Production of dyestuffs from indole derivatives by naphthalene dioxygenase and toluene dioxygenase

AIMS: To isolate and characterize the phorate [O,O-diethyl-S-(ethylthio)methyl phosphoradiothioate] degrading bacteria from agricultural soil, and their assessment for multifarious biological activities of environmental and agronomic significance. METHODS AND RESULTS: Based on their morphological and biochemical characteristics, the selected isolates PS-1, PS-2 and PS-3 were presumptively identified as Rhizobium, Pseudomonas and Proteus species, respectively. The HPLC analysis of phorate in bioaugmented soil revealed its complete disappearance within 40 days. The degradation isotherms of the isolates PS-1, PS-2 and PS-3 suggested time-dependent disappearance of phorate following the first-order rate kinetics at the corresponding rate constants of 0.04, 0.05 and 0.04 d-1. Besides, the isolates concurrently exhibited substantial phosphate solubilization, indole acetic acid (IAA) and siderophore production, as well as limited biocontrol activity against fungal phytopathogens. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The data revealed the potential of isolates for collateral plant growth promotion, biocontrol and bioremediation. The selected strains may serve as an important bioresource for development of effective super-bioinoculants.     

Episodic sitewise positive selection on the signal recognition particle protein Ffh in Actinobacteria

In this work, we report a case of episodic sitewise positive selection acting on the highly conserved SRP protein Ffh in Actinobacteria. An elevated non-synonymous to synonymous mutation ratio (ω) was observed along the non-terminal branches of the species tree, which contained 11 Actinobacteria species, where positively selected residues were frequently observed within the signal sequence-binding domain. Together with the estimated lineage-specific ω ratios for several core components in the major protein translocation systems, our data suggest that this positive selection might be partially driven by the diversification of signal sequences.     

The role of active-site residues in naphthalene dioxygenase

The three-component naphthalene dioxygenase enzyme system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. A member of a large family of bacterial Rieske non-heme iron oxygenases, naphthalene dioxygenase is known to oxidize over 60 different aromatic compounds, and many of the products are enantiomerically pure. The crystal structure of the oxygenase component revealed the enzyme to be an α₃β₃ hexamer and identified the amino acids located near the active site. Site-directed mutagenesis studies have identified the residues involved in electron transfer and those responsible for controlling the regioselectivity and enantioselectivity of the enzyme. The results of these studies suggest that naphthalene dioxygenase can be engineered to catalyze a new and extended range of useful reactions.     

Involvement of naphthalene dioxygenase in indole-3-acetic acid biosynthesis by Pseudomonas putida

Two variants of plant growth-promoting strain Pseudomonas putida BS1380 harboring the naphthalene degradative plasmid pBS2 and the recombinant plasmid pNAU64 that contains the genes encoding for naphthalene dioxygenase were constructed by conjugation. The ability of this strain to produce phytohormone indole-3-acetic acid from different carbon sources was studied. Indole-3-acetic acid synthesis by these transconjugants was 15-30 times as much in contrast to a wild-type strain with glucose as the sole carbon source. No difference was observed in other carbon or nitrogen sources. It is suggested that naphthalene dioxygenase is involved in the conversion of indole-3-pyruvic acid to indole-3-acetic acid.     

Structural insight into the dioxygenation of nitroarene compounds: the crystal structure of nitrobenzene dioxygenase

Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring. However, the bacterium Comamonas sp. strain JS765 can grow with nitrobenzene as a sole source of carbon, nitrogen and energy. Biodegradation is initiated by the nitrobenzene dioxygenase (NBDO) system. We have determined the structure of NBDO, which has a hetero-hexameric structure similar to that of several other Rieske non-heme iron dioxygenases. The catalytic subunit contains a Rieske iron-sulfur center and an active-site mononuclear iron atom. The structures of complexes with substrates nitrobenzene and 3-nitrotoluene reveal the structural basis for its activity with nitroarenes. The substrate pocket contains an asparagine residue that forms a hydrogen bond to the nitro-group of the substrate, and orients the substrate in relation to the active-site mononuclear iron atom, positioning the molecule for oxidation at the nitro-substituted carbon.     

Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase

Abstract Naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4 and biphenyl dioxygenase from Beijerinckia sp. B8/36 oxidized the aromatic N-heterocycle carbazole to 3-hydroxycarbazole. Toluene dioxygenase from Pseudomonas putida F39/D did not oxidize carbazole. Transformations were carried out by mutant strains which oxidize naphthalene and biphenyl to cis -dihydrodiols, and with a recombinant E. coli strain expressing the structural genes of naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4. 3-Hydroxycarbazole is presumed to result from the dehydration of an unstable cis -dihydrodiol.     

