Genetic and structural organization of the aminophenol catabolic operon and its implication for evolutionary process

The aminophenol (AP) catabolic operon in Pseudomonas putida HS12 mineralizing nitrobenzene was found to contain all the enzymes responsible for the conversion of AP to pyruvate and acetyl coenzyme A via extradiol meta cleavage of 2-aminophenol. The sequence and functional analyses of the corresponding genes of the operon revealed that the AP catabolic operon consists of one regulatory gene, nbzR, and the following nine structural genes, nbzJCaCbDGFEIH, which encode catabolic enzymes. The NbzR protein, which is divergently transcribed with respect to the structural genes, possesses a leucine zipper motif and a MarR homologous domain. It was also found that NbzR functions as a repressor for the AP catabolic operon through binding to the promoter region of the gene cluster in its dimeric form. A comparative study of the AP catabolic operon with other meta cleavage operons led us to suggest that the regulatory unit (nbzR) was derived from the MarR family and that the structural unit (nbzJCaCbDGFEIH) has evolved from the ancestral meta cleavage gene cluster. It is also proposed that these two functional units assembled through a modular type gene transfer and then have evolved divergently to acquire specialized substrate specificities (NbzCaCb and NbzD) and catalytic function (NbzE), resulting in the creation of the AP catabolic operon. The evolutionary process of the AP operon suggests how bacteria have efficiently acquired genetic diversity and expanded their metabolic capabilities by modular type gene transfer.     

The role of mobile genetic elements in bacterial adaptation to xenobiotic organic compounds

Retrospective studies clearly indicate that mobile genetic elements (MGEs) play a major role in the in situ spread and even de novo construction of catabolic pathways in bacteria, allowing bacterial communities to rapidly adapt to new xenobiotics. The construction of novel pathways seems to occur by an assembly process that involves horizontal gene transfer: different appropriate genes or gene modules that encode different parts of the novel pathway are recruited from phylogenetically related or distant hosts into one single host. Direct evidence for the importance of catabolic MGEs in bacterial adaptation to xenobiotics stems from observed correlations between catabolic gene transfer and accelerated biodegradation in several habitats and from studies that monitor catabolic MGEs in polluted sites.     

Enhanced degradation of phenoxyacetic acid in soil by horizontal transfer of the tfdA gene encoding a 2,4-dichlorophenoxyacetic acid dioxygenase

Few studies have investigated the possible impact of in situ gene transfer on the degradation of xenobiotic compounds in natural environments. In this work we showed that horizontal transfer of the tfdA gene, carried on plasmid pRO103, to phenol degrading recipient strains significantly increased the degradation rate of phenoxyacetic acid in sterile and non-sterile soil microcosms. The tfdA gene encodes a 2,4-dichlorophenoxyacetic acid/2-oxoglutarate dioxygenase and by complementation with the phenol degradation pathway an expanded catabolic substrate range, now including phenoxyacetic acid, is evolved. Presence of selective pressure had a positive effect on the emergence of transconjugants. However, even in the absence of phenoxyacetic acid transconjugant populations were detected and were kept at a constant level throughout the experimental period. The residuesphere (interface between decaying plant material and soil matrix) of dry leaves of barley was shown to be a hot-spot for gene transfer and presence of barley straw increased the conjugation frequencies in soil microcosms to the same extent as presence of organic nutrients. The results of this study indicate that dissemination of catabolic plasmids is a possible mechanism of genetic adaptation to degradation of xenobiotic compounds in natural environments, and that complementation of catabolic pathways possibly plays an important role in the evolution of new degradative capabilities. The application of horizontal gene transfer as a possible tool in bioremediation of contaminated sites is discussed.     

