**3. Genomics**

to the great potential of contamination of the environment. In addition, antibiotic treatment does not produce satisfactory results due to difficult penetration into the abscess capsule, making the treatment unfeasible, emphasizing that prophylaxis is the best method to combat

*C. ulcerans* has been described as the etiological agent of several infections in animals, such as goats, dogs, cats and cattle. The contact with affected animals is the main form of transmission of *C. ulcerans* to human hosts, causing diphtheria of zoonotic nature. The first cases of human infections were related to the consumption of milk contaminated by this microorganism. In the 1990s, it was presented as an emerging pathogen in countries of large animal production,

As for its biochemical characterization, *C. ulcerans* presents features such as the production of the enzyme gelatinase, inability to reduce nitrate and virulence factors, including toxic lipids associated with the cell wall, which may mediate bacterial resistance to phagocyte attack. *C. ulcerans* is capable of producing PLD, as well as *C. pseudotuberculosis* [22]. The third virulence factor for *C. ulcerans* is the production of the diphtheria toxin. *C. ulcerans* strains infected by bacteriophage carriers of the *tox* gene are the major responsible for clinical cases in humans

The diseases related to these strains show symptoms as frequent nasal bleeding, skin lesions similar to cutaneous diphtheria, necrosis and mucosal ulceration, granulomatous pulmonary

Although diphtheria by *C. ulcerans* is associated with its TD production, vaccination using the diphtheria toxoid has an unknown efficacy. This fact is due to the knowledge limitation of the bacterium molecular epidemiology. This limitation is mainly related to the structure of the *tox* gene, which has specific differences when compared to both interspecific (*C. diphthe-*

Bacterial strains belonging to *C. glutamicum* species are commonly found in the environment, in habitats such as soil. This bacterium is rod-shaped, capable of reducing nitrate to nitrite, facultative, mesophilic anaerobic and capable of carbohydrates fermentation. As a generally recognized as safe (GRAS) microorganism, it is widely used in the biotechnology industry, for its ability to produce amino acids like L-glutamate and L-lysine, used as flavor promoters and food additives [25]. More than 2.5 million tons of lysine is produced annually by mutant strains of *C. glutamicum*, for animal nutrition, applications in the pharmaceutical, cosmetics,

The nutrients used for industrial fermentation by *C. glutamicum* include glucose, fructose and sucrose, derived from corn starch, cassava or wheat, as well as cane molasses and beet molasses. Obtaining sugar from raw materials and agroindustry wastes is very common in

nodules, lymph node involvement and the occurrence of cellular death [24].

the disease [3].

and animals [23].

**2.3.** *Corynebacterium ulcerans*

62 Basic Biology and Applications of Actinobacteria

such as England, Japan, Germany, Denmark, and Brazil [21].

*riae tox* gene) and intraspecific in *C. ulcerans* [21].

**2.4.** *Corynebacterium glutamicum*

fuel and polymer industries [26].

#### **3.1. The impact of next sequencing technologies on genomics of the genus**  *Corynebacterium*

Forty years ago, the advent of DNA sequencing by Sanger method began to revolutionize genome studies [28]. The first genomes to be sequenced were viruses and organelles. In 1995, Craig Venter and colleagues published the two first complete bacterial genomes: *Haemophilus influenzae* and *Mycoplasma genitalium* [29, 30]. Later, several sequencing projects were created, which transformed the biology as a whole, by means of allowing to decipher complete genes and later whole genomes using the methodology developed by Sanger and colleagues in 1977 [31].

The publication of the first draft of the human genome in 2001 prompted companies to develop new sequencers that would provide more speed and accuracy, as well as cost and labor savings [32]. Since 2005, new sequencing technologies, called next-generation sequencing (NGS) or high-performance sequencers, have been able to generate gigabases (Gb) of data in a few days, (e.g. Illumina, Ion Torrent, Single Molecule Real Time-SMRT, PacBio, and Oxford Nanopore) [33]. Hence, the public domain databases have had, since registered the emergence of NGS platforms, an exponential increase in the number of deposited biological sequences, with more than 144,000 bacterial genomes already registered [34].

Currently, the genus *Corynebacterium* has more than 265 genome projects registered in public databases. According to the GOLD website, a database that provides project information in all three domains of life, *Corynebacterium* genome deposits date back to 2007 [35]. Since then, the increase of these data positively impacted the development of studies with transcriptomic and proteomic approaches, in order to provide a better understanding of several molecular processes from different corynebacterial species.

### **3.2. Comparative genomics studies**

The remarkable growth of the number of complete genomes provided the advance in the comparative analyzes between genomes, allowing studies in large scale. Comparative genomics provide a global understanding of the gene repertoire of a given species or genus, in order to elucidate the essential genes that are involved in processes such as replication, transcription and translation, in addition to the genes considered as accessory, that are also important for the characterization of variabilities in their genetic patterns, as well as allows the analysis of the genomic plasticity [36].

