Isolation and characterization of drought-tolerant ACC deaminase and exopolysaccharide-producing fluorescent Pseudomonas sp.

The enzyme 1-aminocyclopropane-1-carboxylate deaminase catalyzes the degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of the plant hormone ethylene, into α-ketobutyrate and ammonia. The enzyme has been detected in a limited number of bacteria and plays a significant role in sustaining plant growth and development under biotic and abiotic stress conditions by reducing stress-induced ethylene production in plants. We have screened 32 fluorescent Pseudomonas sp. isolated from rhizosphere and non-rhizosphere soils of different crop production systems for drought tolerance using polyethylene glycol 6000 (PEG 6000). Nine of these isolates were tolerant to a substrate metric potential of ?0.30 MPa (15 % PEG 6000) and therefore considered to be drought-tolerant. All of these drought-tolerant isolates were screened for ACC deaminase activity using ACC as the sole nitrogen source, and one (SorgP4) was found to be positive for ACC, producing 3.71?±?0.025 and 1.42?±?0.039 μM/mg protein/h of α-ketobutyrate under the non-stress and drought stress condition, respectively. The isolate SorgP4 also showed other plant growth-promoting traits, such as indole acetic acid production, phosphate solubilization, siderophore and hydrogen cyanide production. The ACC deaminase gene (acdS) from the isolate SorgP4 was amplified, and the nucleotide sequence alignment of the acdS gene showed significant homology with acdS genes of NCBI Genbank. The 16S rRNA gene sequencing analysis identified the isolate as Pseudomonas fluorescens. Both sequences have been submitted to the NCBI GenBank under the accession numbers JX885767 and KC192771 respectively.     

Transformation of Azospirillum brasilense Cd with an ACC deaminase gene from enterobacter cloacae UW4 fused to the Tet r gene promoter improves its fitness and plant growth promoting ability

It has been reported that PGPB, containing ACC deaminase, can cleave the plant ethylene precursor ACC and thereby lower ethylene concentration in a developing or stressed plant, protecting it against the deleterious effects of stress ethylene and facilitating the formation of longer roots. In a previous work we have demonstrated expression of the ACC deaminase gene (acdS) from Enterobacter cloacae UW4 under the control of the lac promoter in Azospirillum brasilense Cd. With the inference that a construct including the ACC deaminase gene under the control of a constitutive promoter weaker than the lac promoter might impose less metabolic load on Azospirillum and improve its fitness, it was decided to clone acdS under the control of a tetracycline resistance gene promoter. The ACC deaminase structural gene was fused to the Tet ^r gene promoter by overlap extension using PCR, cloned in pRK415, and transferred into A. brasilense Cd. The resulting transformants showed lower ACC deaminase activity than those with the lac promoter controlled acdS gene. However, acdS under the control of the Tet ^r gene promoter imposed lesser metabolic load on Azospirillum brasilense Cd. The result was significantly increased IAA synthesis and greater bacterial growth rate, as well as increased ability to survive on the surface of tomato leaves and to promote the growth of tomato seedlings.     

Expression of an Exogenous 1-Aminocyclopropane-1-Carboxylate Deaminase Gene in Sinorhizobium meliloti Increases Its Ability To Nodulate Alfalfa

1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant growth-promoting rhizobacteria, including rhizobia. This enzyme degrades ACC, the immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene in higher plants. The ACC deaminase of Rhizobium leguminosarum bv. viciae 128C53K was previously reported to be able to enhance nodulation of peas. The ACC deaminase structural gene (acdS) and its upstream regulatory gene, a leucine-responsive regulatory protein (LRP)-like gene (lrpL), from R. leguminosarum bv. viciae 128C53K were introduced into Sinorhizobium meliloti, which does not produce this enzyme, in two different ways: through a plasmid vector and by in situ transposon replacement. The resulting ACC deaminase-producing S. meliloti strains showed 35 to 40% greater efficiency in nodulating Medicago sativa (alfalfa), likely by reducing ethylene production in the host plants. Furthermore, the ACC deaminase-producing S. meliloti strain was more competitive in nodulation than the wild-type strain. We postulate that the increased competitiveness might be related to utilization of ACC as a nutrient within the infection threads.     

Enhanced chickpea growth-promotion ability of a Mesorhizobium strain expressing an exogenous ACC deaminase gene

Aims

The main goal of the study reported herein was to assess the nodulation performance of a Mesorhizobium strain transformed with an exogenous ACC deaminase gene (acdS), and its subsequent ability to increase chickpea plant growth under normal and waterlogged conditions.

