Arthrobacter aurescens TC1 Atrazine Catabolism Genes trzN, atzB, and atzC Are Linked on a 160-Kilobase Region and Are Functional in Escherichia coli

Arthrobacter aurescens strain TC1 metabolizes atrazine to cyanuric acid via TrzN, AtzB, and AtzC. The complete sequence of a 160-kb bacterial artificial chromosome clone indicated that trzN, atzB, and atzC are linked on the A. aurescens genome. TrzN, AtzB, and AtzC were shown to be functional in Escherichia coli. Hybridization studies localized trzN, atzB, and atzC to a 380-kb plasmid in A. aurescens strain TC1.     

Combined metabolic activity within an atrazine-mineralizing community enriched from agrochemical factory soil

The main objective of this work was to characterize an atrazine-mineralizing community originating from agrochemical factory soil, especially to elucidate the catabolic pathway and individual metabolic and genetic potentials of culturable members. A stable four-member bacterial community, characterized by colony morphology and 16S rDNA sequencing, was rapidly able to mineralize atrazine to CO₂ and NH₃. Two primary organisms were identified as Arthrobacter species (ATZ1 and ATZ2) and two secondary organisms (CA1 and CA2) belonged to the genera Ochrobactrum and Pseudomonas, respectively. PCR assessment of atrazine-degrading genetic potential of the community, revealed the presence of trzN, trzD, atzB and atzC genes. Isolates ATZ1 and ATZ2 were capable of dechlorinating atrazine to hydroxyatrazine and contained the trzN gene. ATZ2 further degraded hydroxyatrazine to cyanuric acid and contained atzB and atzC genes whereas ATZ1 contained atzC but not atzB. Isolates CA1 and CA2 grew on cyanuric acid and contained the trzD gene. Complete atrazine degradation was a result of the combined metabolic attack on the atrazine molecule, and complex interactions may exist between the community members sharing carbon and nitrogen from atrazine mineralization.Scientific relevance: Despite numerous reports on atrazine degradation by pure bacterial cultures, the pathways and the atrazine-degrading gene combinations harboured by bacterial communities are only poorly described. In this work, we characterized a four-member atrazine-mineralizing community enriched from an agrochemical factory soil, which was capable of rapidly metabolizing atrazine to CO₂. This study will contribute towards better understanding of the genetic potential and metabolic activities of atrazine-degrading communities, which are generally considered to be responsible for atrazine mineralization in the natural environment.     

Evidence of atrazine mineralization in a soil from the Nile Delta: Isolation of Arthrobacter sp. TES6, an atrazine-degrading strain

The s-triazine herbicide atrazine was rapidly mineralized (i.e., about 60% of ¹⁴C-ring-labelled atrazine released as ¹⁴CO₂ within 21 days) by an agricultural soil from the Nile Delta (Egypt) that had been cropped with corn and periodically treated with this herbicide. Seven strains able to degrade atrazine were isolated by enrichment cultures of this soil. DNA fingerprint and phylogenetic studies based on 16S rRNA analysis showed that the seven strains were identical and belonged to the phylogeny of the genus Arthrobacter (99% similarity with Arthrobacter sp. AD38, EU710554). One strain, designated Arthrobacter sp. strain TES6, degraded atrazine and mineralized the ¹⁴C-chain-labelled atrazine. However, it was unable to mineralize the ¹⁴C-ring-labelled atrazine. Atrazine biodegradation ended in a metabolite that co-eluted with cyanuric acid in HPLC. This was consistent with its atrazine-degrading genetic potential, shown to be dependent on the trzN, atzB, and atzC gene combination. Southern blot analysis revealed that the three genes were located on a large plasmid of about 175 kb and clustered on a 22-kb SmaI fragment. These results reveal for the first time the adaptation of a North African agricultural soil to atrazine mineralization and raise interesting questions about the pandemic dispersion of the trzN, atzBC genes among atrazine-degrading bacteria worldwide.     