Sucrose utilisation in bacteria: genetic organisation and regulation

Sucrose is the most abundant disaccharide in the environment because of its origin in higher plant tissues, and many Eubacteria possess catalytic enzymes, such as the sucrose-6-phosphate hydrolases and sucrose phosphorylases, that enable them to metabolise this carbohydrate in a regulated manner. This review describes the range of gene architecture, uptake systems, catabolic activity and regulation of the sucrose-utilisation regulons that have been reported in the Eubacteria to date. Evidence is presented that, although there are many common features to these gene clusters and high conservation of the proteins involved, there has been a certain degree of gene shuffling. Phylogenetic analyses of these proteins supports the hypothesis that these clusters have been acquired through horizontal gene transfer via mobile elements and transposons, and this may have enabled the recipient bacteria to colonise sucrose-rich environmental niches.Electronic Supplementary Material Supplementary material is available for this article at .     

Dehalogenases: environmental defence mechanism and model of enzyme evolution

Studies on the fate of halogenated organic compounds in the environment from the 1950s onwards have led to the conclusion that the main method of degradation of these compounds is via microbial metabolism, and that this is mediated by enzymes which remove the halogen substituents. The enzymes are called dehalogenases. The study of these enzymes has yielded much useful information about the evolution of catabolic enzymes in general and represents an ideal tool for the investigation and illustration of many key concepts in enzymology including parallel evolution, convergent evolution, gene transfer, determination of reaction mechanisms and structure—activity relationships. This review summarises the key principles of microbial dehalogenation and illustrates the applications that these studies may have in teaching more generalised enzymological concepts.     

Evolution of arginine deiminase (ADI) pathway genes

We have analyzed the evolution of the three genes encoding structural enzymes of the arginine deiminase (ADI) pathway, arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) in a wide range of organisms, including Archaea, Bacteria, and Eukarya. This catabolic route was probably present in the last common ancestor to all the domains of life. The results obtained indicate that these genes have undergone a complex evolutionary history, including horizontal transfer events, duplications, and losses. Therefore, these genes are not adequate to infer organismal relationships at deep branching levels, but they provide an insight into how catabolic genes evolved and were assembled into metabolic pathways. Our results suggest that the three genes evolved independently and were later assembled into a single cluster with functional interdependence, thus, providing support for the gene recruitment hypothesis. Furthermore, the molecular phylogenetic analysis of OTC suggests a new classification of these genes into three subfamilies.     

The evolution of IncP catabolic plasmids

The recent adoption of whole plasmid genome sequencing as a routine analytical technique has provided the basis for cataloging the historical events through which plasmids are assembled from the available families of modular plasmid components. Horizontal gene transfer mediated by plasmids plays an important role in the adaptation of bacteria to the presence of specific metabolizable compounds, including man-made chemicals, in the surrounding environment. Bacterial plasmid genome sequence comparisons indicate that plasmids have complex genetic histories resulting from transposition, homologous recombination, and illegitimate recombinational events. Evidence from IncP plasmid genome sequences indicates that cryptic plasmid backbones acquire diverse catabolic pathways through gene capture and horizontal gene transfer.     

Cloning and Characterization of Benzoate Catabolic Genes in the Gram-Positive Polychlorinated Biphenyl Degrader Rhodococcus sp. Strain RHA1

Benzoate catabolism is thought to play a key role in aerobic bacterial degradation of biphenyl and polychlorinated biphenyls (PCBs). Benzoate catabolic genes were cloned from a PCB degrader, Rhodococcus sp. strain RHA1, by using PCR amplification and temporal temperature gradient electrophoresis separation. A nucleotide sequence determination revealed that the deduced amino acid sequences encoded by the RHA1 benzoate catabolic genes, benABCDK, exhibit 33 to 65% identity with those of Acinetobacter sp. strain ADP1. The gene organization of the RHA1 benABCDK genes differs from that of ADP1. The RHA1 benABCDK region was localized on the chromosome, in contrast to the biphenyl catabolic genes, which are located on linear plasmids. Escherichia coli cells containing RHA1 benABCD transformed benzoate to catechol via 2-hydro-1,2-dihydroxybenzoate. They transformed neither 2- nor 4-chlorobenzoates but did transform 3-chlorobenzoate. The RHA1 benA gene was inactivated by insertion of a thiostrepton resistance gene. The resultant mutant strain, RBD169, neither grew on benzoate nor transformed benzoate, and it did not transform 3-chlorobenzoate. It did, however, exhibit diminished growth on biphenyl and growth repression in the presence of a high concentration of biphenyl (13 mM). These results indicate that the cloned benABCD genes could play an essential role not only in benzoate catabolism but also in biphenyl catabolism in RHA1. Six rhodococcal benzoate degraders were found to have homologs of RHA1 benABC. In contrast, two rhodococcal strains that cannot transform benzoate were found not to have RHA1 benABC homologs, suggesting that many Rhodococcus strains contain benzoate catabolic genes similar to RHA1 benABC.     

