From genotype to phenotype: computational approaches for inferring microbial traits relevant to the food industry.

When selecting microbial strains for the production of fermented foods, various microbial phenotypes need to be taken into account to achieve target product characteristics, such as biosafety, flavor, texture, and health-promoting effects. Through continuous advances in sequencing technologies, microbial whole-genome sequences of increasing quality can now be obtained both cheaper and faster, which increases the relevance of genome-based characterization of microbial phenotypes. Prediction of microbial phenotypes from genome sequences makes it possible to quickly screen large strain collections in silico to identify candidates with desirable traits. Several microbial phenotypes relevant to the production of fermented foods can be predicted using knowledge-based approaches, leveraging our existing understanding of the genetic and molecular mechanisms underlying those phenotypes. In the absence of this knowledge, data-driven approaches can be applied to estimate genotype-phenotype relationships based on large experimental datasets. Here, we review computational methods that implement knowledge- and data-driven approaches for phenotype prediction, as well as methods that combine elements from both approaches. Furthermore, we provide examples of how these methods have been applied in industrial biotechnology, with special focus on the fermented food industry.

Genome-Scale Metabolic Modeling Combined with Transcriptome Profiling Provides Mechanistic Understanding of Streptococcus thermophilus CH8 Metabolism.

Streptococcus thermophilus is a lactic acid bacterium adapted toward growth in milk and is a vital component of starter cultures for milk fermentation. Here, we combine genome-scale metabolic modeling and transcriptome profiling to obtain novel metabolic insights into this bacterium. Notably, a refined genome-scale metabolic model (GEM) accurately representing S. thermophilus CH8 metabolism was developed. Modeling the utilization of casein as a nitrogen source revealed an imbalance in amino acid supply and demand, resulting in growth limitation due to the scarcity of specific amino acids, in particular sulfur amino acids. Growth experiments in milk corroborated this finding. A subtle interdependency of the redox balance and the secretion levels of the key metabolites lactate, formate, acetoin, and acetaldehyde was furthermore identified with the modeling approach, providing a mechanistic understanding of the factors governing the secretion product profile. As a potential effect of high expression of arginine biosynthesis genes, a moderate secretion of ornithine was observed experimentally, augmenting the proposed hypothesis of ornithine/putrescine exchange as part of the protocooperative interaction between S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in yogurt. This study provides a foundation for future community modeling of food fermentations and rational development of starter strains with improved functionality. IMPORTANCE Streptococcus thermophilus is one the main organisms involved in the fermentation of milk and, increasingly, also in the fermentation of plant-based foods. The construction of a functional high-quality genome-scale metabolic model, in conjunction with in-depth transcriptome profiling with a focus on metabolism, provides a valuable resource for the improved understanding of S. thermophilus physiology. An example is the model-based prediction of the most significant route of synthesis for the characteristic yogurt flavor compound acetaldehyde and identification of metabolic principles governing the synthesis of other flavor compounds. Moreover, the systematic assessment of amino acid supply and demand during growth in milk provides insights into the key challenges related to nitrogen metabolism that is imposed on S. thermophilus and any other organism associated with the milk niche.

Regulation and distinct physiological roles of manganese in bacteria.

Manganese (Mn2+) is an essential trace element within organisms spanning the entire tree of life. In this review, we provide an overview of Mn2+ transport and the regulation of its homeostasis in bacteria, with a focus on its functions beyond being a cofactor for enzymes. Crucial differences in Mn2+ homeostasis exist between bacterial species that can be characterized to have an iron- or manganese-centric metabolism. Highly iron-centric species require minimal Mn2+ and mostly use it as a mechanism to cope with oxidative stress. As a consequence, tight regulation of Mn2+ uptake is required, while organisms that use both Fe2+ and Mn2+ need other layers of regulation for maintaining homeostasis. We will focus in detail on manganese-centric bacterial species, in particular lactobacilli, that require little to no Fe2+ and use Mn2+ for a wider variety of functions. These organisms can accumulate extraordinarily high amounts of Mn2+ intracellularly, enabling the nonenzymatic use of Mn2+ for decomposition of reactive oxygen species while simultaneously functioning as a mechanism of competitive exclusion. We further discuss how Mn2+ accumulation can provide both beneficial and pathogenic bacteria with advantages in thriving in their niches.

Global responses to oxytetracycline treatment in tetracycline-resistant Escherichia coli.

We characterized the global transcriptome of Escherichia coli MG1655:: tetA grown in the presence of ½ MIC (14 mg/L) of OTC, and for comparison WT MG1655 strain grown with 1//2 MIC of OTC (0.25 mg/L OTC). 1646 genes changed expression significantly (FDR > 0.05) in the resistant strain, the majority of which (1246) were also regulated in WT strain. Genes involved in purine synthesis and ribosome structure and function were top-enriched among up-regulated genes, and anaerobic respiration, nitrate metabolism and aromatic amino acid biosynthesis genes among down-regulated genes. Blocking of the purine-synthesis- did not affect resistance phenotypes (MIC and growth rate with OTC), while blocking of protein synthesis using low concentrations of chloramphenicol or gentamicin, lowered MIC towards OTC. Metabolic-modeling, using a novel model for MG1655 and continuous weighing factor that reflected the degree of up or down regulation of genes encoding a reaction, identified 102 metabolic reactions with significant change in flux in MG1655:: tetA when grown in the presence of OTC compared to growth without OTC. These pathways could not have been predicted by simply analyzing functions of the up and down regulated genes, and thus this work has provided a novel method for identification of reactions which are essential in the adaptation to growth in the presence of antimicrobials.