Isolation and characterization of novel atrazine-degrading microorganisms from an agricultural soil

Six previously undescribed microorganisms capable of atrazine degradation were isolated from an agricultural soil that received repeated exposures of the commonly used herbicides atrazine and acetochlor. These isolates are all Gram-positive and group with microorganisms in the genera Nocardioides and Arthrobacter, both of which contain previously described atrazine degraders. All six isolates were capable of utilizing atrazine as a sole nitrogen source when provided with glucose as a separate carbon source. Under the culture conditions used, none of the isolates could utilize atrazine as the sole carbon and nitrogen source. We used several polymerase-chain-reaction-based assays to screen for the presence of a number of atrazine-degrading genes and verified their identity through sequencing. All six isolates contain trzN and atzC, two well-characterized genes involved in the conversion of atrazine to cyanuric acid. An additional atrazine-degrading gene, atzB, was detected in one of the isolates as well, yet none appeared to contain atzA, a commonly encountered gene in atrazine impacted soils and atrazine-degrading isolates. Interestingly, the deoxyribonucleic acid sequences of trzN and atzC were all identical, implying that their presence may be the result of horizontal gene transfer among these isolates.     

Isolation and characterization of atrazine-degrading Arthrobacter sp. strains from Argentine agricultural soils

Three bacterial strains capable of degrading atrazine were isolated from Manfredi soils (Argentine) using enrichment culture techniques. These soils were used to grow corn and were treated with atrazine for weed control during 3 years. The strains were nonmotile Gram-positive bacilli which formed cleared zones on atrazine solid medium, and the 16S rDNA sequences indicated that they were Arthrobacter sp. strains. The atrazine-degrading activity of the isolates was characterized by the ability to grow with atrazine as the sole nitrogen source, the concomitant herbicide disappearance, and the chloride release. The atrazine-degrader strain Pseudomonas sp. ADP was used for comparative purposes. According to the results, all of the isolates used atrazine as sole source of nitrogen, and sucrose and sodium citrate as the carbon sources for growth. HPLC analyses confirmed herbicide clearance. PCR analysis revealed the presence of the atrazine catabolic genes trzN, atzB, and atzC. The results of this work lead to a better understanding of microbial degradation activity in order to consider the potential application of the isolated strains in bioremediation of atrazine-polluted agricultural soils in Argentina.     

Characterization of an Atrazine-Degrading Pseudaminobacter sp. Isolated from Canadian and French Agricultural Soils

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Fourteen bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from soils obtained from two farms in Canada and two farms in France. These strains were indistinguishable from each other based on repetitive extragenic palindromic PCR genomic fingerprinting performed with primers ERIC1R, ERIC2, and BOXA1R. Based on 16S rRNA sequence analysis of one representative isolate, strain C147, the isolates belong to the genus Pseudaminobacter in the family Rhizobiaceae. Strain C147 did not form nodules on the legumes alfalfa (Medicago sativa L.), birdsfoot trefoil (Lotus corniculatus L.), red clover (Trifolium pratense L.), chickpea (Cicer arietinum L.), and soybean (Glycine max L.). A number of chloro-substituted s-triazine herbicides were degraded, but methylthio-substituted s-triazine herbicides were not degraded. Based on metabolite identification data, the fact that oxygen was not required, and hybridization of genomic DNA to the atzABC genes, atrazine degradation occurred via a series of hydrolytic reactions initiated by dechlorination and followed by dealkylation. Most strains could mineralize [ring-U-¹⁴C]atrazine, and those that could not mineralize atrazine lacked atzB or atzBC. The atzABC genes, which were plasmid borne in every atrazine-degrading isolate examined, were unstable and were not always clustered together on the same plasmid. Loss of atzB was accompanied by loss of a copy of IS1071. Our results indicate that an atrazine-degrading Pseudaminobacter sp. with remarkably little diversity is widely distributed in agricultural soils and that genes of the atrazine degradation pathway carried by independent isolates of this organism are not clustered, can be independently lost, and may be associated with a catabolic transposon. We propose that the widespread distribution of the atrazine-degrading Pseudaminobacter sp. in agricultural soils exposed to atrazine is due to the characteristic ability of this organism to utilize alkylamines, and therefore atrazine, as sole sources of carbon when the atzABC genes are acquired.     

