Eddy J. Smid

Microbe-microbe interactions in mixed culture food fermentations.

Most known natural and industrial food fermentation processes are driven by either simple or complex communities of microorganisms. Obviously, these fermenting microbes will not only interact with the fermentable substrate but also with each other. These microbe-microbe interactions are complex but thought to be crucial for obtaining the desired product characteristics. Microbial interactions are mediated through a variety of molecular and physiological mechanisms. Examples of interaction mechanisms which have an impact on the outcome of food fermentation processes will be discussed. Finally, the technological and scientific challenges associated with the production and propagation of complex mixed starter cultures are briefly addressed. Research on the composition and functionality of complex microbial consortia is gaining momentum and will open new avenues for controlling and improving food fermentation processes, and developing new applications for mixed cultures.

A comprehensive metatranscriptome analysis pipeline and its validation using human small intestine microbiota datasets

BackgroundNext generation sequencing (NGS) technologies can be applied in complex microbial ecosystems for metatranscriptome analysis by employing direct cDNA sequencing, which is known as RNA sequencing (RNA-seq). RNA-seq generates large datasets of great complexity, the comprehensive interpretation of which requires a reliable bioinformatic pipeline. In this study, we focus on the development of such a metatranscriptome pipeline, which we validate using Illumina RNA-seq datasets derived from the small intestine microbiota of two individuals with an ileostomy.ResultsThe metatranscriptome pipeline developed here enabled effective removal of rRNA derived sequences, followed by confident assignment of the predicted function and taxonomic origin of the mRNA reads. Phylogenetic analysis of the small intestine metatranscriptome datasets revealed a strong similarity with the community composition profiles obtained from 16S rDNA and rRNA pyrosequencing, indicating considerable congruency between community composition (rDNA), and the taxonomic distribution of overall (rRNA) and specific (mRNA) activity among its microbial members. Reproducibility of the metatranscriptome sequencing approach was established by independent duplicate experiments. In addition, comparison of metatranscriptome analysis employing single- or paired-end sequencing methods indicated that the latter approach does not provide improved functional or phylogenetic insights. Metatranscriptome functional-mapping allowed the analysis of global, and genus specific activity of the microbiota, and illustrated the potential of these approaches to unravel syntrophic interactions in microbial ecosystems.ConclusionsA reliable pipeline for metatransciptome data analysis was developed and evaluated using RNA-seq datasets obtained for the human small intestine microbiota. The set-up of the pipeline is very generic and can be applied for (bacterial) metatranscriptome analysis in any chosen niche.

Health benefits of fermented foods: microbiota and beyond

Fermented foods and beverages were among the first processed food products consumed by humans. The production of foods such as yogurt and cultured milk, wine and beer, sauerkraut and kimchi, and fermented sausage were initially valued because of their improved shelf life, safety, and organoleptic properties. It is increasingly understood that fermented foods can also have enhanced nutritional and functional properties due to transformation of substrates and formation of bioactive or bioavailable end-products. Many fermented foods also contain living microorganisms of which some are genetically similar to strains used as probiotics. Although only a limited number of clinical studies on fermented foods have been performed, there is evidence that these foods provide health benefits well-beyond the starting food materials.

Diversity of human small intestinal Streptococcus and Veillonella populations

Molecular and cultivation approaches were employed to study the phylogenetic richness and temporal dynamics of Streptococcus and Veillonella populations in the small intestine. Microbial profiling of human small intestinal samples collected from four ileostomy subjects at four time points displayed abundant populations of Streptococcus spp. most affiliated with S. salivarius, S. thermophilus, and S. parasanguinis, as well as Veillonella spp. affiliated with V. atypica, V. parvula, V. dispar, and V. rogosae. Relative abundances varied per subject and time of sampling. Streptococcus and Veillonella isolates were cultured using selective media from ileostoma effluent samples collected at two time points from a single subject. The richness of the Streptococcus and Veillonella isolates was assessed at species and strain level by 16S rRNA gene sequencing and genetic fingerprinting, respectively. A total of 160 Streptococcus and 37 Veillonella isolates were obtained. Genetic fingerprinting differentiated seven Streptococcus lineages from ileostoma effluent, illustrating the strain richness within this ecosystem. The Veillonella isolates were represented by a single phylotype. Our study demonstrated that the small intestinal Streptococcus populations displayed considerable changes over time at the genetic lineage level because only representative strains of a single Streptococcus lineage could be cultivated from ileostoma effluent at both time points.

Development of a minimal growth medium for Lactobacillus plantarum

Aim:  A medium with minimal requirements for the growth of Lactobacillus plantarum WCFS was developed. The composition of the minimal medium was compared to a genome‐scale metabolic model of L. plantarum.

