CHAPTER 2. Phylogeny Trumps Chemotaxonomy
2.1. Introduction
Actinobacteria is one of the major bacterial phyla, consisting of gram-positive bacteria with the high G+C content genome. Actinobacteria inhabit various environments such as soil, ocean and the human body. Many pathogens such as Mycobacterium tuberculosis and Nocardia asteroids belong to Actinobacteria.
Tuberculosis is the disease that causes more than one million death per year, which caused by infection of Mycobacterium tuberculosis complex (MTBC), and the emergence of antibiotic-resistant strains has emerged as a new problem in tuberculosis treatment (Sandhu, 2011).
The order Corynebacteriales, an order in Actinobacteria, circumscribes which are important in clinical, industrial, and environmental aspects (Goodfellow and Jones, 2015; Lehmann and Neumann, 1896). This taxon is widely known as the presence of mycolic acids (MA), a special type of branched fatty acid consists of two long chains. MAs are known to act as a cell wall permeability barricade that argues to antibiotics and phagocytes (Gebhardt et al., 2007). The length and double bonds in MAs are employed as substantial chemotaxonomic markers for demarcating between genera classified in Corynebacteriales (Bernard et al., 2010;
Marrakchi et al., 2014), except for some MA-lacked species (Collins et al., 1998;
Funke et al., 1994; Wiertz et al., 2013). The classification of MA-containing taxon groups was directed by a combination of 16S rRNA gene sequence and chemotaxonomic characteristics. Based on those criteria, several new genera such as Hoyosella (Jurado et al., 2009) and Lawsonella (Bell et al., 2016) were described. However, the absence of agreement between these sets of data in some
taxon needs to use the extra clue to signify this circumstance.
The classification of the genus Turicella is such a case. The type and only species in this genus, Turicella otitidis, was originally proposed to harbor bacterial strains isolated from the ear of an otitis media patient (Funke et al., 1994). In this study, the type strain of T. otitidis was reclaimed as a sister taxon to Corynebacterium based on 16S rRNA tree and two distinct chemotaxonomic markers; 1) the presence of fully unsaturated menaquinones (MK-10 and MK-11) as contradicting to partially saturated MKs in Corynebacterium [MK-8(H2) and MK-9(H2)], and 2) lack of MA whereas most Corynebacterium species contain MA.
However, succeeding studies using more 16S rRNA gene sequences demonstrated that Turicella formed a phyletic lineage within the Corynebacterium clade (Goyache et al., 2003; Hall et al., 2003).
Two major fatty acid biosynthesis pathways, FAS-I (Fatty acid synthesis-I) and FAS-II (Fatty acid synthesis-II) cycles are known in Actinobacteria (Marrakchi et al., 2014). One gene (3-oxoacyl-ACP synthase; fas) is charged in whole steps in FAS-I pathway (Bloch and Vance, 1977), whereas four essential genes are burden in FAS-II pathway, which are beta-ketoacyl-ACP synthase (kasA) (Bhatt et al., 2005), beta-ketoacyl-ACP reductase (mabA; Parish et al., 2007), (3R)-hydroxy acyl-ACP dehydratase subunit B (hadB; Brown et al., 2007; Sacco et al., 2007) and (NADH)dependent trans-2-enoyl-ACP reductase (inhA; Vilcheze et al., 2000).
Mycobacterium species are known to possess both FAS-I and FAS-II pathway, whereas most Corynebacterium species contain only FAS-I pathway. It is revealed that Corynebacterium jeikeium and Corynebacterium urealyticum have neither the FAS-I nor FAS-II cycle and absorb fatty acids from the exogenous environment (Tauch et al., 2005; Tauch et al., 2008).
MAs are synthesized by conjugation of two long chain fatty acids, which are carboxylated
α
-branch fatty acid and meromycolic acid. The carboxylatedα
-branch fatty acid was formed by acyl-CoA carboxylase (accD4) and acetyl-CoA carboxylase (Gande et al., 2007). The meromycolic acid was formed after modification steps including desaturation (NADPH-dependent stearoyl-CoA 9- desaturase; desA3; Cole et al., 1998), Then, long-chain fatty acid AMP ligase (fadD32) leads to the synthesis of the meromycolic acid (Portevin et al., 2005).Eventually, MAs are formed by merging two fatty acids by polyketide synthase 13 (pks13) (Portevin et al., 2004). It is known that a single operon consists of three genes (fadD32-pks13-accD4) is substantial for MA formation (Portevin et al., 2005). Also, two beta subunits of carboxylases (accD4 and accD5) are also necessary for MA forming (Gande et al., 2004).
Isoprenoid quinone plays the role of electron and proton transporter in the electron transport system of photosynthesis and cellular respiration in many species.
Two types of quinones in prokaryote cell are naphthoquinone and benzoquinone, MK corresponds to former, and UQ corresponds to the latter. Gram-positive bacteria have MK, while gram-negative bacteria have UQ (Nowicka and Kruk, 2010). Some species such as Escherichia coli have both MK and UQ (Meganathan and Kwon, 2009). In Actinobacteria, most species have MK as a respiratory quinone. Species in Nocardia, Skermania, and Smaragdicoccus have cyclic MK, having a cyclized ring at the end of the isoprenoid chain (Adachi et al., 2007; Chun et al., 1997). Some species produce other types of quinone instead of MK. Species belonging to Bifidobacterium don’t have respiratory quinone. They obtain quinone from the host or the surrounding environment (Ramotar et al., 1984).
Classical MK pathway synthesizes MK via isochorismate (Bentley and Meganathan, 1982). An alternative pathway produces MK via futalosine (Seto et al., 2008). Among the species belonging to Actinobacteria, MK biosynthesis pathway through isochorismate was discovered in M. tuberculosis (Dhiman et al., 2009), while Streptomyces coelicolor has MK biosynthesis pathway via futalosine (Hiratsuka et al., 2008).
The length and saturated sites in isoprenoid chain of MK are factors for species identification (Collins and Jones, 1981). Also, the enzyme menaquinone reductase (menJ) was discovered (Upadhyay et al., 2015), which confers the saturation of MK in M. tuberculosis. The deletion of this gene guides to the production of fully unsaturated MK (MK-9) instead of normal partially saturated MK [MK-9(H2)].
In this study, we re-examine the opaque taxonomic status of the genus Turicella using genome-based phylogenetics and comparative genomics of genes charged in synthesizing or modifying chemotaxonomic markers. Based on the genomic evidence, Turicella otitidis need to be classified in the genus Corynebacterium, reclassify as Corynebacterium otitidis.