Metabolic ability and gene characteristics of Arthrobacter sp. strain DNS10, the sole atrazine-degrading strain in a consortium isolated from black soil

Strain DNS10 was the only member that could utilize atrazine as the sole nitrogen source for growth in an atrazine-degrading consortium which was isolated from black soil previously in our laboratory. It belongs to the genus Arthrobacter according to the sequence of 16S rRNA gene and is designated as Arthrobacter sp. DNS10. 16S rRNA gene phylogenetic analysis showed that strain DNS10 was located in a different evolutionary branch comparing with other Arthrobacter sp. atrazine-degrading strains. The degrading genes such as trzN, atzB and atzC harbored in strain DNS10 revealed high sequence similarity with those in Arthrobacter aurescens TC1 and Pseudomonas sp. ADP. These genes enabled the strain DNS10 to decompose atrazine to cyanuric acid. This was further proved by the results that the strain DNS10 (10⁸ CFU mL⁻¹) could degrade the whole atrazine (100 mg L⁻¹) in the medium within 24 h at 30 °C and there was 66.13 ± 2.11 mg L⁻¹ cyanuric acid accumulated at 24 h. These results imply that the strain DNS10 seems to be an excellent atrazine-degrading strain. Furthermore, this paper helps us in the better understanding of the strain evolution by comparing the metabolic ability and gene characteristics of strain DNS10 with other geographically distinct atrazine-degrading strains.     

Isolation and characterization of Arthrobacter sp. strain MCM B-436, an atrazine-degrading bacterium, from rhizospheric soil

Atrazine is one of the most environmentally prevalent s-triazine-ring herbicides. The widespread use of atrazine and its toxicity necessitates search for remediation technology. As atrazine is still used in India as a major herbicide, exploration of atrazine-degrading bacterial community is of immense importance. Considering lack of reports on well characterized atrazine-degrading bacterial cultures from India and wide diversity and density of microorganisms in rhizosphere, soil sample from rhizosphere of atrazine-resistant plant was studied. Arthrobacter sp. strain isolated in this investigation utilizes atrazine as the sole nitrogen source. In addition, the bacterium degrades other triazines such as ametryn, cyanizine, propazine and simazine. PCR analysis confirms the presence of atzBCD and triazine hydrolase (trzN) genes on chromosomal DNA. Sequencing of the trzN gene reveals high sequence similarity with trzN from Nocardioides sp. C190. An inducible and intracellular atrazine chlorohydrolase enzyme was isolated and partially purified from this isolate. This study confirms the presence of atrazine-degrading microbial population in Indian soils and could be used efficiently for remediation of contaminated soils. Presence of trzN gene indicates possible presence of bacterial community with more efficient and novel enzymatic capabilities. Comparison of enzyme and gene structure of this isolate with other geographically distinct atrazine-degrading strains will help us in the better understanding of gene transfer and evolution.     

Enzymatic Degradation of Chlorodiamino-s-Triazine

2-Chloro-4,6-diamino-s-triazine (CAAT) is a metabolite of atrazine biodegradation in soils. Atrazine chlorohydrolase (AtzA) catalyzes the dechlorination of atrazine but is unreactive with CAAT. In this study, melamine deaminase (TriA), which is 98% identical to AtzA, catalyzed deamination of CAAT to produce 2-chloro-4-amino-6-hydroxy-s-triazine (CAOT). CAOT underwent dechlorination via hydroxyatrazine ethylaminohydrolase (AtzB) to yield ammelide. This represents a newly discovered dechlorination reaction for AtzB. Ammelide was subsequently hydrolyzed by N-isopropylammelide isopropylaminohydrolase to produce cyanuric acid, a compound metabolized by a variety of soil bacteria.     