Genomic and functional analysis of the IncP-9 naphthalene-catabolic plasmid NAH7 and its transposon Tn4655 suggests catabolic gene spread by a tyrosine recombinase

The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.     

Towards a systems biology approach to study type II/IV secretion systems

Many gram-negative bacteria produce thin protein filaments, named pili, which extend beyond the confines of the outer membrane. The importance of these pili is illustrated by the fact that highly complex, multi-protein pilus-assembly machines have evolved, not once, but several times. Their many functions include motility, adhesion, secretion, and DNA transfer, all of which can contribute to the virulence of bacterial pathogens or to the spread of virulence factors by horizontal gene transfer. The medical importance has stimulated extensive biochemical and genetic studies but the assembly and function of pili remains an enigma. It is clear that progress in this field requires a more holistic approach where the entire molecular apparatus that forms the pilus is studied as a system. In recent years systems biology approaches have started to complement classical studies of pili and their assembly. Moreover, continued progress in structural biology is building a picture of the components that make up the assembly machine. However, the complexity and multiple-membrane spanning nature of these secretion systems pose formidable technical challenges, and it will require a concerted effort before we can create comprehensive and predictive models of these remarkable molecular machines.     

Towards a systems biology approach to study type II/IV secretion systems

Many gram-negative bacteria produce thin protein filaments, named pili, which extend beyond the confines of the outer membrane. The importance of these pili is illustrated by the fact that highly complex, multi-protein pilus-assembly machines have evolved, not once, but several times. Their many functions include motility, adhesion, secretion, and DNA transfer, all of which can contribute to the virulence of bacterial pathogens or to the spread of virulence factors by horizontal gene transfer. The medical importance has stimulated extensive biochemical and genetic studies but the assembly and function of pili remains an enigma. It is clear that progress in this field requires a more holistic approach where the entire molecular apparatus that forms the pilus is studied as a system. In recent years systems biology approaches have started to complement classical studies of pili and their assembly. Moreover, continued progress in structural biology is building a picture of the components that make up the assembly machine. However, the complexity and multiple-membrane spanning nature of these secretion systems pose formidable technical challenges, and it will require a concerted effort before we can create comprehensive and predictive models of these remarkable molecular machines.     

Examination of four HLA class I pseudogenes. Common events in the evolution of HLA genes and pseudogenes.

The HLA class I gene family in lymphoblastoid cell line 721 has been studied in detail and a number of sequences in addition to the classical genes have been identified. The cloning, characterization, and nucleotide sequences of four sequences, all full length HLA class I pseudogenes, are described in this report. These pseudogenes, contained within 5.4-, 5.9-, 7.0-, and 9.2-kb HindIII fragments, each have the class I exon-intron structure as well as class I homology in their 5' and 3' flanking regions. However, all four sequences have one or more substitutions that perturb the coding region, leaving little doubt that they are in fact pseudogenes. Comparisons among these sequences and the HLA class I genes revealed that their homology with the class I genes is patchwork. Thus, although some regions have diverged, other contiguous intron-exon sequences are highly conserved. Comparisons in the 5' regions indicate that the pseudogene promoters more closely resemble the classical HLA promoters than the nonclassical promoters as none of the unique structural features found in the HLA-E, -F, or -G regulatory regions are present in any of the pseudogene promoters. Further comparisons revealed that at least two putative gene conversion events, similar to those hypothesized to have occurred in the evolution of some HLA genes, may have occurred in the evolution of some of the pseudogenes. These and other hypothetical events in the evolution of the class I gene family are discussed.     