Constraint-based modeling in microbial food biotechnology.

Genome-scale metabolic network reconstruction offers a means to leverage the value of the exponentially growing genomics data and integrate it with other biological knowledge in a structured format. Constraint-based modeling (CBM) enables both the qualitative and quantitative analyses of the reconstructed networks. The rapid advancements in these areas can benefit both the industrial production of microbial food cultures and their application in food processing. CBM provides several avenues for improving our mechanistic understanding of physiology and genotype-phenotype relationships. This is essential for the rational improvement of industrial strains, which can further be facilitated through various model-guided strain design approaches. CBM of microbial communities offers a valuable tool for the rational design of defined food cultures, where it can catalyze hypothesis generation and provide unintuitive rationales for the development of enhanced community phenotypes and, consequently, novel or improved food products. In the industrial-scale production of microorganisms for food cultures, CBM may enable a knowledge-driven bioprocess optimization by rationally identifying strategies for growth and stability improvement. Through these applications, we believe that CBM can become a powerful tool for guiding the areas of strain development, culture development and process optimization in the production of food cultures. Nevertheless, in order to make the correct choice of the modeling framework for a particular application and to interpret model predictions in a biologically meaningful manner, one should be aware of the current limitations of CBM.

Adaptive responses to cefotaxime treatment in ESBL-producing Escherichia coli and the possible use of significantly regulated pathways as novel secondary targets.

OBJECTIVES: The aim of the study was to determine how ESBL-producing Escherichia coli change the expression of metabolic and biosynthesis genes when adapting to inhibitory concentrations of cefotaxime. Secondly, it was investigated whether significantly regulated pathways constitute putative secondary targets that can be used to combat the resistant bacteria. METHODS: Strains of E. coli MG1655 encoding blaCTX-M-1 from an IncI1 plasmid and from the chromosome were challenged with cefotaxime corresponding to inhibitory concentrations, and transcriptional patterns were compared with growth without or with very low concentrations of cefotaxime by RNA sequencing. Significantly regulated pathways were inhibited with suitable inhibitors, or genes encoding the enzymes of the regulated pathways were knocked out. The ability of the bacteria to grow in the presence of cefotaxime was determined. Chequerboard assays were utilized to confirm synergies between treatments. RESULTS: Genes belonging to 16 different functional gene classes were significantly regulated. Protein and peptidoglycan syntheses were up-regulated and low concentrations of chloramphenicol or d-cycloserine, targeting these systems, strongly reduced the MIC of cefotaxime (>32-fold). Inhibition and/or mutations in other genes that were significantly regulated, belonging to energy synthesis, purine synthesis, proline uptake or potassium uptake, also rendered the resistant bacteria more susceptible to cefotaxime. CONCLUSIONS: The results show that ESBL-producing E. coli adapt to treatment with cefotaxime by changing their gene expression patterns and furthermore that targeting regulated adaptive pathways may be a suitable way to identify targets for drugs that will specifically inhibit the resistant bacteria.

Deletion and acquisition of genomic content during early stage adaptation of Pseudomonas aeruginosa to a human host environment.

Adaptation of bacterial pathogens to a permanently host-associated lifestyle by means of deletion or acquisition of genetic material is usually examined through comparison of present-day isolates to a distant theoretical ancestor. This limits the resolution of the adaptation process. We conducted a retrospective study of the dissemination of the P.aeruginosa DK2 clone type among patients suffering from cystic fibrosis, sequencing the genomes of 45 isolates collected from 16 individuals over 35 years. Analysis of the genomes provides a high-resolution examination of the dynamics and mechanisms of the change in genetic content during the early stage of host adaptation by this P.aeruginosa strain as it adapts to the cystic fibrosis (CF) lung of several patients. Considerable genome reduction is detected predominantly through the deletion of large genomic regions, and up to 8% of the genome is deleted in one isolate. Compared with in vitro estimates the resulting average deletion rates are 12- to 36-fold higher. Deletions occur through both illegitimate and homologous recombination, but they are not IS element mediated as previously reported for early stage host adaptation. Uptake of novel DNA sequences during infection is limited as only one prophage region was putatively inserted in one isolate, demonstrating that early host adaptation is characterized by the reduction of genomic repertoire rather than acquisition of novel functions. Finally, we also describe the complete genome of this highly adapted pathogenic strain of P.aeruginosa to strengthen the genetic basis, which serves to help our understanding of microbial evolution in a natural environment.