Identification of Functional Differences in Metabolic Networks Using Comparative Genomics and Constraint-Based Models

A genome-scale network reconstruction (GENRE) is a knowledgebase for an organism and has various applications. Available genome sequences have risen in recent years, but the number of curated GENREs has not kept pace. Existing yeast GENREs contain significant commission and omission errors. Current practices limit the quantity and quality of GENREs. An open and transparent phylogenomic-driven framework is outlined to address these issues. The method is demonstrated with 33 yeasts and fungi in Dikarya. A pan-fungal metabolic network called FYRMENT (Fungal and Yeast Metabolic Network) (https://github.com/LMSE/FYRMENT) is created, and annotated with ortholog groups from AYbRAH (https://github.com/LMSE/AYbRAH). Metabolic models for lower-level taxons are compiled. The fungal pan-GENRE contains 1553 orthologs, 2759 reactions, 2251 metabolites. The GENREs have higher genomic and metabolic coverage than existing yeast and fungal GENREs created with other methods. Metabolic simulations show the maximum amino acid yields from glucose differs between yeast lineages, indicating metabolic networks have evolved. Curating genomes and reactions at higher taxonomic-levels increases the quantity and quality of GENREs than conventional approaches. This approach can scale to other branches in the tree of life.

Chapter twenty-one - Whole-Genome Metabolic Network Reconstruction and Constraint-Based Modeling ⋆

GEnome-scale Network REconstructions (GENREs) mathematically describe metabolic reactions of an organism or a specific cell type. GENREs can be used with a number of constraint-based reconstruction and analysis (COBRA) methods to make computational predictions on how a system changes in different environments. We created a simplified GENRE (referred to as iSIM) that captures central energy metabolism with nine metabolic reactions to illustrate the use of and promote the understanding of GENREs and constraint-based methods. We demonstrate the simulation of single and double gene deletions, flux variability analysis (FVA), and test a number of metabolic tasks with the GENRE. Code to perform these analyses is provided in Python, R, and MATLAB. Finally, with iSIM as a guide, we demonstrate how inaccuracies in GENREs can limit their use in the interrogation of energy metabolism.

ABSTRACTSalmonella enterica serovar Typhimurium definitive type 2 (DT2) is host restricted to Columba livia (rock or feral pigeon) but is also closely related to S. Typhimurium isolates that circulate in livestock and cause a zoonosis characterized by gastroenteritis in humans. DT2 isolates formed a distinct phylogenetic cluster within S. Typhimurium based on whole-genome-sequence polymorphisms. Comparative genome analysis of DT2 94-213 and S. Typhimurium SL1344, DT104, and D23580 identified few differences in gene content with the exception of variations within prophages. However, DT2 94-213 harbored 22 pseudogenes that were intact in other closely related S. Typhimurium strains. We report a novel in silico approach to identify single amino acid substitutions in proteins that have a high probability of a functional impact. One polymorphism identified using this method, a single-residue deletion in the Tar protein, abrogated chemotaxis to aspartate in vitro. DT2 94-213 also exhibited an altered transcriptional profile in response to culture at 42°C compared to that of SL1344. Such differentially regulated genes included a number involved in flagellum biosynthesis and motility.

Helicobacter pylori enhances the risk for ulcer disease and gastric cancer, yet only a minority of H. pylori-colonized individuals develop disease. We examined the ability of two H. pylori isolates to induce differential host responses in vivo or in vitro, and then used an H. pylori whole genome microarray to identify bacterial determinants related to pathogenesis. Gastric ulcer strain B128 induced more severe gastritis, proliferation, and apoptosis in gerbil mucosa than did duodenal ulcer strain G1.1, and gastric ulceration and atrophy occurred only in B128+ gerbils. In vitro, gerbil-passaged B128 derivatives significantly increased IL-8 secretion and apoptosis compared with G1.1 strains. DNA hybridization to the microarray identified several strain-specific differences in gene composition including a large deletion of the cag pathogenicity island in strain G1.1. Partial and complete disruption of the cag island in strain B128 attenuated induction of IL-8 in vitro and significantly decreased gastric inflammation in vivo. These results indicate that the ability of H. pylori to regulate epithelial cell responses related to inflammation depends on the presence of an intact cag pathogenicity island. Use of an H pylori whole genome microarray is an effective method to identify differences in gene content between H. pylori strains that induce distinct pathological outcomes in a rodent model of H. pylori infection.

Genomic studies of Candida auris are underpinned by the generation of high-quality genome assemblies. These reference genomes have been essential for investigations of the evolution and epidemiology of this emerging fungal pathogen. In addition to genomic epidemiology studies of local outbreaks and analysis of the global emergence of this species, comparisons of genomes of isolates from the five major clades have revealed differences in gene content and genomic structure. Here, we provide a detailed protocol for generating complete genome assemblies for C. auris.

The development of baculovirus expression vector systems has accompanied a rapid expansion of our knowledge about the genes, their function and regulation in insect cells. Classification of these viruses has also been refined as we learn more about differences in gene content between isolates, how this affects virus structure and their replication in insect larvae. Baculovirus gene expression occurs in an ordered cascade, regulated by early, late and very late gene promoters. There is now a detailed knowledge of these promoter elements and how they interact first with host cell-encoded RNA polymerases and later with virus-encoded enzymes. The composition of this virus RNA polymerase is known. The virus replication process culminates in the very high level expression of both polyhedrin and p10 gene products in the latter stages of infection. It has also been realized that the insect host cell has innate defenses against baculoviruses in the form of an apoptotic response to virus invasion. Baculoviruses counter this by encoding apoptotic-suppressors, which also appear to have a role in determining the host range of the virus. Also of importance to our understanding of baculovirus expression systems is how the virus can accumulate mutations within genes that affect recombinant protein yield in cell culture. The summary in this chapter is not exhaustive, but should provide a good preparation to those wishing to use this highly successful gene expression system.