Molecular evolution of animal antimicrobial peptides: widespread moderate positive selection

An increasing number of studies in both vertebrates and invertebrates show that the evolution of antimicrobial peptides is driven by positive selection. Because these diverse molecules show potential for therapeutic applications, they are currently the targets of much structural and functional research, providing extensive background data for evolutionary studies. In this paper, patterns of molecular evolution in antimicrobial peptide genes are reviewed. Evidence for positive selection on antimicrobial peptides includes an excess of nonsynonymous nucleotide substitutions, an excess of charge-changing amino acid substitutions, nonneutral patterns of allelic variation, and functional assays in vivo and in vitro that show improved antimicrobial effects for derived sequence variants. Positive selection on antimicrobial peptides may be as common as, but perhaps weaker than, selection on the best-known example of adaptively evolving immunity genes, the major histocompatibility complex. Thus, antimicrobial peptides present a useful and underutilized model for the study of adaptive molecular evolution.     

Episodic evolution in the stomach lysozymes of ruminants

Summary By sequencing lysozymesc from deer and pig stomachs and comparing them to the known amino acid sequences of other lysozymesc, it was possible to examine the rate of sequence change during and after the period in which this enzyme acquired a new function. Evolutionary tree analysis suggests that the rate went up while lysozyme was being recruited to function as a digestive enzyme in the stomach of early ruminants. Later, presumably after lysozyme was well adapted for functioning in the new environment, which contains acid, pepsin, and fermentation products, the rate of amino acid replacement became subnormal.     

Choice of microbial host for the naphthalene dioxygenase bioconversion

The use of whole cell biotransformations for single and multistep enzyme conversions is gaining widespread application. In this study the naphthalene dioxygenasenah A gene was transferred intoPseudomonas aeruginosa PAC 1R,Escherichia coli JM107 andPseudomonas putida PpG 277. The effect of ethanol on these genetically engineered Gram-negative bacteria was studied by measurement of enzyme activity, stability and cell integrity. Ethanol has been used in biotransformations as a co-substrate carbon source for co-factor recycling and as a co-solvent increasing dissolved substrate and product levels. Ethanol increased the dissolved substrate (naphthalene) concentration slightly and dissolved product ((+)-cis-(1R, 2S)-dihydroxy-1,2-dihydronaphthalene) by approximately 30% at 4% (w/v) ethanol. BothP. aeruginosa PAC 1R andP. putida PpG 277 showed decreased activity with increasing ethanol concentration whilstE. coli enzyme activity increased with increasing ethanol concentration being comparable to that when glucose was used as a carbon source. This project highlighted the many factors involved in the selection of microbial hosts for whole cell biotransformation processes.     

Positive natural selection has driven the evolution of the Hsp70s in Diguetia spiders

Hsp70s are a ubiquitous family of highly conserved proteins. Hsp70s are chaperones and have important roles in both protein folding and thermotolerance. It has been widely assumed that Hsp70 sequence evolution is governed by the strong functional constraints imposed by its crucial cellular functions. In this study of cytosolic heat-inducible Hsp70s from three spider families, we have found clear evidence of positive natural selection altering Hsp70s in desert-dwelling and heat-loving Diguetidae spiders. These spiders are a small family restricted to deserts. They display heat-tolerant behaviours not seen in their closest relatives, the Pholcidae and Plectreuridae.     

Sexual selection and the adaptive evolution of mammalian ejaculate proteins

An elevated rate of substitution characterizes the molecular evolution of reproductive proteins from a wide range of taxa. Although the selective pressures explaining this rapid evolution are yet to be resolved, recent evidence implicates sexual selection as a potentially important explanatory factor. To investigate this hypothesis, we sought evidence of a high rate of adaptive gene evolution linked to postcopulatory sexual selection in muroid rodents, a model vertebrate group displaying a broad range of mating systems. Specifically, we sequenced 7 genes from diverse rodents that are expressed in the testes, prostate, or seminal vesicles, products of which have the potential to act in sperm competition. We inferred positive Darwinian selection in these genes by estimation of the ratio of nonsynonymous (d(N), amino acid changing) to synonymous (d(S), amino acid retaining) substitution rates (omega = d(N)/d(S)). Next, we tested whether variation in this ratio among lineages could be attributed to interspecific variation in mating systems, as inferred from the variation in these rodents' relative testis sizes (RTS). Four of the 7 genes examined (Prm1, Sva, Acrv1, and Svs2, but not Svp2, Msmb, or Spink3) exhibit unambiguous evidence of positive selection. One of these, the seminal vesicle-derived protein Svs2, also shows some evidence for a concentration of positive selection in those lineages in which sperm competition is common. However, this was not a general trend among all the rodent genes we examined. Using the same methods, we then reanalyzed previously published data on 2 primate genes, SEMG1 and SEMG2. Although SEMG2 also shows evidence of positive selection concentrated in lineages subject to high levels of sperm competition, no such trend was found for SEMG1. Overall, despite a high rate of positive selection being a feature of many ejaculate proteins, these results indicate that the action of sexual selection potentially responsible for elevated evolutionary rates may be difficult to detect on a gene-by-gene basis. Although the extreme diversity of reproductive phenotypes exhibited in nature attests to the power of sexual selection, the extent to which this force predominates in driving the rapid molecular evolution of reproductive genes therefore remains to be determined.     