Molecular Basis of a Bacterial Consortium: Interspecies Catabolism of Atrazine

Pseudomonas sp. strain ADP contains the genes, atzA, -B, and -C, that encode three enzymes which metabolize atrazine to cyanuric acid. Atrazine-catabolizing pure cultures isolated from around the world contain genes homologous to atzA, -B, and -C. The present study was conducted to determine whether the same genes are present in an atrazine-catabolizing bacterial consortium and how the genes and metabolism are subdivided among member species. The consortium contained four or more bacterial species, but two members, Clavibacter michiganese ATZ1 and Pseudomonas sp. strain CN1, collectively mineralized atrazine. C. michiganese ATZ1 released chloride from atrazine, produced hydroxyatrazine, and contained a homolog to the atzA gene that encoded atrazine chlorohydrolase. C. michiganese ATZ1 stoichiometrically metabolized hydroxyatrazine to N-ethylammelide and contained genes homologous to atzB and atzC, suggesting that either a functional AtzB or -C catalyzed N-isopropylamine release from hydroxyatrazine. C. michiganese ATZ1 grew on isopropylamine as its sole carbon and nitrogen source, explaining the ability of the consortium to use atrazine as the sole carbon and nitrogen source. A second consortium member, Pseudomonas sp. strain CN1, metabolized the N-ethylammelide produced by C. michiganese ATZ1 to transiently form cyanuric acid, a reaction catalyzed by AtzC. A gene homologous to the atzC gene of Pseudomonas sp. strain ADP was present, as demonstrated by Southern hybridization and PCR. Pseudomonas sp. strain CN1, but not C. michiganese, metabolized cyanuric acid. The consortium metabolized atrazine faster than did C. michiganese individually. Additionally, the consortium metabolized a much broader set of triazine ring compounds than did previously described pure cultures in which the atzABC genes had been identified. These data begin to elucidate the genetic and metabolic bases of catabolism by multimember consortia.     

Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport

Several Frankia strains have been shown to be copper-tolerant. The mechanism of their copper tolerance was investigated for Frankia sp. strain EuI1c. Copper binding was shown by binding studies. Unusual globular structures were observed on the surface of the bacterium. These globular structures were composed of aggregates containing many relatively smaller “leaf-like” structures. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDAX) analysis of these structures indicated elevated copper and phosphate levels compared to the control cells. Fourier transform infrared spectroscopy (FTIR) analysis indicated an increase in extracellular phosphate on the cell surface of copper-stressed cells. Bioinformatics’ analysis of the Frankia sp. strain EuI1c genome revealed five potential cop genes: copA, copZ, copC, copCD, and copD. Experiments with Frankia sp. strain EuI1c using qRT-PCR indicated an increase in messenger RNA (mRNA) levels of the five cop genes upon Cu²⁺ stress. After 5 days of Cu²⁺ stress, the copA, copZ, copC, copCD, and copD mRNA levels increased 25-, 8-, 18-, 18-, and 25-fold, respectively. The protein profile of Cu²⁺-stressed Frankia sp. strain EuI1c cells revealed the upregulation of a 36.7 kDa protein that was identified as FraEuI1c_1092 (sulfate-binding periplasmic transport protein). Homologues of this gene were only present in the genomes of the Cu²⁺-resistant Frankia strains (EuI1c, DC12, and CN3). These data indicate that copper tolerance by Frankia sp. strain EuI1c involved the binding of copper to the cell surface and transport proteins.     

