Summary: Actinobacteria constitute one of the largest phyla among Bacteria and represent gram-positive bacteria with a high G+C content in their DNA. This bacterial group includes microorganisms exhibiting a wide spectrum of morphologies, from coccoid to fragmenting hyphal forms, as well as possessing highly variable physiological and metabolic properties. Furthermore, Actinobacteria members have adopted different lifestyles, and can be pathogens (e.g., Corynebacterium, Mycobacterium, Nocardia, Tropheryma, and Propionibacterium), soil inhabitants (Streptomyces), plant commensals (Leifsonia), or gastrointestinal commensals (Bifidobacterium). The divergence of Actinobacteria from other bacteria is ancient, making it impossible to identify the phylogenetically closest bacterial group to Actinobacteria. Genome sequence analysis has revolutionized every aspect of bacterial biology by enhancing the understanding of the genetics, physiology, and evolutionary development of bacteria. Various actinobacterial genomes have been sequenced, revealing a wide genomic heterogeneity probably as a reflection of their biodiversity. This review provides an account of the recent explosion of actinobacterial genomics data and an attempt to place this in a biological and evolutionary context.

Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum

The Perigord black truffle ($Tuber\ melanosporum$ Vittad.) and the Piedmont white truffle dominate today's truffle market. The hypogeous fruiting body of $T.\ melanosporum$ is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal $Laccaria\ bicolor$, the expansion of gene families may have acted as a 'symbiosis toolbox'. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of $T.\ melanosporum$, which at $\sim$125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for $\sim$58% of the genome. In contrast, this genome only contains $\sim$7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that $T.\ melanosporum$ degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both $L.\ bicolor$ and $T.\ melanosporum$, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis $-$'the symbiosis toolbox'$-$ evolved along different ways in ascomycetes and basidiomycetes

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Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis

Bacteria belonging to the genus Lactobacillus are members of the lactic acid bacteria (LAB), a broadly defined group characterized by the formation of lactic acid as the sole or main end product of carbohydrate metabolism. They can be found in plants or material of plant origin, silage, fermented food (yogurt, cheese, olives, pickles, salami, etc.), as well as in the oral cavities, gastrointestinal tracts (GIT), and vaginas of humans and animals (31). In particular, the Lactobacillus species found in the GIT have received tremendous attention due to their health-promoting properties. They are commonly used as probiotics, which are defined by the FAO/WHO as live microorganisms that when administered in adequate amounts confer a health benefit on the host. The economic success and exciting prospects of probiotic products have accelerated research on intestinal lactobacilli. Genomics of Lactobacillus species is booming, and the genomes of five strains that belong to species commonly found in human fecal samples have recently been sequenced (50). Several comparative and functional genomic investigations have been conducted to gain information about the functionality of lactobacilli in the GIT (69). Unfortunately, a major misconception regarding the ecological role of lactobacilli in the intestinal tract has been embraced by many scientists working in the field. Specifically, there has been a general and persistent assumption that a large number of Lactobacillus species form stable and numerically significant populations in the human intestinal tract, especially in the small intestine, where they are presumed to form epithelial associations (101). Considering how widespread and accepted this perception is, there is surprisingly little experimental evidence that supports it. Ecological observations for the prevalence and dynamics of fecal Lactobacillus populations and the findings obtained with comparative genomics do indicate now that the ecological role of most types of intestinal lactobacilli, and their relationship with the human host, should be reconsidered. In this review, evidence is summarized that suggests that only a small number of Lactobacillus species are true inhabitants of the mammalian intestinal tract and that most lactobacilli present are allochthonous members derived from fermented food, the oral cavity, or more proximal parts of the GIT. It is further explained why this knowledge provides information valuable for selecting strains for fundamental research of the ecological role of lactobacilli in the GIT, for their use as probiotics in foods and supplements, and for pharmaceutical applications.

