**3.2. Nitroaromatic degradation pathway as a role model for study the gene evolution**

A role model to study evolution of microbial pathways is to study the degradation of nitroar‐ omatic compounds in different bacteria.

Nitroaromatic compounds are relatively new to the environment, but bacterial systems have already evolved the ability to metabolize them. This cannot be possible only by spontaneous, independent evolution of several new enzymes in a single bacterium. Horizontal gene transfer has to play a key role in combination with the mutagenesis of the existing enzymes to facilitate rapid evolution of new pathways. Evolution of diverse pathways for the degradation of different nitroaromatics thus stands testament to this.

A good example of this is the evolution of chloronitrobenzene dioxygenase system from a chloronitrobenzene degrading strain *Pseudomonas stutzeri* [127]. It has several insertion sequences embedded between the gene clusters, which proves such involvement in its evolution. In its dioxygenase enzyme system, reductase and ferredoxin seems to have come from different origins because its reductase and ferredoxin share maximum identity with anthranilate dioxygenase, which is a type IV oxygenase, whereas its oxygenase subunits show a maximum identity with nag (naphthalene degradation gene) and nitroarene dioxygenase, which falls under type III oxygenase systems. This enzyme system thus best illustrates the evolution of catabolic genes best because its upper pathway enzymes seem to have originated from a nitroarene degradation pathway and its lower pathway genes have evolved from some chloroaromatic compound degradation pathway. The genes responsible for these pathways are present in a patchwork like assembly in *P. stutzeri* [127]. The presence of insertion elements in the gene cluster confirms its role in the formation of a modular assembly and its role in evolution of the gene cluster.

**3. Evolution of genes for nitroaromatic degradation**

evolution of its degradation system at a genetic level.

the new environment by bacteria [123, 121].

*3.1.2. Genetic rearrangement within a cell*

catabolic gene transfer processes [126].

omatic compounds in different bacteria.

different nitroaromatics thus stands testament to this.

*3.1.3. Horizontal gene transfer*

**3.1. Modes of gene evolution**

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*3.1.1. Mutational drift*

Nitroaromatics are recent compounds present in the environment and bacterial strains that adapted themselves for the removal of these compounds. This was possible only through

Substrate profile of an existing enzyme may be altered by point mutations in its corresponding genes [124]. The reasons for changes in primary DNA sequences are slippage of DNA poly‐ merase while replications, erroneous DNA repair, and gene conversion [122]. However, results of these changes are relatively smaller. These alone cannot be accountable for adaptation to

The rearrangement of genes for the development of new pathway may take place by the help of cells own recombination system. Gene segments can be exchanged between two positions flanked by homologous sequences, insertion elements, transposons, and even sequence

Horizontal gene transfer is reported as the main source of evolution of pathways in bacteria [123, 124]. Sequencing results of genomes from different bacterial strains have revealed the presence of acquired genes in mosaic like fashion throughout bacterial genomes. Their presence varies from almost negligible (*Rickettsia prowazekii* and *Mycobacterium genitalium*) to about 17% (in *Synechocystis* strain) [123]. Plasmids also play a major role in carrying catabolic genes during horizontal gene transfer [125]. Transposons are also known to facilitate the

**3.2. Nitroaromatic degradation pathway as a role model for study the gene evolution**

A role model to study evolution of microbial pathways is to study the degradation of nitroar‐

Nitroaromatic compounds are relatively new to the environment, but bacterial systems have already evolved the ability to metabolize them. This cannot be possible only by spontaneous, independent evolution of several new enzymes in a single bacterium. Horizontal gene transfer has to play a key role in combination with the mutagenesis of the existing enzymes to facilitate rapid evolution of new pathways. Evolution of diverse pathways for the degradation of

identities of four base pairs are sufficient to facilitate this process [122].