Methods

The Mesorhizobium ciceri strain LMS-1 was transformed with the acdS gene of Pseudomonas putida UW4 by triparental conjugation using plasmid pRKACC. A plant growth assay was conducted to verify the plant growth promotion ability of the LMS-1 (pRKACC) transformed strain under normal and waterlogging conditions. Bacterial ACC deaminase and nitrogenase activity was measured.

Results

By expressing the exogenous acdS gene, the transformed strain LMS-1 showed a 127% increased ability to nodulate chickpea and a 125% promotion of the growth of chickpea compared to the wild-type strain, under normal conditions. Plants inoculated with the LMS-1 wild-type strain showed a higher nodule number under waterlogging stress than under control conditions, suggesting that waterlogging increases nodulation in chickpea. No significant relationship was found between ACC deaminase and nitrogenase activity.

Conclusions

The results obtained in this study show that the use of rhizobial strains with improved ACC deaminase activity might be very important for developing microbial inocula for agricultural purposes.     

Detection and Characterization of ACC Deaminase in Plant Growth Promoting Rhizobacteria

The enzyme 1-aminocyclopropane-1-carboxylate deaminase converts ACC, the precursor of the plant hormone ethylene to α-ketobutyrate and ammonium. The enzyme has been identified in few soil bacteria, and is proposed to play a key role in plant growth promotion. In this study, the isolates of plant growth promoting rhizobacteria were screened for ACC deaminase activity based on their ability to grow on ACC as a sole nitrogen source. The selected isolates showed the presence of other plant growth promoting characteristics such as IAA production, phosphate solubilization and siderophore production. The role of ACC deaminase in lowering ethylene production under cadmium stress condition was also studied by measuring in vitro ethylene evolution by wheat seedlings treated with ACC deaminase positive isolates. Nucleic acid hybridization confirmed the presence of ACC deaminase gene (acdS) in the bacterial isolates.     

Rhizobium leguminosarum Biovar viciae 1-Aminocyclopropane-1-Carboxylate Deaminase Promotes Nodulation of Pea Plants

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.     

1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Genes in Rhizobia from Southern Saskatchewan

A collection of 233 rhizobia strains from 30 different sites across Saskatchewan, Canada was assayed for 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, with 27 of the strains displaying activity. When all 27 strains were characterized based on 16S rRNA gene sequences, it was noted that 26 strains are close to Rhizobium leguminosarum and one strain is close to Rhizobium gallicum. Polymerase chain reaction (PCR) was used to rapidly isolate ACC deaminase structural genes from the above-mentioned 27 strains; 17 of them have 99% identities with the previously characterized ACC deaminase structural gene (acdS) from R. leguminosarum bv. viciae 128C53K, whereas the other ten strains are 84% identical (864~866/1,020 bp) compared to the acdS from strain 128C53K. Southern hybridization showed that each strain has only one ACC deaminase gene. Using inverse PCR, the region upstream of the ACC deaminase structural genes was characterized for all 27 strains, and 17 of these strains were shown to encode a leucine-responsive regulatory protein. The results are discussed in the context of a previously proposed model for the regulation of bacterial ACC deaminase in R. leguminosarum 128C53K. An erratum to this article can be found at     

An ACC Deaminase Minus Mutant of Enterobacter cloacae UW4No Longer Promotes Root Elongation

The ACC deaminase gene (acdS) from Enterobacter cloacae UW4 was replaced by homologous recombination with the acdS gene with a tetracycline resistance gene inserted within the coding region. Upon characterization of this AcdS minus mutant, it was determined that both ACC deaminase activity and the ability to promote the elongation of canola roots under gnotobiotic conditions were greatly diminished. This result is consistent with a previously postulated model that suggests that a major mechanism utilized by plant growth-promoting bacteria involves the lowering of plant ethylene levels, and hence ethylene inhibition of root elongation, by bacterial ACC deaminase. Received: 20 January 2000 / Accepted: 22 February 2000     

Comparative effects of ethylene inhibitors on Agrobacterium-mediated transformation of drought-tolerant wild watermelon