Molecular population genetics and the search for adaptive evolution in plants

The first papers on plant molecular population genetics were published approximately 10 years ago. Since that time, well over 50 additional studies of plant nucleotide polymorphism have been published, and many of these studies focused on detecting the signature of balancing or positive selection at a locus. In this review, we discuss some of the theoretical and statistical issues surrounding the detection of selection, with focus on plant populations, and we also summarize the empirical plant molecular population genetics literature. At face value, the literature suggests that a history of balancing or positive selection in plant genes is rampant. In two well-studied taxa (maize and Arabidopsis) over 20% of studied genes have been interpreted as containing the signature of selection. We argue that this is probably an overstatement of the prevalence of natural selection in plant genomes, for two reasons. First, demographic effects are difficult to incorporate and have generally not been well integrated into the plant population genetics literature. Second, the genes studied to date are not a random sample, so selected genes may be overrepresented. The next generation of studies in plant molecular population genetics requires additional sampling of local populations, explicit comparisons among loci, and improved theoretical methods to control for demography. Eventually, candidate loci should be confirmed by explicit consideration of phenotypic effects.     

Major role of positive selection in the evolution of conservative segments of Drosophila proteins

Slow evolution of conservative segments of coding and non-coding DNA is caused by the action of negative selection, which removes new mutations. However, the mode of selection that affects the few substitutions that do occur within such segments remains unclear. Here, we show that the fraction of allele replacements that were driven by positive selection, and the strength of this selection, is the highest within the conservative segments of Drosophila protein-coding genes. The McDonald-Kreitman test, applied to the data on variation in Drosophila melanogaster and in Drosophila simulans, indicates that within the most conservative protein segments, approximately 72 per cent (approx. 80%) of allele replacements were driven by positive selection, as opposed to only approximately 44 per cent (approx. 53%) at rapidly evolving segments. Data on multiple non-synonymous substitutions at a codon lead to the same conclusion and additionally indicate that positive selection driving allele replacements at conservative sites is the strongest, as it accelerates evolution by a factor of approximately 40, as opposed to a factor of approximately 5 at rapidly evolving sites. Thus, random drift plays only a minor role in the evolution of conservative DNA segments, and those relatively rare allele replacements that occur within such segments are mostly driven by substantial positive selection.     

Frequent false detection of positive selection by the likelihood method with branch-site models

Positive Darwinian selection promotes fixations of advantageous mutations during gene evolution and is probably responsible for most adaptations. Detecting positive selection at the DNA sequence level is of substantial interest because such information provides significant insights into possible functional alterations during gene evolution as well as important nucleotide substitutions involved in adaptation. Efficient detection of positive selection, however, has been difficult because selection often operates on only a few sites in a short period of evolutionary time. A likelihood-based method with branch-site models was recently introduced to overcome such difficulties. Here I examine the accuracy of the method using computer simulation. I find that the method detects positive selection in 20%-70% of cases when the DNA sequences are generated by computer simulation under no positive selection. Although the frequency of such false detection varies depending on, among other things, the tree topology, branch length, and selection scheme, the branch-site likelihood method generally gives misleading results. Thus, detection of positive selection by this method alone is unreliable. This unreliability may have resulted from its over-sensitivity to violations of assumptions made in the method, such as certain distributions of selective strength among sites and equal transition/transversion ratios for synonymous and nonsynonymous substitutions.     

Evidence for positive selection on the floral scent gene isoeugenol-O-methyltransferase

Isoeugenol-O-methyltransferase (IEMT) is an enzyme involved in the production of the floral volatile compounds methyl eugenol and methyl isoeugenol in Clarkia breweri (Onagraceae). IEMT likely evolved by gene duplication from caffeic acid-O-methyltransferase followed by amino acid divergence, leading to the acquisition of its novel function. To investigate the selective context under which IEMT evolved, maximum likelihood methods that estimate variable d(N)/d(S) ratios among lineages, among sites, and among a combination of both lineages and sites were utilized. Statistically significant support was obtained for a hypothesis of positive selection driving the evolution of IEMT since its origin. Subsequent Bayesian analyses identified several sites in IEMT that have experienced positive selection. Most of these positions are in the active site of IEMT and have been shown by site-directed mutagenesis to have large effects on substrate specificity. Although the selective agent is unknown, the adaptive evolution of this gene may have resulted in increased effectiveness of pollinator attraction or herbivore repellence.     