Metabolic models for rational improvement of lactic acid bacteria as cell factories

Lactic acid bacteria (LAB) are microbes that are used all over the world in a variety of fermentations. Beside their most important application, which is undoubtedly in the dairy industry, LAB are also applied at an industrial scale in the fermentation of other food-raw materials like meat and vegetables. LAB have a relatively simple carbon and energy metabolism which is characterized by the rapid glycolytic conversion of sugars into lactic acid. Many examples of successful metabolic engineering approaches in LAB focus on re-routing of the pyruvate metabolism. Recently, LAB have also been used for the engineering of complex biosynthetic pathways leading to the production of valuable metabolites with health benefits for the consumers (Hugenholtz and Smid 2002). Engineering complex biosynthetic pathways such as for vitamin or polysaccharide biosynthesis, often leads to unexpected phenotypes which can only be understood if genome-wide metabolic models of the microorganism are available. Here we describe the construction of metabolic models of Lactobacillus plantarum based on the availability of genome sequence information. After prediction of gene function, we have focused on the development and improvement of methods and tools to go from genome sequence to gene annotation, to pathway reconstruction and to prediction of phenotype through metabolic models. We have set up different bioinformatics tools, including web-interfaced databases and simulation software. This paper describes some of these tools, and how they are used and combined with experimental data to arrive at a model of the metabolic network of L. plantarum. The use of these types of models and the type of questions that can be addressed will be discussed.

Comparative Genomics Analysis of Streptococcus Isolates from the Human Small Intestine Reveals their Adaptation to a Highly Dynamic Ecosystem

The human small-intestinal microbiota is characterised by relatively large and dynamic Streptococcus populations. In this study, genome sequences of small-intestinal streptococci from S. mitis, S. bovis, and S. salivarius species-groups were determined and compared with those from 58 Streptococcus strains in public databases. The Streptococcus pangenome consists of 12,403 orthologous groups of which 574 are shared among all sequenced streptococci and are defined as the Streptococcus core genome. Genome mining of the small-intestinal streptococci focused on functions playing an important role in the interaction of these streptococci in the small-intestinal ecosystem, including natural competence and nutrient-transport and metabolism. Analysis of the small-intestinal Streptococcus genomes predicts a high capacity to synthesize amino acids and various vitamins as well as substantial divergence in their carbohydrate transport and metabolic capacities, which is in agreement with observed physiological differences between these Streptococcus strains. Gene-specific PCR-strategies enabled evaluation of conservation of Streptococcus populations in intestinal samples from different human individuals, revealing that the S. salivarius strains were frequently detected in the small-intestine microbiota, supporting the representative value of the genomes provided in this study. Finally, the Streptococcus genomes allow prediction of the effect of dietary substances on Streptococcus population dynamics in the human small-intestine.

Comparative Analysis of Lactobacillus plantarum WCFS1 Transcriptomes by Using DNA Microarray and Next-Generation Sequencing Technologies

ABSTRACT RNA sequencing is starting to compete with the use of DNA microarrays for transcription analysis in eukaryotes as well as in prokaryotes. The application of RNA sequencing in prokaryotes requires additional steps in the RNA preparation procedure to increase the relative abundance of mRNA and cannot employ the poly(T)-primed approach in cDNA synthesis. In this study, we aimed to validate the use of RNA sequencing (direct cDNA sequencing and 3′-untranslated region [UTR] sequencing) using Lactobacillus plantarum WCFS1 as a model organism, employing its established microarray platform as a reference. A limited effect of mRNA enrichment on genome-wide transcript quantification was observed, and comparative transcriptome analyses were performed for L. plantarum WCFS1 grown in two different laboratory media. Microarray analyses and both RNA sequencing methods resulted in similar depths of analysis and generated similar fold-change ratios of differentially expressed genes. The highest overall correlation was found between microarray and direct cDNA sequencing-derived transcriptomes, while the 3′-UTR sequencing-derived transcriptome appeared to deviate the most. Overall, a high similarity between patterns of transcript abundance and fold-change levels of differentially expressed genes was detected by all three methods, indicating that the biological conclusions drawn from the transcriptome data were consistent among the three technologies.

High-resolution amplified fragment length polymorphism typing of Lactococcus lactis strains enables identification of genetic markers for subspecies-related phenotypes

ABSTRACT A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers.

Physiological and transcriptional responses of different industrial microbes at near-zero specific growth rates

ABSTRACT The current knowledge of the physiology and gene expression of industrially relevant microorganisms is largely based on laboratory studies under conditions of rapid growth and high metabolic activity. However, in natural ecosystems and industrial processes, microbes frequently encounter severe calorie restriction. As a consequence, microbial growth rates in such settings can be extremely slow and even approach zero. Furthermore, uncoupling microbial growth from product formation, while cellular integrity and activity are maintained, offers perspectives that are economically highly interesting. Retentostat cultures have been employed to investigate microbial physiology at (near-)zero growth rates. This minireview compares information from recent physiological and gene expression studies on retentostat cultures of the industrially relevant microorganisms Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger. Shared responses of these organisms to (near-)zero growth rates include increased stress tolerance and a downregulation of genes involved in protein synthesis. Other adaptations, such as changes in morphology and (secondary) metabolite production, were species specific. This comparison underlines the industrial and scientific significance of further research on microbial (near-)zero growth physiology.