Ethylene (C₂H₄), a phytohormone that is produced in response to both abiotic and biotic stresses, is an important factor influencing the efficiency of Agrobacterium-mediated transformation. In this study, effects of various ethylene inhibitors on the efficiency of Agrobacterium-mediated genetic transformation in drought-tolerant wild watermelon was comparatively examined. Consequently, in comparison to the application of chemical inhibitors such as AgNO₃ and aminoethoxyvinylglycine (AVG), lower ethylene level was observed when the infecting Agrobacterium contained a gene for 1-aminocyclopropane-carboxylic acid (ACC) deaminase (acdS), which cleaves ethylene precursor ACC into α-ketobutyrate and ammonia. GUS histochemical and spectrophotometric enzyme assays showed that acdS was more effective in enhancing gene transfer than the chemical ethylene inhibitors. Efficiency of transgenic shoots formation was higher in acdS- and AVG-treated explants. These observations demonstrated that controlling the ethylene level during co-cultivation and shoot formation, particularly using the acdS-harboring Agrobacterium, is advantageous for enhancing the transformation efficiency in this plant.     

Azospirillum lipoferum strain AZm5 containing 1-aminocyclopropane-1-carboxylic acid deaminase improves early growth of tomato seedlings under nitrogen deficiency

In this study we evaluated the ability of two wild strains of Azospirillum, A. lipoferum AZm5 and A. brasilense VS9, to produce ACC deaminase. We tested the effects of a deficiency and medium doses of nitrogenous fertilizers on the growth and physiology of tomato plants (Lycopersicon esculentum Mill cv. ACE VF55) inoculated with both Azospirillum strains independently. Tomato plants were evaluated by root elongation assay and grown in pot soil culture with different nitrogen levels (0 kg N ha^–1 and 170 kg N ha^–1). The root:shoot ratio (R:S) and some ecophysiological traits were determined after 42 days of plant growth. Results showed very different physiological characteristics in both strains. We found three relevant aspects related to the AZm5 strain: it produces high amounts of cytokinins, it contains the gene acdS, which encodes ACC deaminase, and it promotes plant growth. We conclude that AZm5 maybe useful to increase N uptake in N-deficient soil by production of cytokinins and the promotion of ACC deaminase activity, which favored leaf expansion and higher leaf N investment. Therefore, for tomato culture, a simultaneous biofertilization with AZm5 and a relatively low fertilization with N (170 kg N ha^–1) to promote AZm5 activity could be advantageous.     

ACC deaminase from plant growth-promoting bacteria affects crown gall development

In addition to the well-known roles of indoleacetic acid and cytokinin in crown gall formation, the plant hormone ethylene also plays an important role in this process. Many plant growth-promoting bacteria (PGPB) encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which can degrade ACC, the immediate precursor of ethylene in plants, to alpha-ketobutyrate and ammonia and thereby lower plant ethylene levels. To study the effect of ACC deaminase on crown gall development, an ACC deaminase gene from the PGPB Pseudomonas putida UW4 was introduced into Agrobacterium tumefaciens C58, so that the effect of ACC deaminase activity on tumour formation in tomato and castor bean plants could be assessed. Plants were also coinoculated with A. tumefaciens C58 and P. putida UW4 or P. putida UW4-acdS- (an ACC deaminase minus mutant strain). In both types of experiments, it was observed that the presence of ACC deaminase generally inhibited tumour development on both tomato and castor bean plants.     

Bacterial community compositions of tomato (Lycopersicum esculentum Mill.) seeds and plant growth promoting activity of ACC deaminase producing Bacillus subtilis (HYT-12-1) on tomato seedlings

Study of endophytic bacteria within plant seeds is very essential and meaningful on account of their heritability and versatility. This study investigated Bacillus bacterial communities within the seeds of four commercial tomato varieties, by 16S rRNA gene PCR-RFLP (restriction fragment length polymorphism). Phylogenetic analysis of 16S rRNA gene sequences indicated that the 22 representative isolates belonged to five species of genus Bacillus and the bacterial compositions showed remarkable differences among tomato varieties. Isolates exhibited multiple plant growth promoting (PGP) traits: 37 % of indole-3-acetic acid production; 37 % of phosphate solubilization; 24 % of siderophores production; 85 % of potential nitrogen fixation and 6 % of 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Isolate HYT-12-1 was shown to have highest ACC deaminase activity (112.02 nmol α-ketobutyrate mg^?1 protein h^?1) among the five ACC deamiase producing strains. 16S rRNA gene sequencing indicated that the isolate HYT-12-1 shared the highest sequence similarity (100 %) with B. subtilis. PGP experiments under gnotobiotic and greenhouse conditions revealed the ability of strain HYT-12-1 to enhance the growth of tomato seedlings. This is the first study to describe endophytic Bacillus communities within tomato seeds, and the results suggest that B. subtilis strain HYT-12-1 would have a great potential for industrial application as biofertilizer in the future.     