Another example is the origin of 2,4-DNT degradation pathway in *B. cepacia* R34 where enzymes for the degradation pathway have originated from at least three different sources [128]. The first enzyme in the degradation pathway (DNTDO), which removes the first nitrite from 2,4-DNT, seems to have originated from naphthalene degradation pathway like in *Ralstonia* sp. strain U2. [129]. However, 4-methyl-5-nitrocatechol mono-oxygenase, which facilitates the removal of the second nitrite, appears to be derived from a pathway for degra‐ dation for chloroaromatic compounds [128]. The last enzyme of the pathway could have originated from a gene cluster for amino acid degradation. This is known as progressive compaction of the genes. However, the presence of ORFs without any known role in 2,4-DNT degradation and truncated transposons in the regions suggests that compaction is probably in an intermediate stage in the evolution of such an optimal system with the genetic materials from different bacterial origin.

A good example of similar type of evolution is reported in diphenylamine degrader *Burkholde‐ ria* sp. strain JS665 [130]. In this pathway, diphenylamine is converted to catechol and aniline. An analysis of this sequence of diphenylamine degrading enzyme system showed that it has evolved by recruiting two pathway enzymes, one of which is from dioxygenase and the other is from nitroaniline degradation pathway enzymes in a much more recent evolution event [127].

Another example of evolution of genes for nitrotoluenes degradation is the evolution of 3NTDO in *Diaphorobacter* sp. strain DS2. Five complete ORFs were identified by probable ORF finding program and by homology to polypeptide sequences from several previously reported multicomponent dioxygenase systems. The predicted translation products from ORFs were designated as a putative regulatory protein, a ferredoxin reductase subunit, a ferredoxin subunit, an oxygenase large, and a small subunit based on their homology. A 571-bp DNA stretch was present in between reductase and ferredoxin subunits. In its gene structure, the regulatory protein is divergently transcribed from the other four ORFs. The organization of gene cluster and its similarity with other known dioxygenases is shown in Figure 15.

It has been suggested in several reports that *Ralstonia* sp. strain U2 [129] is the progenitor of all the nitroarene dioxygenases because it has the entire functional gene in its gene assembly. This seems true for the 3NTDO as well because the sequence present in between the reductase and the ferredoxin is truncated into parts of two functional ORFs present in the *Ralstonia* sp. strain U2. The regulatory protein sequence of 3NTDO (MntR) differs only at three amino acid positions from NagR of *Ralstonia* sp. strain U2 out of which two are uniquely present in strain DS2 only.

**Figure 15.** Comparison of gene arrangements in various multicomponent dioxygenases.

The different components (reductase, ferredoxin, and oxygenase) of 3NTDO show different levels of sequence identity with components from similar multicomponent enzyme systems of different organisms. Its reductase subunit (MntAa) shares a high amino acid sequence identity with those of DNTDO from *B. cepacia* [128] and NDO of *Ralstonia* sp. strain U2 (99%) [129], but its ferredoxin subunit (MntAb) is 100% identical to the ferredoxin of dinitrotoluene dioxygenase from *Burkholderia* sp. strain DNT and *Burkholderia* sp. strain R34. Its large oxygenase subunit (MntAc) showed more identity with chloronitrobenzene dioxygenase (CnbAc, 96%) from *P. stutzeri* ZWLR2-1, whereas the small oxygenase subunit (MntAd) showed more identity with PahAd of *Comamonas testosteroni* (96%) and NTDO from *Acidovor‐ ax* sp. strain JS42. It is known that oxygenase large subunit controls substrate specificity. If we compare important active site residues in oxygenase large subunit (MntAc) of strain DS2 with well-characterized oxygenase systems, it contains amino acid combinations of other systems, in which the sequence retains His293, which is present in NDO system of *P. putida* 9816-4, *Ralstonia* U2, *C. testosteroni* H, and *Burkholderia* sp. C3. Position 350 is occupied by Valine, which is reported in the DNTDO of *Burkholderia* sp. strain R34. Thus, the above facts seem to indicate that 3NTDO gene in *Diaphorobacter* sp. strain DS2 came through a horizontal gene transfer from ancestors common to strains like *Ralstonia* U2 or *Burkholderia* sp. strain R34, and then its catalytic subunit has been diversely evolved to degrade other nitroaromatic compounds.