Mechanisms That Shape Microbial Pangenomes.

Two sequenced nodulation regions of lupin Bradyrhizobium sp. WM9 carried the majority of genes involved in the Nod factor production. The nod region I harbored: nolA, nodD, nodA, nodB, nodC, nodS, nodI, nodJ, nolO, nodZ, fixR, nifA, fixA, nodM, nolK and noeL. This gene arrangement resembled that found in the nodulation region of Bradyrhizobium japonicum USDA110, however strain WM9 harbored only one nodD gene copy, while the nodM, nolK and noeL genes had no counterparts in the 410 kb symbiotic region of strain USDA110. Region II harbored nolL and nodW, but lacked an nodV gene. Both regions carried ORFs that lacked similarity to the published USDA110 sequences, though they had homologues in symbiotic regions of Rhizobium etli, Sinorhizobium sp. NGR234 and Mesorhizobium loti. These differences in gene content, as well as a low average sequence identity (70%) of symbiotic genes with respect to B. japonicum USDA110 were in contrast with the phylogenetic relationship of USDA110 and WM9 revealed by the analysis of 16S rDNA and dnaK sequences. This most likely reflected an early divergence of symbiotic loci, and possible co-speciation with distinct legumes. During this process the loss of a noel gene and the acquisition of a nolL gene could be regarded as an adaptation towards these legumes that responded to Nod factors carrying 4-O-acetylfucose rather than 2-O-methylfucose. This explained various responses of lupins and serradella plants to infection by mutants in nodZ and nolL genes, knowing that serradella is a stringent legume while lupins are more promiscuous legumes.

The Baculovirus 10-kDa Protein

Lactobacillus plantarum is a lactic acid bacterium that promotes animal intestinal health as a probiotic and is found in a wide variety of habitats. Here, we investigated the genomic features of different clusters of L. plantarum strains via pan-genomic analysis. We compared the genomes of 108 L. plantarum strains that were available from the NCBI GenBank database. These genomes were 2.9-3.7 Mbp in size and 44-45% in G+C content. A total of 8,847 orthologs were collected, and 1,709 genes were identified to be shared as core genes by all the strains analyzed. On the basis of SNPs from the core genes, 108 strains were clustered into five major groups (G1-G5) that are different from previous reports and are not clearly associated with habitats. Analysis of group-specific enriched or depleted genes revealed that G1 and G2 were rich in genes for carbohydrate utilization (L-arabinose, L-rhamnose, and fructooligosaccharides) and that G3, G4, and G5 possessed more genes for the restriction-modification system and MazEF toxin-antitoxin. These results indicate that there are critical differences in gene content and survival strategies among genetically clustered L. plantarum strains, regardless of habitats.

Komagataeibacter strains are important bacterial cellulose producers, yet closely related isolates can differ in cellulose yield, pellicle properties, and genetic stability during propagation. Such variability suggests that lineage structure and mobile genetic elements both contribute to strain-level genomic divergence. Here, complete genome comparisons were used to integrate vertical relatedness, gene-content structure, cellulose-associated signatures, and mobilome heterogeneity across 22 closed Komagataeibacter assemblies. A maximum likelihood phylogeny inferred from 642 single copy core genes provided the lineage scaffold. An anvi'o pangenome analysis defined a constant core gene cluster component across genomes and a noncore fraction that accounted for most of the genome differences in gene content. Targeted features linked to cellulose biosynthesis and local c-di-GMP-associated context were extracted from each genome. These features captured differences in bcs neighborhood composition and the presence of nearby GGDEF and EAL domain signals. The resulting feature matrix was projected by principal component analysis to summarize between-genome variation. Mobilome profiles were strongly strain dependent. Plasmid homology clustering identified 12 clusters comprising 36 plasmids from 13 genomes, including two dominant clusters of seven and six plasmids. Mash-based distance summaries further distinguished clusters consistent with conserved backbones from clusters consistent with heterogeneous, module-driven relationships. Prophage sequences, assessed as VIBRANT-predicted regions, were widespread but sparse per genome and dominated by medium length fragments. Insertion sequence burden ranged from 50 to 181 elements per genome, indicating substantial differences in transposition-associated sequence content. Pairwise association tests did not support robust cross module covariation beyond expected relationships among pangenome composition metrics at the current sampling depth. Overall, these results provide a complete genome reference framework linking lineage structure and mobilome heterogeneity, and they define reusable resources for comparative studies in bacterial cellulose biotechnology.

A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism.

Microarray analysis of gene expression patterns in immature ear, seedling, and embryo tissues from the maize inbred lines B73 and Mo17 identified numerous genes with variable expression. Some genes had detectable expression in only one of the two inbreds; most of these genes were detected in the genomic DNA of both inbreds, indicating that the expression differences are likely caused by differential regulation rather than by differences in gene content. Gene expression was also monitored in the reciprocal F1 hybrids B73xMo17 and Mo17xB73. The reciprocal F1 hybrid lines did not display parental effects on gene expression levels. Approximately 80% of the differentially expressed genes displayed additive expression patterns in the hybrids relative to the inbred parents. The approximately 20% of genes that display nonadditive expression patterns tend to be expressed at levels within the parental range, with minimal evidence for novel expression levels greater than the high parent or less than the low parent. Analysis of allele-specific expression patterns in the hybrid suggested that intraspecific variation in gene expression levels is largely attributable to cis-regulatory variation in maize. Collectively, our data suggest that allelic cis-regulatory variation between B73 and Mo17 dictates maintenance of inbred allelic expression levels in the F1 hybrid, resulting in additive expression patterns.

Functional and comparative genomics of pathogenic bacteria