Codon deviation studies can aid in the understanding of the evolutionary molecular basis through parameters such as gene expression, amino acid conservation and codon-anticodon interaction. These factors reveal the type of selective pressure in eukaryotic and prokaryotic genes. In order to understand the molecular evolution of the genus *Corynebacterium*, comparative analyzes of G + C content and codon use were carried out relating different species, revealing evolutionary relationships that allowed divergence between the groups of patho-

The Genus *Corynebacterium* in the Genomic Era http://dx.doi.org/10.5772/intechopen.80445 65

The genomic approach allowed to know the sequence of DNA of a certain organism, though, only this knowledge does not define the gene function to external stimuli. A protein to be synthesized primarily needs the DNA to be transcribed into an RNA molecule, later translated into a protein molecule. However, the genes are not active all the time in the cell, and they are expressed when necessary to act in cellular biological process. The set of genes are expressed in a cell under a certain physiological condition or stage of development at a specific time is

Studies that address the transcriptome technique aim the analysis of the collection of all transcripts and provide information about the regulation of the genes, too allow inferring functions of uncharacterized genes, helping to understand the biology of the organism analyzed. One of the applications obtained by this approach is the usage of the data generated to provide more information about the host defense response to the survival and proliferation of bacterial pathogens, which enables an understanding of the pathogenesis of infectious

Due to the diverse applications of transcriptomics, new technologies and high-throughput methods have been developed for large-scale analysis, such as hybridization-based method

Microarray technology is considered a large-scale method because it generates the expression profile of thousands of transcripts simultaneously. Studies with microarray technology have identified clusters of genes that are involved in specific physiological responses, through the variations of environmental conditions faced by microorganisms [48], such as ammonia limitation. This compound is used as a source of nitrogen that is essential for almost all complex macromolecules in bacteria. A study analyzed the response of *C. glutamicum* in ammonia-limiting medium, demonstrating that there was alteration in the expression of 285 genes, many of which encode transport proteins and proteins involved in metabolism, nitrogen regulation,

Other studies with *C. glutamicum* were carried out aiming to evaluate the level of gene expression essential to the survival of the bacteria in stress environments. The transcriptional profile of this species under growth conditions with citrate as a source of carbon and energy compared to glucose demonstrated that *citM* and *tctCBA* encoding citrate uptake systems were induced, while the *ptsG, ptsS* and *ptsF* genes encoding the glucose capping system were

(Microarray) and sequencing-based methods such as RNA sequencing [47].

genic and nonpathogenic corynebacteria [40].

energy generation and protein turnover [49].

**4. Transcriptomics**

called transcriptome [45].

diseases [46].

In another aspect, comparative analyzes between different strains within the same phylogenetic clade make it possible to recognize similarities and differences among genomes, to clarify which sequences are capable of diverging phenotypic changes in organisms, and to elucidate the mechanisms of virulence among pathogenic organisms or in in the case of environmental microorganisms. From this premise, the pan-genome concept emerged [37].

Regarding *C. ulcerans*, a study was conducted with 19 strains identifying 4120 genes composing the pan-genome, of which 1405 were present in the core genome and 2715 present in the accessory genome, where proteins involved in the pili formation and the *tox* gene were found in a large part of the genomes. Furthermore, variations between the transmembrane proteins and proteins secreted among the different species have been identified, contributing to the variability of the pathogenicity between them. This study made a greater understanding possible, regarding the knowledge around the virulence of this emerging pathogen [38].

The pan-genome is constituted by the core genome, which configures the genes present among all analyzed strains; the accessory genome that shares genes between two or more, but not all, strains and includes the genes the bacteria needs to survive in a specific environment, in addition to species-specific genes belonging to a single lineage, which can be acquired via horizontal transfer [37, 39]. The representatives of the genus *Corynebacterium* become an interesting object of studies of comparative genomics and evolution, due to its diverse lifestyles [40].

This approach was used in *C. jeikeium* by comparing 17 plasmids from different clinical isolates, which identified that plasmid pK43 can act as a natural vehicle for gene transfer conferring antimicrobial resistance between multiresistant strains and possibly between other members of the corynebacteria group, such as *C. diphtheriae* [41].

In *C. pseudotuberculosis*, the pan-genome of 15 strains revealed differences between the biovars of this species, in which the biovar *ovis* presented clonal behavior, while the *equi* group has a greater genetic diversity [42]. Recently, a study with strains isolated from equines was analyzed and corroborated the diversity of the biovar, also presenting a wide repertoire of resistance genes and virulence factors such as: beta-lactamases, recombination endonucleases and phage integrase [43].

In a comparative analysis between *Corynebacterium jeikeium, Corynebacterium urealyticum, Corynebacterium kroppenstedtii, Corynebacterium resistens* and *Corynebacterium variabile,* it was possible to identify 83 regulatory genes, being 56 of transcriptional DNA binding regulators and nine sigma factors. Furthermore, 44 regulatory proteins were identified that were present in the core genome. These genes shared by the strains are involved in the generation of short-chain volatile acids, which are related to the odor formation process of the human body, showing the importance of this approach in lipophilic corynebacteria [44].

Codon deviation studies can aid in the understanding of the evolutionary molecular basis through parameters such as gene expression, amino acid conservation and codon-anticodon interaction. These factors reveal the type of selective pressure in eukaryotic and prokaryotic genes. In order to understand the molecular evolution of the genus *Corynebacterium*, comparative analyzes of G + C content and codon use were carried out relating different species, revealing evolutionary relationships that allowed divergence between the groups of pathogenic and nonpathogenic corynebacteria [40].
