casei, Brevibacterium aurantiacum, and Arthrobacter arilaitensis and 26S rRNA and beta tubulin gene transcripts of Geotrichum candidum and Debaryomyces hansenii could be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed.

Genome sequence of Staphylococcus equorum subsp. equorum Mu2, isolated from a French smear-ripened cheese.

Staphylococcus equorum subsp. equorum is a member of the coagulase-negative staphylococcus group and is frequently isolated from fermented food products and from food-processing environments. It contributes to the formation of aroma compounds during the ripening of fermented foods, especially cheeses and sausages. Here, we report the draft genome sequence of Staphylococcus equorum subsp. equorum Mu2 to provide insights into its physiology and compare it with other Staphylococcus species.

Growth of aerobic ripening bacteria at the cheese surface is limited by the availability of iron.

The microflora on the surface of smear-ripened cheeses is composed of various species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. The objective of the present study was to show that iron availability is a limiting factor in the growth of typical aerobic ripening bacteria in cheese. For that purpose, we investigated the effect of iron or siderophore addition in model cheeses that were coinoculated with a yeast and a ripening bacterium. Both iron and the siderophore desferrioxamine B stimulated the growth of ripening bacteria belonging to the genera Arthrobacter, Corynebacterium, and Brevibacterium. The extent of stimulation was strain dependent, and generally, the effect of desferrioxamine B was greater than that of iron. Measurements of the expression of genes related to the metabolism of iron by Arthrobacter arilaitensis Re117 by real-time reverse transcription-PCR showed that these genes were transcribed during growth in cheese. The addition of desferrioxamine B increased the expression of two genes encoding iron-siderophore ABC transport binding proteins. The addition of iron decreased the expression of siderophore biosynthesis genes and of part of the genes encoding iron-siderophore ABC transport components. It was concluded that iron availability is a limiting factor in the growth of typical cheese surface bacteria. The selection of strains with efficient iron acquisition systems may be useful for the development of defined-strain surface cultures. Furthermore, the importance of iron metabolism in the microbial ecology of cheeses should be investigated since it may result in positive or negative microbial interactions.

Genome sequence of Corynebacterium casei UCMA 3821, isolated from a smear-ripened cheese.

Corynebacterium casei is one of the most prevalent species present on the surfaces of smear-ripened cheeses, where it contributes to the production of the desired organoleptic properties. Here, we report the draft genome sequence of Corynebacterium casei UCMA 3821 to provide insights into its physiology.

The arthrobacter arilaitensis Re117 genome sequence reveals its genetic adaptation to the surface of cheese.

Arthrobacter arilaitensis is one of the major bacterial species found at the surface of cheeses, especially in smear-ripened cheeses, where it contributes to the typical colour, flavour and texture properties of the final product. The A. arilaitensis Re117 genome is composed of a 3,859,257 bp chromosome and two plasmids of 50,407 and 8,528 bp. The chromosome shares large regions of synteny with the chromosomes of three environmental Arthrobacter strains for which genome sequences are available: A. aurescens TC1, A. chlorophenolicus A6 and Arthrobacter sp. FB24. In contrast however, 4.92% of the A. arilaitensis chromosome is composed of ISs elements, a portion that is at least 15 fold higher than for the other Arthrobacter strains. Comparative genomic analyses reveal an extensive loss of genes associated with catabolic activities, presumably as a result of adaptation to the properties of the cheese surface habitat. Like the environmental Arthrobacter strains, A. arilaitensis Re117 is well-equipped with enzymes required for the catabolism of major carbon substrates present at cheese surfaces such as fatty acids, amino acids and lactic acid. However, A. arilaitensis has several specificities which seem to be linked to its adaptation to its particular niche. These include the ability to catabolize D-galactonate, a high number of glycine betaine and related osmolyte transporters, two siderophore biosynthesis gene clusters and a high number of Fe(3+)/siderophore transport systems. In model cheese experiments, addition of small amounts of iron strongly stimulated the growth of A. arilaitensis, indicating that cheese is a highly iron-restricted medium. We suggest that there is a strong selective pressure at the surface of cheese for strains with efficient iron acquisition and salt-tolerance systems together with abilities to catabolize substrates such as lactic acid, lipids and amino acids.