Isolation and characterization of an unusual 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4

A genomic library of the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth-promoting bacterium Enterobacter cloacae UW4 in pUC19 in Escherichia coli was screened for the ability to utilize ACC as a sole source of nitrogen. One of the clones that was isolated contained a plasmid with an insert of approximately 0.8 kb that conferred ACC deaminase activity. Sequence analysis revealed that this DNA fragment contains an open-reading frame of 696 nucleotides predicted to encode a protein of 232 amino acids, a member of the amidohydrolase protein superfamily, i.e., a deaminase that contains a mononuclear or binuclear metal center as compared to the canonical ACC deaminase which contains pyridoxal phosphate as a co-factor.     

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.     

Evidence for the involvement of ACC deaminase from Pseudomonas putida UW4 in the biocontrol of pine wilt disease caused by Bursaphelenchus xylophilus

Pine wilt disease, caused by the nematode Bursaphelenchus xylophilus, is responsible for devastation of pine forests worldwide. Until now, there are no effective ways of dealing with this serious threat. The use of 1-aminocyclopropane-1-carboxylate (ACC) deaminase (encoded by the acdS gene)-producing plant growth-promoting bacteria has been shown to be a useful strategy to reduce the damage due to biotic and abiotic stresses. Pinus pinaster seedlings inoculated with the ACC deaminase-producing bacterium Pseudomonas putida strain UW4 showed an increased root and shoot development and reduction of B. xylophilus induced symptoms. In contrast, a P. putida UW4 acdS mutant was unable to promote pine seedling growth or to decrease B. xylophilus induced symptoms. This is the first report on the use of ACC deaminase-producing bacteria as a potential biological control agent for a tree disease, thus suggesting that the inoculation of pine seedlings grown in a tree nursery might constitute a novel strategy to obtain B. xylophilus resistant pine trees.     

An efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of “Egusi” melon (<Emphasis Type="Italic">Colocynthis citrullus</Emphasis> L.)

Cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were co-cultivated with Agrobacterium tumefaciens strain EHA101 carrying either plasmid pIG121-Hm harbouring genes coding for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII) or plasmid pBBRacdS harbouring these same genes along with a gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Six weeks after co-cultivation, more than 35% of explants produced shoots in both cultivars. A DNA fragment corresponding to the gus gene or the selection marker nptII was amplified from genomic DNA extracted from leaves of regenerated plant clones rooted on hormone-free MS medium containing 100 mg/l kanamycin, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of one to three copies of the gus gene. Transformation efficiencies of cultivar NHC1-130 with EHA101(pIG121-Hm) and EHA101(pIG121-Hm, pBBRacdS) were 3.8% and 10%, respectively, which were higher than those obtained for cultivar ‘Ejagham’ of 2.4% and 5.7%, respectively. Co-cultivation medium containing 5 mg/l BA was effective for obtaining high transformation efficiency for both cultivars as compared with that without it.     

Distribution of 1-aminocyclopropane-1-carboxylate deaminase and <Emphasis Type="SmallCaps">d</Emphasis>-cysteine desulfhydrase genes among type species of the genus <Emphasis Type="Italic">Methylobacterium</Emphasis>

The presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase determines the ability of bacteria to increase the resistance of plants to various types of stress. The genes of ACC deaminase (acdS) and the closely related enzyme d-cysteine desulfhydrase (dcyD) were searched in type strains of various representatives of the genus Methylobacterium. Using PCR screening and in silico searching in the available complete genome sequences of type strains, the genes were found in 28 of 48 species of the genus. Phylogenetic analysis of amino acid sequences of proteins revealed two large groups of sequences of the AcdS protein and one of the DcyD protein. The distribution of these groups correlates well with the phylogenetic tree based on the sequences of the 16S rRNA genes, which apparently indicates a different evolutionary adaptation to association with plants in the representatives of these groups. For the first time for aerobic methylotrophs it was demonstrated that the gene dcyD encodes d-cysteine desulfhydrase by cloning and recombinant protein characterization.