Phn Genes Research Articles

Bacterial isolates from Argentine Pampas and their ability to degrade glyphosate

Glyphosate is a synthetic phosphonate compound characterized by a carbon‑phosphorus bond. Glyphosate based herbicides (GBH) are widely distributed in most of the economically productive lands in which crop production is mainly based on glyphosate-resistant genetically modified plants. Naturally, glyphosate is remediated by soil microorganisms, which accelerate its degradation. Technology based on microorganisms is considered highly efficient, low-cost and eco-friendly to remediate contaminated environments, denoting the importance of characterizing new bacterial strains able to degrade glyphosate to perform its bioremediation. In this work, 13 different bacterial strains able to grow in GBH as only phosphorous source were isolated from different environmental samples from the Argentine vastly productive glyphosate-resistant soybean crop area. These strains were identified and they belong to the genera Acinetobacter, Achromobacter, Agrobacterium, Ochrobactrum, Pantoea and Pseudomonas. Their ability to grow and consume GBH, glyphosate or the aminomethylphosphonic acid (AMPA), another phosphonate derived from glyphosate degradation, was evaluated. The best degradation performance was observed for bacteria from the genera Achromobacter, Agrobacterium and Ochrobactrum. The genome of the highly efficient GBH degrader Agrobacterium tumefaciens CHLDO was sequenced revealing the presence of a phn cluster, responsible for phosphonate metabolization. Expression analysis of A. tumefaciens CHLDO phn genes in the presence of 1.5 mM GBH compared to inorganic phosphorous showed that most of them are highly expressed during growth in the presence of the herbicide, suggesting a strong participation of phn cluster in GBH degradation. The importance of discovering new bacterial strains and the value of deciphering molecular determinants of GBH degradation give promising tools for bioremediation techniques to be used in glyphosate-contaminated environments is discussed.

Bacterial Community Composition and Genes for Herbicide Degradation in a Stormwater Wetland Collecting Herbicide Runoff

Stormwater wetlands collect and attenuate runoff-related herbicides, limiting their transport into aquatic ecosystems. Knowledge on wetland bacterial communities with respect to herbicide dissipation is scarce. Previous studies showed that hydrological and hydrochemical conditions, including pesticide removal capacity, may change from spring to summer in stormwater wetlands. We hypothesized that these changes alter bacterial communities, which, in turn, influence pesticide degradation capacities in stormwater wetland. Here, we report on bacterial community changes in a stormwater wetland exposed to pesticide runoff, and the occurrence of trz, atz, puh, and phn genes potentially involved in the biodegradation of simazine, diuron, and glyphosate. Based on T-RFLP analysis of amplified 16S rRNA genes, a response of bacterial communities to pesticide exposure was not detected. Changes in stormwater wetland bacterial community mainly followed seasonal variations in the wetland. Hydrological and hydrochemical fluctuations and vegetation development in the wetland presumably contributed to prevent detection of effects of pesticide exposure on overall bacterial community. End point PCR assays for trz, atz, phn, and puh genes associated with herbicide degradation were positive for several environmental samples, which suggest that microbial degradation contributes to pesticide dissipation. However, a correlation of corresponding genes with herbicide concentrations could not be detected. Overall, this study represents a first step to identify changes in bacterial community associated with the presence of pesticides and their degradation in stormwater wetland.

In silico analysis of bacterial arsenic islands reveals remarkable synteny and functional relatedness between arsenate and phosphate

In order to construct a more universal model for understanding the genetic requirements for bacterial AsIII oxidation, an in silico examination of the available sequences in the GenBank was assessed and revealed 21 conserved 5–71 kb arsenic islands within phylogenetically diverse bacterial genomes. The arsenic islands included the AsIII oxidase structural genes aioBA, ars operons (e.g., arsRCB) which code for arsenic resistance, and pho, pst, and phn genes known to be part of the classical phosphate stress response and that encode functions associated with regulating and acquiring organic and inorganic phosphorus. The regulatory genes aioXSR were also an island component, but only in Proteobacteria and orientated differently depending on whether they were in α-Proteobacteria or β-/γ-Proteobacteria. Curiously though, while these regulatory genes have been shown to be essential to AsIII oxidation in the Proteobacteria, they are absent in most other organisms examined, inferring different regulatory mechanism(s) yet to be discovered. Phylogenetic analysis of the aio, ars, pst, and phn genes revealed evidence of both vertical inheritance and horizontal gene transfer (HGT). It is therefore likely the arsenic islands did not evolve as a whole unit but formed independently by acquisition of functionally related genes and operons in respective strains. Considering gene synteny and structural analogies between arsenate and phosphate, we presumed that these genes function together in helping these microbes to be able to use even low concentrations of phosphorus needed for vital functions under high concentrations of arsenic, and defined these sequences as the arsenic islands.