Ecological Role of Lactobacilli in the Gastrointestinal Tract: Implications for Fundamental and Biomedical Research

Members of the genus Bifidobacterium can be found as components of the gastrointestinal microbiota, and are believed to play an important role in maintaining and promoting human health by eliciting a number of beneficial properties. Bifidobacteria can utilize a diverse range of dietary carbohydrates that escape degradation in the upper parts of the intestine, many of which are plant-derived oligo- and polysaccharides. The gene content of a bifidobacterial genome reflects this apparent metabolic adaptation to a complex carbohydrate-rich gastrointestinal tract environment as it encodes a large number of predicted carbohydrate-modifying enzymes. Different bifidobacterial strains may possess different carbohydrate utilizing abilities, as established by a number of studies reviewed here. Carbohydrate-degrading activities described for bifidobacteria and their relevance to the deliberate enhancement of number and/or activity of bifidobacteria in the gut are also discussed in this review.

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Carbohydrate metabolism in Bifidobacteria.

Recent advances in ultra-high-throughput sequencing technology and metagenomics have led to a paradigm shift in microbial genomics from few genome comparisons to large-scale pan-genome studies at different scales of phylogenetic resolution. Pan-genome studies provide a framework for estimating the genomic diversity of the dataset, determining core (conserved), accessory (dispensable) and unique (strain-specific) gene pool of a species, tracing horizontal gene-flux across strains and providing insight into species evolution. The existing pan genome software tools suffer from various limitations like limited datasets, difficult installation/requirements, inadequate functional features etc. Here we present an ultra-fast computational pipeline BPGA (Bacterial Pan Genome Analysis tool) with seven functional modules. In addition to the routine pan genome analyses, BPGA introduces a number of novel features for downstream analyses like core/pan/MLST (Multi Locus Sequence Typing) phylogeny, exclusive presence/absence of genes in specific strains, subset analysis, atypical G + C content analysis and KEGG & COG mapping of core, accessory and unique genes. Other notable features include minimum running prerequisites, freedom to select the gene clustering method, ultra-fast execution, user friendly command line interface and high-quality graphics outputs. The performance of BPGA has been evaluated using a dataset of complete genome sequences of 28 Streptococcus pyogenes strains.

/pdf/bpga-an-ultra-fast-pan-genome-analysis-pipeline-1ewx3j7b92.pdf

BPGA- an ultra-fast pan-genome analysis pipeline

Lactic acid bacteria (LAB) are a group of gram-positive, lactic acid producing Firmicutes. They have been extensively used in food fermentations, including the production of various dairy products. The proteolytic system of LAB converts proteins to peptides and then to amino acids, which is essential for bacterial growth and also contributes significantly to flavor compounds as end-products. Recent developments in high-throughput genome sequencing and comparative genomics hybridization arrays provide us with opportunities to explore the diversity of the proteolytic system in various LAB strains. We performed a genome-wide comparative genomics analysis of proteolytic system components, including cell-wall bound proteinase, peptide transporters and peptidases, in 22 sequenced LAB strains. The peptidase families PepP/PepQ/PepM, PepD and PepI/PepR/PepL are described as examples of our in silico approach to refine the distinction of subfamilies with different enzymatic activities. Comparison of protein 3D structures of proline peptidases PepI/PepR/PepL and esterase A allowed identification of a conserved core structure, which was then used to improve phylogenetic analysis and functional annotation within this protein superfamily. The diversity of proteolytic system components in 39 Lactococcus lactis strains was explored using pangenome comparative genome hybridization analysis. Variations were observed in the proteinase PrtP and its maturation protein PrtM, in one of the Opp transport systems and in several peptidases between strains from different Lactococcus subspecies or from different origin. The improved functional annotation of the proteolytic system components provides an excellent framework for future experimental validations of predicted enzymatic activities. The genome sequence data can be coupled to other "omics" data e.g. transcriptomics and metabolomics for prediction of proteolytic and flavor-forming potential of LAB strains. Such an integrated approach can be used to tune the strain selection process in food fermentations.