Rapid evolution by positive Darwinian selection in the extracellular domain of the abundant lymphocyte protein CD45 in primates

CD45, encoded by PTPRC in humans, is the most abundantly expressed protein on the surface of many lymphocytes. We investigated whether the extracellular region of CD45 was under positive selection in Old World primates, and whether there was differential selection across this region, particularly on exons that were involved in alternative splicing and those that were not alternatively spliced. The results show extraordinarily strong and consistent positive Darwinian selection on the extracellular part of CD45 throughout the evolution of Old World monkeys, apes and humans. Positive selection is concentrated in exons 9 and 14, which code for the previously neglected linker and fibronectin III domains. These exons have a high rate of evolution at nonsynonymous sites that is roughly twice as high as that of the intronic rate in this gene. In contrast, alternatively spliced exons 4-6, which code for the variable domains, are under weaker positive selection and are evolving more slowly than the intronic rate. These data provide a striking example of positive selection in a well-known gene that should provide an impetus for further functional studies to elucidate its species-specific function.     

Multiple mutations at the active site of naphthalene dioxygenase affect regioselectivity and enantioselectivity

The importance of five amino acids at the active site of the multicomponent naphthalene dioxygenase (NDO) system was determined by generating site-directed mutations in various combinations. The substrate specificities of the mutant enzymes were tested with the substrates indole, indoline, 2-nitrotoluene (2NT), naphthalene, biphenyl, and phenanthrene. Transformation of these substrates measured the ability of the mutant enzymes to catalyze dioxygenation, monooxygenation, and desaturation reactions. In addition, the position of oxidation and the enantiomeric composition of products were characterized. All enzymes with up to three amino acid substitutions were able to catalyze dioxygenation reactions. A subset of these enzymes could also catalyze the monooxygenation of 2NT and desaturation of indoline. Single amino acid substitutions at positions 352 and 206 had the most profound effects on product formation. Of the single mutations made, only changes at position 352 affected the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene, but in the presence of the F352I mutation, changes at positions 206 and 295 also affected enantioselectivity. Major shifts in regioselectivity with biphenyl and phenanthrene resulted with several of the singly, doubly, and triply mutated enzymes. A new product not formed by the wild-type enzyme, phenanthrene cis-9,10-dihydrodiol, was formed as a major product from phenanthrene by enzymes with two (A206I/F352I) or three amino acid substitutions (A206I/F352I/H295I). The results indicate that a variety of amino acid substitutions are tolerated at the active site of NDO. Journal of Industrial Microbiology & Biotechnology (2001) 27, 94–103. Received 25 September 2000/ Accepted in revised form 29 June 2001     

Compound tests for the detection of hitchhiking under positive selection

Many statistical tests have been developed for detecting positive selection. Most of these tests draw conclusions based on significant deviations from the patterns of polymorphism predicted by the neutral model. However, many non-equilibrium forces may cause similar deviations, and thus the tests usually have low statistical specificity to positive selection. The main challenge is hence to construct test statistics that are reasonably powerful in detecting positive selection, but are relatively insensitive to other forces. Recently, Zeng et al. (2006) proposed a new test, DH, which is a compound of Tajima's D and Fay and Wu's H, and showed that DH has reasonably high statistical specificity to positive selection. In this report, we expand the idea of a compound test by combining Fay and Wu's H or DH with the Ewens-Watterson (EW) test. We refer to these 2 new tests as HEW and DHEW, respectively. Compared to the DH test, HEW and DHEW are more robust against the presence of recombination, and are also more powerful in detecting positive selection. Furthermore, the DHEW test, similar to DH, is also relatively insensitive to background selection and demography. The HEW test, on the other hand, tends to be somewhat less conservative than DH and DHEW in some cases.     

Evidence for positive selection on Drosophila melanogaster seminal fluid protease homologs

Proteins present in the seminal fluid of Drosophila melanogaster (accessory gland proteins Acps) contribute to female postmating behavioral changes, sperm storage, sperm competition, and immunity. Consequently, male-female coevolution and host-pathogen interactions are thought to underlie the rapid, adaptive evolution that characterizes several Acp-encoding genes. We propose that seminal fluid proteases are likely targets of selection due to their demonstrated or potential roles in between-sex interactions and immune processes. We use within- and between-species sequence data for 5 predicted protease-encoding Acp loci to test this hypothesis. Our polymorphism-based analyses find evidence for positive selection at 2 genes, both of which encode predicted serine protease homologs. One of these genes, CG6069, also shows evidence for consistent selection on a subset of codons over a deeper evolutionary time scale. The second gene, CG9997, was previously shown to be essential for normal sperm usage, suggesting that sexual selection may underlie its history of adaptation.