The phn Island: A New Genomic Island Encoding Catabolism of Polynuclear Aromatic Hydrocarbons

Bacteria are key in the biodegradation of polycyclic aromatic hydrocarbons (PAH), which are widespread environmental pollutants. At least six genotypes of PAH degraders are distinguishable via phylogenies of the ring-hydroxylating dioxygenase (RHD) that initiates bacterial PAH metabolism. A given RHD genotype can be possessed by a variety of bacterial genera, suggesting horizontal gene transfer (HGT) is an important process for dissemination of PAH-degrading genes. But, mechanisms of HGT for most RHD genotypes are unknown. Here, we report in silico and functional analyses of the phenanthrene-degrading bacterium Delftia sp. Cs1-4, a representative of the phnAFK2 RHD group. The phnAFK2 genotype predominates PAH degrader communities in some soils and sediments, but, until now, their genomic biology has not been explored. In the present study, genes for the entire phenanthrene catabolic pathway were discovered on a novel ca. 232 kb genomic island (GEI), now termed the phn island. This GEI had characteristics of an integrative and conjugative element with a mobilization/stabilization system similar to that of SXT/R391-type GEI. But, it could not be grouped with any known GEI, and was the first member of a new GEI class. The island also carried genes predicted to encode: synthesis of quorum sensing signal molecules, fatty acid/polyhydroxyalkanoate biosynthesis, a type IV secretory system, a PRTRC system, DNA mobilization functions and >50 hypothetical proteins. The 50% G + C content of the phn gene cluster differed significantly from the 66.7% G + C level of the island as a whole and the strain Cs1-4 chromosome, indicating a divergent phylogenetic origin for the phn genes. Collectively, these studies added new insights into the genetic elements affecting the PAH biodegradation capacity of microbial communities specifically, and the potential vehicles of HGT in general.

Phosphorus-related gene content is similar in Prochlorococcus populations from the North Pacific and North Atlantic Oceans

Coleman and Chisholm (1) analyzed shotgun metagenomic data and concluded that populations of marine Prochlorococcus were indistinguishable between the North Atlantic and the North Pacific Oceans except in their phosphorus (P) utilization genes. They attributed these findings to recent horizontal gene transfer (HGT) of P genes in the genome of the Atlantic Prochlorococcus, favored by the stronger P-limitation in the Atlantic vs. the Pacific Ocean. We analyzed the datasets in their article (1) and found that the most abundant surface Prochlorococcus ecotype is indistinguishable between the two sampling sites [Hawaii Ocean Time Series (HOT) for Pacific and Bermuda Atlantic Time Series (BATS) for Atlantic] in most, if not all, of the P-utilization genes identified previously to be more abundant at BATS (1). In particular, the two corresponding populations encode the phoBR genes (phosphate two-component response regulators), whereas they both lack the phn operon (phosphonate utilization) (Table 1). phn genes become relatively more abundant with higher depth at BATS, which accounts for the results reported previously (1), but this is likely associated with non-Prochlorococcus populations. For instance, the reads of the BATS-50m small-insert library encoding Prochlorococcus-like phn genes have sister reads matching more frequently non-Prochlorococcus than Prochlorococcus genomes. Similarly, the higher abundance of phoBR in deeper (but not surface) waters at BATS vs. HOT is associated with non-Prochlorococcus taxa (Table 1) and the presence of a mixture of Prochlorococcus ecotypes, which are not all shared between BATS and HOT (Fig. 1). Only phoA [putative alkaline phosphatase (2)] clearly shows higher abundance at BATS-20m vs. HOT-25m; however, this is inconsistent with the results for the remaining pho genes, all cells encode an alternative dedA-type alkaline phosphatase, and many phoA-encoding reads likely originate from non-Prochlorococcus organisms, including viruses (e.g., note that phoA is twice as abundant at BATS-50m vs. BATS-100m, although total Prochlorococcus signal is similar).