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The proteolytic system of lactic acid bacteria revisited: a genomic comparison

Lactic acid bacteria (LAB) have been widely used as starter or nonstarter cultures in the dairy industry for over a thousand years. They play an essential role in flavor formation during the fermentation of dairy products. Several metabolic routes can lead to the formation of flavor compounds when

Comparative Genomics of Enzymes in Flavor-Forming Pathways from Amino Acids in Lactic Acid Bacteria

Lactobacillus bulgaricus and Streptococcus thermophilus, used in yogurt starter cultures, are well known for their stability and protocooperation during their coexistence in milk. In this study, we show that a close interaction between the two species also takes place at the genetic level. We performed an in silico analysis, combining gene composition and gene transfer mechanism-associated features, and predicted horizontally transferred genes in both L. bulgaricus and S. thermophilus. Putative horizontal gene transfer (HGT) events that have occurred between the two bacterial species include the transfer of exopolysaccharide (EPS) biosynthesis genes, transferred from S. thermophilus to L. bulgaricus, and the gene cluster cbs-cblB(cglB)-cysE for the metabolism of sulfur-containing amino acids, transferred from L. bulgaricus or Lactobacillus helveticus to S. thermophilus. The HGT event for the cbs-cblB(cglB)-cysE gene cluster was analyzed in detail, with respect to both evolutionary and functional aspects. It can be concluded that during the coexistence of both yogurt starter species in a milk environment, agonistic coevolution at the genetic level has probably been involved in the optimization of their combined growth and interactions.

https://aem.asm.org/content/aem/75/12/4120.full.pdf

In silico prediction of horizontal gene transfer events in Lactobacillus bulgaricus and Streptococcus thermophilus reveals protocooperation in yogurt manufacturing.

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Time and Bayesian Networks

The incompleteness of genome-scale metabolic models is a major bottleneck for systems biology approaches, which are based on large numbers of metabolites as identified and quantified by metabolomics. Many of the revealed secondary metabolites and/or their derivatives, such as flavor compounds, are non-essential in metabolism, and many of their synthesis pathways are unknown. In this study, we describe a novel approach, Reverse Pathway Engineering (RPE), which combines chemoinformatics and bioinformatics analyses, to predict the "missing links" between compounds of interest and their possible metabolic precursors by providing plausible chemical and/or enzymatic reactions. We demonstrate the added-value of the approach by using flavor-forming pathways in lactic acid bacteria (LAB) as an example. Established metabolic routes leading to the formation of flavor compounds from leucine were successfully replicated. Novel reactions involved in flavor formation, i.e. the conversion of alpha-hydroxy-isocaproate to 3-methylbutanoic acid and the synthesis of dimethyl sulfide, as well as the involved enzymes were successfully predicted. These new insights into the flavor-formation mechanisms in LAB can have a significant impact on improving the control of aroma formation in fermented food products. Since the input reaction databases and compounds are highly flexible, the RPE approach can be easily extended to a broad spectrum of applications, amongst others health/disease biomarker discovery as well as synthetic biology.

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Combining chemoinformatics with bioinformatics: in silico prediction of bacterial flavor-forming pathways by a chemical systems biology approach "reverse pathway engineering".

Mengjin Liu

Papers

The proteolytic system of lactic acid bacteria revisited: a genomic comparison

Comparative Genomics of Enzymes in Flavor-Forming Pathways from Amino Acids in Lactic Acid Bacteria

In silico prediction of horizontal gene transfer events in Lactobacillus bulgaricus and Streptococcus thermophilus reveals protocooperation in yogurt manufacturing.

Time and Bayesian Networks

Combining chemoinformatics with bioinformatics: in silico prediction of bacterial flavor-forming pathways by a chemical systems biology approach "reverse pathway engineering".