Pan-genome is defined as the set of orthologous and unique genes of a specific group of organisms. The pan-genome is composed by the core genome, accessory genome, and species- or strain-specific genes. The pan-genome is considered open or closed based on the alpha value of the Heap law. In an open pan-genome, the number of gene families will continuously increase with the addition of new genomes to the analysis, while in a closed pan-genome, the number of gene families will not increase considerably. The first step of a pan-genome analysis is the homogenization of genome annotation. The same software should be used to annotate genomes, such as GeneMark or RAST. Subsequently, several software are used to calculate the pan-genome such as BPGA, GET_HOMOLOGUES, PGAP, among others. This review presents all these initial steps for those who want to perform a pan-genome analysis, explaining key concepts of the area. Furthermore, we present the pan-genomic analysis of 9 bacterial species. These are the species with the highest number of genomes deposited in GenBank. We also show the influence of the identity and coverage parameters on the prediction of orthologous and paralogous genes. Finally, we cite the perspectives of several research areas where pan-genome analysis can be used to answer important issues.

Since the publication of one of the first studies using 2D gel electrophoresis by Patrick H. O’Farrell in 1975, several other studies have used that method to evaluate cellular responses to different physicochemical variations. In environmental microbiology, bacterial adaptation to cold environments is a “hot topic” because of its application in biotechnological processes. As in other fields, gel-based and gel-free proteomic methods have been used to determine the molecular mechanisms of adaptation to cold of several psychrotrophic and psychrophilic bacterial species. In this review, we aim to describe and discuss these main molecular mechanisms of cold adaptation, referencing proteomic studies that have made significant contributions to our current knowledge in the area. Furthermore, we use Exiguobacterium antarcticum B7 as a model organism to present the importance of integrating genomic, transcriptomic, and proteomic data. This species has been isolated in Antarctica and previously studied at all three omic levels. The integration of these data permitted more robust conclusions about the mechanisms of bacterial adaptation to cold.

Seven genomes of Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, generating high-quality scaffolds over 2.35 Mbp. This bacterium is the causative agent of disease known as “pigeon fever” which commonly affects horses worldwide. The pangenome of biovar equi was calculated and two phylogenomic approaches were used to identify clustering patterns within Corynebacterium genus. Furthermore, other comparative analyses were performed including the prediction of genomic islands and prophages, and SNP-based phylogeny. In the phylogenomic tree, C. pseudotuberculosis was divided into two distinct clades, one formed by nitrate non-reducing species (biovar ovis) and another formed by nitrate-reducing species (biovar equi). In the latter group, the strains isolated from California were more related to each other, while the strains CIP 52.97 and 1/06-A formed the outermost clade of the biovar equi. A total of 1,355 core genes were identified, corresponding to 42.5% of the pangenome. This pangenome has one of the smallest core genomes described in the literature, suggesting a high genetic variability of biovar equi of C. pseudotuberculosis. The analysis of the similarity between the resistance islands identified a higher proximity between the strains that caused more severe infectious conditions (infection in the internal organs). Pathogenicity islands were largely conserved between strains. Several genes that modulate the pathogenicity of C. pseudotuberculosis were described including peptidases, recombination enzymes, micoside synthesis enzymes, bacteriocins with antimicrobial activity and several others. Finally, no genotypic differences were observed between the strains that caused the three different types of infection (external abscess formation, infection with abscess formation in the internal organs, and ulcerative lymphangitis). Instead, it was noted that there is a higher phenetic correlation between strains isolated at California compared to the other strains. Additionally, high variability of resistance islands suggests gene acquisition through several events of horizontal gene transfer.

Mangroves provide a unique ecological environment for complex microbial communities, which play important roles in biogeochemical cycles, such as those for carbon, sulfur, and nitrogen. Microbial diversity analyses of these ecosystems help us understand the changes caused by external influences. Amazonian mangroves occupy an area of 9000 km², corresponding to 70% of the mangroves in Brazil, on which studies of microbial biodiversity are extremely scarce. The present study aimed to determine changes in microbial community structure along the PA-458 highway, which fragmented a mangrove zone. Mangrove samples were collected from three zones, (i) degraded, (ii) in the process of recovery, and (iii) preserved. Total DNA was extracted and submitted for 16S rDNA amplification and sequencing on an MiSeq platform. Subsequently, reads were processed for quality control and biodiversity analyses. The most abundant phyla were Proteobacteria, Firmicutes, and Bacteroidetes in all three mangrove locations, but in significantly different proportions. We observed a considerable reduction in diversity in the degraded zone. Important genera involved in sulfur, carbon, and nitrogen metabolism were absent or dramatically reduced in this zone. Our results show that human impact in the mangrove areas, caused by the construction of the PA-458 highway, has resulted in a loss of biodiversity.

Exiguobacterium and Psychrobacter are bacterial genera with several cold-adapted species. These extremophiles are commonly isolated from the same habitats in Earth’s cryosphere and have great ecological and biotechnological relevance. Thus, through comparative genomic analyses, it was possible to understand the functional diversity of these psychrotrophic and psychrophilic species and present new insights into the microbial adaptation to cold. The nucleotide identity between Exiguobacterium genomes was >90%. Three genomic islands were identified in the E. antarcticum B7 genome. These islands contained genes involved in flagella biosynthesis and chemotaxis, as well as enzymes for carotenoid biosynthesis. Clustering of cold shock proteins by Ka/Ks ratio suggests the occurrence of a positive selection over these genes. Neighbor-joining clustering of complete genomes showed that the E. sibiricum was the most closely related to E. antarcticum. A total of 92 genes were shared between Exiguobacterium and Psychrobacter. A reduction in the genomic content of E. antarcticum B7 was observed. It presented the smallest genome size of its genus and a lower number of genes because of the loss of many gene families compared with the other genomes. In our study, eight genomes of Exiguobacterium and Psychrobacter were compared and analysed. Psychrobacter showed higher genomic plasticity and E. antarcticum B7 presented a large decrease in genomic content without changing its ability to grow in cold environments.

Exiguobacterium antarcticum strain B7 is a Gram-positive psychrotrophic bacterial species isolated in Antarctica. Although this bacteria has been poorly studied, its genome has already been sequenced. Therefore, it is an appropriate model for the study of thermal adaptation. In the present study, we analyzed the transcriptomes and proteomes of E. antarcticum B7 grown at 0°C and 37°C by SOLiD RNA-Seq, Ion Torrent RNA-Seq and two-dimensional difference gel electrophoresis tandem mass spectrometry (2D-DIGE-MS/MS). We found expression of 2,058 transcripts in all replicates from both platforms and differential expression of 564 genes (absolute log2FC ≥1, P-value <0.001) comparing the two temperatures by RNA-Seq. A total of 73 spots were differentially expressed between the two temperatures on 2D-DIGE, 25 of which were identified by MS/MS. Some proteins exhibited patterns of dispersion in the gel that are characteristic of post-translational modifications. Our findings suggest that the two sequencing platforms yielded similar results and that different omic approaches may be used to improve the understanding of gene expression. To adapt to low temperatures, E. antarcticum B7 expresses four of the six cold-shock proteins present in its genome. The cold-shock proteins were the most abundant in the bacterial proteome at 0°C. Some of the differentially expressed genes are required to preserve transcription and translation, while others encode proteins that contribute to the maintenance of the intracellular environment and appropriate protein folding. The results denote the complexity intrinsic to the adaptation of psychrotrophic organisms to cold environments and are based on two omic approaches. They also unveil the lifestyle of a bacterial species isolated in Antarctica.