Phn and Nag-like dioxygenases metabolize polycyclic aromatic hydrocarbons in Burkholderia sp. C3

Burkholderia sp. C3 can transform polycyclic aromatic hydrocarbons (PAHs), a class of ubiquitous pollutants, through multiple pathways, indicating existence of multiple dioxygenases (Seo et al., in Biodegradation 18:123-131, 2006a). Both phn and nag-like genes in C3 were cloned and identified with the DNA sequence alignment and the gene organization in the clusters. When cloned and expressed in Escherichia coli, either the alpha- and beta-subunits of dioxygenase of the phn genes or the ferredoxin-, alpha- and beta-subunits of the nag-like genes transformed naphthalene, phenanthrene and dibenzothiophene but at different rates. The E. coli transformant containing the phn genes transformed phenanthrene faster than that containing the nag-like genes, which was consistent with higher transcription of the phnAc gene than the nagAc-like gene in C3 in response to phenanthrene. 1-Hydroxy-2-naphthanoic acid (1H2NA) and 2-hydroxy-1-naphthanoic acid (2H1NA) (3,4- and 1,2-dioxygenation metabolites of phenanthrene, respectively) were detected in the culture medium of the phn genes transformed E. coli. The concentration of 1H2NA was 262-fold higher than 2H1NA in the medium of the phn genes transformed E. coli. The results suggested that the phn genes play a major role in 1,2-/3,4-dioxygenation and 3,4-dioxygenation dominates. Twenty-eight PAH degradation-associated enzymes including those encoded by the nag-like and phn genes in phenanthrene-grown C3 cells were identified via alignment of amino acid sequences of the detected polypeptides with those in protein databases. The polypeptides were determined with nano liquid chromatography-ion trap mass spectrometry after tryptic in-gel digestion of the enzymes on 1D SDS-PAGE.

Seasonal Expression of the Picocyanobacterial Phosphonate Transporter Gene phnD in the Sargasso Sea

In phosphorus-limited marine environments, picocyanobacteria (Synechococcus and Prochlorococcus spp.) can hydrolyze naturally occurring phosphonates as a P source. Utilization of 2-aminoethylphosphonate (2-AEP) is dependent on expression of the phn genes, encoding functions required for uptake, and C–P bond cleavage. Prior work has indicated that expression of picocyanobacterial phnD, encoding the phosphonate binding protein of the phosphonate ABC transporter, is a proxy for the assimilation of phosphonates in natural assemblages of Synechococcus spp. and Prochlorococcus spp (Ilikchyan et al., 2009). In this study, we expand this work to assess seasonal phnD expression in the Sargasso Sea. By RT-PCR, our data confirm that phnD expression is constitutive for the Prochlorococcus spp. detected, but in Synechococcus spp. phnD transcription follows patterns of phosphorus availability in the mixed layer. Specifically, our data suggest that phnD is repressed in the spring when P is bioavailable following deep winter mixing. In the fall, phnD expression follows a depth-dependent pattern reflecting depleted P at the surface following summertime drawdown, and elevated P at depth.

Detection and expression of the phosphonate transporter gene phnD in marine and freshwater picocyanobacteria

We describe a PCR-based assay designed to detect expression of the phosphonate assimilation gene phnD from picocyanobacteria. The phnD gene encodes the phosphonate binding protein of the ABC-type phosphonate transporter, present in many of the picocyanobacterial genome sequences. Detection of phnD expression can indicate a capacity of picoplankton to utilize phosphonates, a refractory form of phosphorus that can represent 25% of the high-molecular-weight dissolved organic phosphorus pool in marine systems. Primer sets were designed to specifically amplify phnD sequences from marine and freshwater Synechococcus spp., Prochlorococcus spp. and environmental samples from the ocean and Laurentian Great Lakes. Quantitative RT-PCR from cultured marine Synechococcus sp. strain WH8102 and freshwater Synechococcus sp. ARC-21 demonstrated induction of phnD expression in P-deficient media, suggesting that phn genes are regulated coordinately with genes under phoRB control. Last, RT-PCR of environmental RNA samples from the Sargasso Sea and Pacific Ocean detected phnD expression from the endemic picocyanobacterial population. Synechococcus spp. phnD expression yielded a depth-dependent pattern following gradients of P bioavailability. By contrast, the Prochlorococcus spp. primers revealed that in all samples tested, phnD expression was constitutive. The method described herein will allow future studies aimed at understanding the utilization of naturally occurring phoshonates in the ocean as well as monitoring the acquisition of synthetic phosphonate herbicides (e.g. glyphosate) by picocyanobacteria in freshwaters.

