**2. Main species of medical and biotechnological importance**

### **2.1.** *Corynebacterium diphtheriae*

Diphtheria is a disease of acute and transmissible evolution within local and systemic manifestations, affecting the upper respiratory tract, and has been one of the main causes of death, especially in children, in different continents, even in countries with immunization programs. *C. diphtheriae* was isolated mainly from humans, however it has been isolated from other hosts, such as horses, cats and dogs [9].

*C. diphtheriae* is a rod-shaped bacterium, and its cells can measure from 0.5 to 2.0 μm in size. Strains belonging to this species do not produce spores and do not present structures such as capsules and flagella [10]. The strains are classified in four biovars: *mitis, gravis, intermedius* and *belfanti*, based on the infection severity, morphological pattern of the colonies, carbohydrate fermentation and hemolysis [9]. These biovars resemble the production of cystinase enzyme (Tinsdale medium), and the fermentation of glucose and maltose. Moreover, these biovars produce neither the enzymes pyrazinamidase nor urease, and also they are not capable of fermenting sucrose. In relation to nitrate reduction, the biovars *mitis*, *gravis and intermedius* present a positive reaction, and the *belfanti* biovar presents a negative reaction [11].

In its original form, *C. diphtheriae* does not cause diseases and its pathogenicity is related to infection by a bacteriophage carrying the *tox* gene encoding TD. Thus, the lysogenic cell carries the *tox* gene, highly conserved in the bacterial chromosome through generations [12]. TD is a potent exotoxin of protein nature capable of acting in all tissues with special tropism for the myocardium, nervous system, kidneys and adrenals [6].

TD acts on the inhibition of protein synthesis causing cell death and it is composed of a single polypeptide chain containing two fragments, A and B, connected by a disulfide bond, and both are required for intoxication of tissue culture cells. The fragment A possesses the active site of TD and it is responsible for the enzymatic activity, while the fragment B is responsible for the fixation of the toxin with receptors in the host cells [13].

#### **2.2.** *Corynebacterium pseudotuberculosis*

similar form and/or pathogenicity mechanisms [2]. Currently, the genus has 110 valid spe-

Frequently, members of the genus *Corynebacterium* have a rod morphology, being Grampositive, immobile, nonsporulated, presenting an aerobic growth, and producers of the catalase enzyme. They are part of the normal skin microbiota and mucous membranes of several hosts, being present also in the environment (soil, water, among others). Bacteria of this genus still share characteristics like G+C content (47–74%), oxygenase enzyme production, and no production of the enzyme collagenase. In addition, their cell wall is thick, and it has the presence of mycolic acids, peptidoglycan and arabinogalactan, also saturated and unsaturated

Bacterial species affiliated with this genus can be classified as: pathogenic, opportunistic and saprophytic. The strains of medical and veterinary interest are commonly divided in two groups: diphtheria and nondiphtheria. The diphtheria group, producer of the diphtheria toxin (TD) encoded by the viral *tox* gene, present in the DNA of β lysogenic bacteriophages [4]. Among this group, we can mention three species: *C. pseudotuberculosis*, *C. diphtheriae* and *C. ulcerans* [5]. Nondiphtheria species, as agents of infection, are considered opportunistic pathogens, because they are present in the skin normal microbiota, and in the human nasopharynx [6]. The species *Corynebacterium jeikeium*, *Corynebacterium urealyticum* and *Corynebacterium resistens* are considered opportunistic [7]. Nonpathogenic strains, such as *Corynebacterium glutamicum*, *Corynebacterium efficiens, Corynebacterium crenatum* and *Corynebacterium variabile*, have biotechnological importance in the production of amino acids

cies, of which 132 species have synonymous species and 11 subspecies [1].

**2. Main species of medical and biotechnological importance**

Diphtheria is a disease of acute and transmissible evolution within local and systemic manifestations, affecting the upper respiratory tract, and has been one of the main causes of death, especially in children, in different continents, even in countries with immunization programs. *C. diphtheriae* was isolated mainly from humans, however it has been isolated from other

*C. diphtheriae* is a rod-shaped bacterium, and its cells can measure from 0.5 to 2.0 μm in size. Strains belonging to this species do not produce spores and do not present structures such as capsules and flagella [10]. The strains are classified in four biovars: *mitis, gravis, intermedius* and *belfanti*, based on the infection severity, morphological pattern of the colonies, carbohydrate fermentation and hemolysis [9]. These biovars resemble the production of cystinase enzyme (Tinsdale medium), and the fermentation of glucose and maltose. Moreover, these biovars produce neither the enzymes pyrazinamidase nor urease, and also they are not capable of fermenting sucrose. In relation to nitrate reduction, the biovars *mitis*, *gravis and intermedius*

present a positive reaction, and the *belfanti* biovar presents a negative reaction [11].

fatty acids [3].

and in the cheese industry [8].