Substrate Specificity of Atrazine Chlorohydrolase and Atrazine-Catabolizing Bacteria

Bacterial atrazine catabolism is initiated by the enzyme atrazine chlorohydrolase (AtzA) in Pseudomonas sp. strain ADP. Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear. Here we begin to address this by investigating the catalytic activity of AtzA by using substrate analogs. Purified AtzA from Pseudomonas sp. strain ADP catalyzed the hydrolysis of an atrazine analog that was substituted at the chlorine substituent by fluorine. AtzA did not catalyze the hydrolysis of atrazine analogs containing the pseudohalide azido, methoxy, and cyano groups or thiomethyl and amino groups. Atrazine analogs with a chlorine substituent at carbon 2 and N-alkyl groups, ranging in size from methyl to t-butyl, all underwent dechlorination by AtzA. AtzA catalyzed hydrolytic dechlorination when one nitrogen substituent was alkylated and the other was a free amino group. However, when both amino groups were unalkylated, no reaction occurred. Cell extracts were prepared from five strains capable of atrazine dechlorination and known to contain atzA or closely homologous gene sequences: Pseudomonas sp. strain ADP, Rhizobium strain PATR, Alcaligenes strain SG1, Agrobacterium radiobacter J14a, and Ralstonia picketti D. All showed identical substrate specificity to purified AtzA from Pseudomonas sp. strain ADP. Cell extracts from Clavibacter michiganensis ATZ1, which also contains a gene homologous to atzA, were able to transform atrazine analogs containing pseudohalide and thiomethyl groups, in addition to the substrates used by AtzA from Pseudomonas sp. strain ADP. This suggests that either (i) another enzyme(s) is present which confers the broader substrate range or (ii) the AtzA itself has a broader substrate range.     

AtzC Is a New Member of the Amidohydrolase Protein Superfamily and Is Homologous to Other Atrazine-Metabolizing Enzymes

Pseudomonas sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding N-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway, atzC, was cloned from a Pseudomonas sp. strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escherichia coli. The atzC gene was further delimited by functional analysis following transposon Tn5 mutagenesis and subcloned as a 2.0-kb EcoRI-AvaI fragment. An E. coli strain containing this DNA fragment expressed N-isopropylammelide isopropylamino hydrolase activity, metabolizing N-isopropylammelide stoichiometrically to cyanuric acid and N-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of E. coli cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.     

Cytokinin Secretion by Frankia sp. HFP ArI3 in Defined Medium

Frankia sp. HFP ArI3 (host plant Alnus rubra Bong.) was grown in defined medium and the culture solution was analyzed for the presence of various cytokinins and related compounds. N⁶- (Δ²-isopentenyl) adenosine was the only cytokinin detected by both high performance liquid chromatography and gas chromatography-mass spectrometry, at levels of approximately 1 ng/ml culture medium.     

Effect of Inoculation and Leaf Litter Amendment on Establishment of Nodule-Forming Frankia Populations in Soil

High-N₂-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [¹⁵N]NO₃ and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N₂ fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N₂ fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% ± 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% ± 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% ± 6%) than by group IV (81% ± 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N₂ fixation rates by ¹⁵N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N₂-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N₂-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

Natural Diversity of Frankia Strains in Actinorhizal Root Nodules from Promiscuous Hosts in the Family Myricaceae

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N₂-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia.     

Substituent Effect on Photosynthesis of N2-α-Substituted Benzyl-N4-alkyl-2,4-diamino-6-chloro-s-triazines and Their Phytotoxic Property