Two C-P lyase operons in Pseudomonas stutzeri and their roles in the oxidation of phosphonates, phosphite, and hypophosphite.

DNA sequencing and analysis of two distinct C-P lyase operons in Pseudomonas stutzeri WM88 were completed. The htxABCDEFGHIJKLMN operon encodes a hypophosphite-2-oxoglutarate dioxygenase (HtxA), whereas the predicted amino acid sequences of HtxB to HtxN are each homologous to the components of the Escherichia coli phn operon, which encodes C-P lyase, although homologs of E. coli phnF and phnO are absent. The genes in the htx operon are cotranscribed based on gene organization, and the presence of the intergenic sequences is verified by reverse transcription-PCR with total RNA. Deletion of the htx locus does not affect the ability of P. stutzeri to grow on phosphonates, indicating the presence of an additional C-P lyase pathway in this organism. To identify the genes comprising this pathway, a Deltahtx strain was mutagenized and one mutant lacking the ability to grow on methylphosphonate as the sole P source was isolated. A ca.-10.6-kbp region surrounding the transposon insertion site of this mutant was sequenced, revealing 13 open reading frames, designated phnCDEFGHIJKLMNP, which were homologous to the E. coli phn genes. Deletion of both the htx and phn operons of P. stutzeri abolishes all growth on methylphosphonate and aminoethylphosphonate. Both operons individually support growth on methylphosphonate; however, the phn operon supports growth on aminoethylphosphonate and phosphite, as well. The substrate ranges of both C-P lyases are limited, as growth on other phosphonate compounds, including glyphosate and phenylphosphonate, was not observed.

Molecular characterization and substrate preference of a polycyclic aromatic hydrocarbon dioxygenase from Cycloclasticus sp. strain A5.

Cycloclasticus sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes. The phnA1, phnA2, phnA3, and phnA4 genes, which encode the alpha and beta subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the alpha and beta subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved. Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the E. coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.

Rhizobium (Sinorhizobium) meliloti phn genes: characterization and identification of their protein products.

In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA from Rhizobium (Sinorhizobium) meliloti that contains homologues of the E. coli phnG, -H, -I, -J, and -K genes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the R. meliloti and E. coli phn genes and their predicted products gave new information about C-P lyase. The putative R. meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by R. meliloti grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.

Molecular biology of carbon-phosphorus bond cleavage. Cloning and sequencing of the phn (psiD) genes involved in alkylphosphonate uptake and C-P lyase activity in Escherichia coli B.

Whereas bacteria such as Escherichia coli have been known for some time to cleave carbon-phosphorus (C-P) bonds in unactivated alkylphosphonates, the enzymes responsible for C-P lyase activity have resisted detection or purification. Genes from E. coli B that support growth on alkylphosphonates as the sole phosphorus source have now been cloned (B. L. Wanner and J. A. Boline, unpublished data). Deletion analysis demonstrated that at least 13 kilobases of DNA information is required for E. coli to express the phosphonate utilization phenotype (Phn+). The complete nucleotide sequence of 15,611 bases has been determined, and the gene structures were examined. Seventeen open reading frames (phnA to phnQ) were identified in one transcriptional direction and five open reading frames in the divergent direction. Sequence homology searches identify PhnC, PhnK, PhnL, and, possibly, PhnN proteins as members of nucleotide-binding proteins of the binding protein-dependent transport systems. Candidates for other membrane components and regulatory proteins are also identified. A Pho box-like promoter sequence is also found upstream of the gene cluster starting at phnA, which is consistent with the observation of phosphate regulation of the Phn+ response. Fourteen repetitive extragenic palindromic sequences are found in the phn DNA: 10 exist in the extragenic region between phnA and phnB, two between phnD and phnE, and two between phnK and phnL. An unusual finding is that one of the repetitive extragenic palindromic sequences actually overlaps with the reading frame of the phnE gene.