This seems true for the 3NTDO as well because the sequence present in between the reductase and the ferredoxin is truncated into parts of two functional ORFs present in the *Ralstonia* sp. strain U2. The regulatory protein sequence of 3NTDO (MntR) differs only at three amino acid positions from NagR of *Ralstonia* sp. strain U2 out of which two are uniquely present in strain

**Figure 15.** Comparison of gene arrangements in various multicomponent dioxygenases.

The different components (reductase, ferredoxin, and oxygenase) of 3NTDO show different levels of sequence identity with components from similar multicomponent enzyme systems of different organisms. Its reductase subunit (MntAa) shares a high amino acid sequence identity with those of DNTDO from *B. cepacia* [128] and NDO of *Ralstonia* sp. strain U2 (99%) [129], but its ferredoxin subunit (MntAb) is 100% identical to the ferredoxin of dinitrotoluene dioxygenase from *Burkholderia* sp. strain DNT and *Burkholderia* sp. strain R34. Its large oxygenase subunit (MntAc) showed more identity with chloronitrobenzene dioxygenase (CnbAc, 96%) from *P. stutzeri* ZWLR2-1, whereas the small oxygenase subunit (MntAd) showed more identity with PahAd of *Comamonas testosteroni* (96%) and NTDO from *Acidovor‐ ax* sp. strain JS42. It is known that oxygenase large subunit controls substrate specificity. If we compare important active site residues in oxygenase large subunit (MntAc) of strain DS2 with well-characterized oxygenase systems, it contains amino acid combinations of other systems, in which the sequence retains His293, which is present in NDO system of *P. putida* 9816-4, *Ralstonia* U2, *C. testosteroni* H, and *Burkholderia* sp. C3. Position 350 is occupied by Valine, which

DS2 only.

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The mechanism by which enzymes for the degradation of synthetic compounds have evolved so rapidly still cannot be explained only by horizontal gene transfer and mutations. It can be explained in part by the term promiscuity. Promiscuity refers to the ability of a protein to perform dual functions using same active site [131, 132]. Protein evolution toward a new function based on promiscuity involves transition of an existing specialized enzyme to a generalized intermediate enzyme and then into a new specialized enzyme (Figure 16). A good example of this is transcriptional regulator found in nitroarene dioxygenases [133].

**Figure 16.** Evolution of new specialized enzymes from existing one. (Modified from Ju et al. [134])

The correct functioning of a pathway depends not only on having enzymes with appropriate catalytic activity but also on regulators, which control the expression of the catabolic genes in response to the compounds to be degraded. For example, ntdR, which controls 2NTDO expression in *Acidovorax* JS42, is supposed to have evolved from an ancestral nagR regulator of naphthalene degradation pathway present in *Ralstonia* U2. NtdR differs only by five amino acids with nagR. Ju et al. [134] showed how ntdR like regulator could be created from nagR by making mutations at each of the five positions separately and in combinations in a stepwise manner. They also showed that each mutation broadened the effectors range in a stepwise manner without losing the original activity. Both NagR and NtdR can activate transcription in the presence of salicylate, which is a natural inducer of naphthalene degradation genes in strain *Ralstonia* sp. U2, but ntdR could have gained a broader effector specificity to recognize several nitroaromatic compounds too [134]. Hence, the evolution of the regulatory system of the 2NTDO is in an intermediate stage because it can be induced in response to several nonmetabolizable compounds. Thus, the selection of ntdR variant with high specificity for 2NT with loss of specificity for salicylate would enable the identification of mutations that can lead to the specialized transcription factor from an intermediate stage. Another regulator was reported by Singh et al. [83], where the regulatory protein sequence of 3NTDO (MntR) differs only at three amino acid positions from NagR of *Ralstonia* sp. strain U2, out of which two are uniquely present in strain DS2 only.

It can be concluded that the gene evolution in these dioxygenase systems cannot be explained by considering only one mode of evolution. All the modes of evolution (like horizontal gene transfer, selective mutation, and promiscuity) are responsible for the evolution of a dioxyge‐ nase system [133, 135]. Further, the presence of truncated ORFs (which is not required for enzyme activity) reveals that gene evolution is in an intermediate stage of the so-called progressive compaction of the genes.