The influence of substituents on the activities of a series of N²-α-substituted benzyl-N⁴-alkyl-2,4-diamino-6-chloro-s-triazines as inhibitors of photosystem II (PSII) was examined, and the phytotoxic differences between them and atrazine, as to the photosynthesis in leaf disks, mesophyll cells, intact chloroplasts and broken chloroplasts of spinach, and as to seedling-growth, were discussed. The inhibitory activity of the N²-α,α-dimethylbenzyl-N⁴-ethyl derivative (6), which was comparable on that of atrazine, was lower than those of the N²-α-alkylbenzyl analogues (1 ~5). The N⁴-?-alkyl-N²-α- methylbenzyl derivatives, in spite of the carbon length of the alkyl group, exhibited more potent activity than atrazine, but an a α β substitution of the N⁴-n-alkyl group caused a decrease in the activity with a few exceptions. These data may imply that the space of the binding site on PSII surrounding both the N² and N⁴ amino groups is relatively large. The binding between the receptor site and the N⁴ amino group, however, is easily influenced by a slight structural change in an inhibitor. The herbicidal compounds, N²-α-methylbenzyl-A^⁴-ethyl (1), A^²-α,α-dimethylbenzyl-N⁴-1-methylpropyl (30) and N²-α-methylbenzyl-N⁴,N⁴-diethyl (42) derivatives, exhibited potent inhibitory activity in the seedling growth test under dark/light conditions, whereas atrazine was very poor. The inhibitory activity of compound (1) toward photosynthesis was poor with leaf disks, compared to atrazine, whereas, the order of their activities was the reverse for plant preparations such as abaxial epidermis peeled leaf disks, mesophyll cells, intact chloroplasts and broken chloroplasts. It was indicated that a change in the phytotoxic symptom in the whole plant assay would be correlated to the permeability of the compound through the plant membrane(s).     

Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2

Elevated expression of the plasma membrane drug efflux pump proteins Cdr1p and Cdr2p was shown to accompany decreased azole susceptibility in Candida albicans clinical isolates. DNA sequence analysis revealed extensive allelic heterozygosity, particularly of CDR2. Cdr2p alleles showed different abilities to transport azoles when individually expressed in Saccharomyces cerevisiae. Loss of heterozygosity, however, did not accompany decreased azole sensitivity in isogenic clinical isolates. Two adjacent non-synonymous single nucleotide polymorphisms (NS-SNPs), G1473A and I1474V in the putative transmembrane (TM) helix 12 of CDR2, were found to be present in six strains including two isogenic pairs. Site-directed mutagenesis showed that the TM-12 NS-SNPs, and principally the G1473A NS-SNP, contributed to functional differences between the proteins encoded by the two Cdr2p alleles in a single strain. Allele-specific PCR revealed that both alleles were equally frequent among 69 clinical isolates and that the majority of isolates (81%) were heterozygous at the G1473A/I1474V locus, a significant (P < 0.001) deviation from the Hardy-Weinberg equilibrium. Phylogenetic analysis by maximum likelihood (Paml) identified 33 codons in CDR2 in which amino acid allelic changes showed a high probability of being selectively advantageous. In contrast, all codons in CDR1 were under purifying selection. Collectively, these results indicate that possession of two functionally different CDR2 alleles in individual strains may confer a selective advantage, but that this is not necessarily due to azole resistance.     

Characterization of IS1474, an insertion sequence of the IS21 family isolated from Pseudomonas alcaligenes NCIB 9867

A new insertion sequence (IS) designated IS1474 was isolated from Pseudomonas alcaligenes NCIB 9867 (P25X). IS1474 is a 2632 bp element which showed a characteristic IS structure with 12 bp inverted repeats (IRs) flanking a 2608 bp central region. IS1474 contained four open reading frames (ORF1–ORF4), two in each orientation. Similarities were detected between ORF1 and ORF2 and the putative transposases of the IS21 family. Sequences upstream from IS1474 were found to display up to 89% homology with IS53 from Pseudomonas syringae suggesting that IS1474 had inserted into another related IS element designated IS1475. An open reading frame, ORF5, located at the junction of IS1474 and IS1475, showed similarities with the IstB protein of IS21 and could possibly be the transposase subunit of IS1475. Transposition assays showed that IS1474 transposed at a relatively low frequency leading to cointegration with target plasmids. Hybridization studies showed that IS1474 is present in at least 13 copies in the chromosome of P25X and one copy on its endogenous plasmid.