Frequent,independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes

Even genetically distant prokaryotes can exchange genes between them, and these horizontal gene transfer events play a central role in adaptation and evolution. While this was long thought to be restricted to prokaryotes, certain eukaryotes have acquired genes of bacterial origin. However, gene acquisitions in eukaryotes are thought to be much less important in magnitude than in prokaryotes. Here, we describe the complex evolutionary history of a bacterial catabolic gene that has been transferred repeatedly from different bacterial phyla to stramenopiles and fungi. Indeed, phylogenomic analysis pointed to multiple acquisitions of the gene in these filamentous eukaryotes—as many as 15 different events for 65 microeukaryotes. Furthermore, once transferred, this gene acquired introns and was found expressed in mRNA databases for most recipients. Our results show that effective inter-domain transfers and subsequent adaptation of a prokaryotic gene in eukaryotic cells can happen at an unprecedented magnitude.     

Molecular mechanisms of genetic adaptation to xenobiotic compounds.

Microorganisms in the environment can often adapt to use xenobiotic chemicals as novel growth and energy substrates. Specialized enzyme systems and metabolic pathways for the degradation of man-made compounds such as chlorobiphenyls and chlorobenzenes have been found in microorganisms isolated from geographically separated areas of the world. The genetic characterization of an increasing number of aerobic pathways for degradation of (substituted) aromatic compounds in different bacteria has made it possible to compare the similarities in genetic organization and in sequence which exist between genes and proteins of these specialized catabolic routes and more common pathways. These data suggest that discrete modules containing clusters of genes have been combined in different ways in the various catabolic pathways. Sequence information further suggests divergence of catabolic genes coding for specialized enzymes in the degradation of xenobiotic chemicals. An important question will be to find whether these specialized enzymes evolved from more common isozymes only after the introduction of xenobiotic chemicals into the environment. Evidence is presented that a range of genetic mechanisms, such as gene transfer, mutational drift, and genetic recombination and transposition, can accelerate the evolution of catabolic pathways in bacteria. However, there is virtually no information concerning the rates at which these mechanisms are operating in bacteria living in nature and the response of such rates to the presence of potential (xenobiotic) substrates. Quantitative data on the genetic processes in the natural environment and on the effect of environmental parameters on the rate of evolution are needed.     

Evolution of mitochondrial-type cytochrome c domains and of the protein machinery for their assembly

Proteins containing mitochondrial-type cytochrome c domains, defined here as protein domains having the mitochondrial cytochrome c fold, are found in organisms from all domains of life, and constitute essential components in several different metabolic pathways. The number of cytochrome c domains present in a given organism as well as their functional roles can vary widely even for quite closely related organisms. In this work, we have analysed in detail the distribution of mitochondrial-type cytochrome c domains along the tree of life and attempted to define the evolutionary relationships among them. In parallel, we have similarly analysed also the occurrence and distribution of the different machineries for cytochrome c assembly. It is found that the first appearance of mitochondrial-type cytochrome c domains has likely happened in the bacterial world, together with the first apparatus for their assembly. Evolution of cytochrome c domains has been extensive, involving several gene duplication and gene transfer events. Of particular relevance are gene transfer events from Bacteria to Eukarya and Archaea. The transfer of genes encoding cytochrome c domains has generally co-occurred with transfer of the assembly machinery. This has occurred also in Eukarya, where however the latter machinery has been subsequently replaced by a new one. It is possible that of the three known enzymatic systems for cytochrome c assembly, system II (found, among others, in cyanobacteria and Gram-positive bacteria) is the most ancient. Archaea have inherited from Bacteria system I or, possibly, an evolutionary intermediate between system II and system I.