Assessment of the anti-listerial activity of microfloras from the surface of smear-ripened cheeses.

The anti-listerial activity of microfloras from the surface of various smear-ripened cheeses was evaluated using four methods that were then compared. Method A measured the anti-listerial potential of supernatants from short-time liquid cultures, whereas in Method B, a model cheese was co-inoculated with the microflora and Listeria innocua test strains. Method C was based on successive propagations of the microfloras on this model cheese, and Method D on successive propagations of the microfloras together with Listeria test strains. Anti-listerial activity considerably depended on the microflora used. Significant correlations were obtained between Methods A and B and Methods C and D. With Methods C and D, the highest anti-listerial activity was obtained with the microflora from a Livarot-type cheese (FC12). To investigate the cause of the anti-listerial activity of FC12, Fourier Transform InfraRed (FTIR) analyses of microbial populations were performed on the original microflora as well as on the microflora after propagations on the model cheese. The composition of FC12 had changed considerably upon propagation, and in the propagated microflora, the population of yeasts was dominated by Yarrowia lipolytica strains, whereas the population of bacteria was dominated by Vagococcus species.

Extraction of RNA from cheese without prior separation of microbial cells.

In situ gene expression studies are promising approaches for improving our understanding of the cheese microbial flora. This requires efficient RNA extraction methods, but studies of cheeses are scarce. The objective of the present study was to determine whether RNA samples compatible with quantitative mRNA transcript analyses can be obtained without separating the cells from the cheese matrix. In the method that we describe, the cellular processes are stopped at the very beginning of the procedure. When cheeses were produced with Lactococcus lactis LD61 as the only starter microorganism, the integrity of the purified RNA was good, even for 2-week-old cheeses that had been incubated at 30 degrees C. In addition, the RNA samples did not contain any traces of RNases, and the amount of genomic DNA was negligible. A good level of reproducibility could also be achieved. When real-time reverse transcription-PCR analyses were normalized to the total RNA concentration, the amounts of 16S and 23S rRNA transcripts were constant during the 2-week incubation period, whereas the amount of tuf mRNA transcripts decreased substantially. RNA samples obtained using the method described in this study were compared to samples obtained using the method described by Ulvé et al. (J. Appl. Microbiol., in press), which is based on separation of the cells from the cheese matrix. For most of the 29 genes investigated, the transcript abundance was the same for both types of samples. Differences were observed mainly for genes whose expression has previously been shown to be modified by heat, acid, or osmotic stresses, such as busAA and glnQ.

Microbial interactions within a cheese microbial community.

The interactions that occur during the ripening of smear cheeses are not well understood. Yeast-yeast interactions and yeast-bacterium interactions were investigated within a microbial community composed of three yeasts and six bacteria found in cheese. The growth dynamics of this community was precisely described during the ripening of a model cheese, and the Lotka-Volterra model was used to evaluate species interactions. Subsequently, the effects on ecosystem functioning of yeast omissions in the microbial community were evaluated. It was found both in the Lotka-Volterra model and in the omission study that negative interactions occurred between yeasts. Yarrowia lipolytica inhibited mycelial expansion of Geotrichum candidum, whereas Y. lipolytica and G. candidum inhibited Debaryomyces hansenii cell viability during the stationary phase. However, the mechanisms involved in these interactions remain unclear. It was also shown that yeast-bacterium interactions played a significant role in the establishment of this multispecies ecosystem on the cheese surface. Yeasts were key species in bacterial development, but their influences on the bacteria differed. It appeared that the growth of Arthrobacter arilaitensis or Hafnia alvei relied less on a specific yeast function because these species dominated the bacterial flora, regardless of which yeasts were present in the ecosystem. For other bacteria, such as Leucobacter sp. or Brevibacterium aurantiacum, growth relied on a specific yeast, i.e., G. candidum. Furthermore, B. aurantiacum, Corynebacterium casei, and Staphylococcus xylosus showed reduced colonization capacities in comparison with the other bacteria in this model cheese. Bacterium-bacterium interactions could not be clearly identified.