60 Basic Biology and Applications of Actinobacteria

**2.1.** *Corynebacterium diphtheriae*

hosts, such as horses, cats and dogs [9].

The bacterium *C. pseudotuberculosis* presents characteristics such as the production of the enzyme nitrate reductase (biovar-dependent) and urease, the fermentation of carbohydrates maltose and glucose and the presence of halos of beta-hemolysis in blood agar. As for their colonies, they present a size ranging from 0.5 to 0.6 × 1.0 to 3.0 μm, with a whitish and viscous appearance [14]. Biovar *ovis*, nitrate reductase negative, affects sheep and goats, and occasionally swine, causing caseous lymphadenitis, in humans may cause chronic subacute lymphadenitis [3]. While biovar *equi*, which the isolates can reduce nitrate to nitrite, mainly infects equines, buffaloes and camelids, causing ulcerative lymphangitis and edematous skin disease [15]. Due to its prevalence in animals of economic importance, diseases associated with *C. pseudotuberculosis* strains cause reduction in meat and milk production, wool depreciation, delay in animal development, deficiencies in reproductive indices of the herd, carcass condemnation, early discarding and occasional death of animals, also high treatment costs and veterinary fees [3].

The virulence of *C. pseudotuberculosis* is related to three main factors: the cell wall structure, its intracellular capacity for macrophage persistence and the production of phospholipase D (PLD) as exotoxin, which is considered the main virulence factor of the species [16]. Although its main virulence factor is already well established, toxigenic strains of this species can also produce TD [17, 18].

The diagnosis of *C. pseudotuberculosis* in infected animals is performed by the macroscopic observation of the superficial abscesses formed, associated to laboratory culture in the selective media of tellurian agar, bacterioscopy, catalase test (positive for *Corynebacterium*) and biochemical tests [3]. In addition to serological tests such as seroneutralization, indirect hemagglutination and Enzyme-Linked Immunosorbent Assay (ELISA), allergic tests and tests based on molecular biology such as the polymerase chain reaction (PCR), through the conserved genes *rrs, rpoB* and *pld* in multiplex PCR [19]. Recently, with addition of the *narG* gene, it is possible to distinguish biovars from the capacity to reduce nitrate [20].

The animals affected by caseous lymphadenitis are usually treated through lymph node drainage and isolation of infected animals. Yet, this practice does not completely eliminate bacteria, due to the possibility of dissemination to viscera and other internal organs, as well 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 the disease [3].

countries with high agricultural production, such as China, the United States and Brazil, its use reduces the industrial production process. Additionally, *C. glutamicum* strains are ideal for large-scale fermentation processes, since they are resistant to oscillations in oxygen tension and in the substrate supply, which often occurs in these industrial processes [25].

The Genus *Corynebacterium* in the Genomic Era http://dx.doi.org/10.5772/intechopen.80445 63

One of the factors considered in the selection of strains is the maximum theoretical yield of a cell within production of lysine from glucose. This yield should turn around 75% conversion of carbohydrate into final product. Metabolic flux analysis, considering the main metabolic pathways that can be used by *C. glutamicum* to produce lysine, indicate that the theoretical yield is increasing, producing more than two million tons of amino acids per year [27].

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

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

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

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

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

sequences, with more than 144,000 bacterial genomes already registered [34].

processes from different corynebacterial species.

**3.2. Comparative genomics studies**

**3. Genomics**

*Corynebacterium*

[31].

#### **2.3.** *Corynebacterium ulcerans*

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

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 and animals [23].

The diseases related to these strains show symptoms as frequent nasal bleeding, skin lesions similar to cutaneous diphtheria, necrosis and mucosal ulceration, granulomatous pulmonary nodules, lymph node involvement and the occurrence of cellular death [24].

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

#### **2.4.** *Corynebacterium glutamicum*

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, fuel and polymer industries [26].

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 countries with high agricultural production, such as China, the United States and Brazil, its use reduces the industrial production process. Additionally, *C. glutamicum* strains are ideal for large-scale fermentation processes, since they are resistant to oscillations in oxygen tension and in the substrate supply, which often occurs in these industrial processes [25].

One of the factors considered in the selection of strains is the maximum theoretical yield of a cell within production of lysine from glucose. This yield should turn around 75% conversion of carbohydrate into final product. Metabolic flux analysis, considering the main metabolic pathways that can be used by *C. glutamicum* to produce lysine, indicate that the theoretical yield is increasing, producing more than two million tons of amino acids per year [27].
