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.     

Structural and enzymatic properties of 1-aminocyclopropane-1-carboxylate deaminase homologue from Pyrococcus horikoshii

1-Aminocyclopropane-l-carboxylate deaminase (ACCD) is a pyridoxal 5/-phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene. ACCD from some soil bacteria has been reported to be able to break the cyclopropane ring of ACC to yield a-ketobutyrate and ammonia. We reported the crystal structure of ACCD from the yeast Hansenula saturnus in the absence/presence of substrate ACC, and proposed its ingenious reaction mechanisms. In order to study the enzyme further, we overexpressed the ACCD homologue protein (phAHP) from the fully decoded hyperthermophilic archearon, Pyrococcus horikoshii OT3. However, phAHP does not show ACCD activity at high temperature as well as at room temperature, though it has significant sequence similarity. Instead of ACCD activity, the GC-MS analysis and enzymatic method show that phAHP has deaminase activity toward L and D-serine. Here, we present the crystal structures of the native and ACC-complexed phAHP. The overall topology of the phAHP structure is very similar to that of ACCD; however, critical differences were observed around the active site. Here, the differences of enzymatic activity between phAHP and ACCD are discussed based on the structural differences of these two proteins. We suggest that the catalytic disagreement between these two enzymes comes from the difference of the residues near the pyridine ring of pyridoxal 5'-phosphate (PLP), not the difference of the catalytic residues themselves. We also propose a condition necessary in the primary sequence to have ACCD activity.     

ACC deaminase positive Enterobacter-mediated mitigation of salinity stress,and plant growth promotion of Cajanus cajan: a lab to field study

Salinity is a major abiotic stress that negatively impacts plant health and soil microbiota. ACC (1-aminocyclopropane carboxylic acid) deaminase producing microorganisms act as natural stress busters that protect plants from different kinds of stresses. The study focused on the isolation of potent, indigenous, multi-trait ACC deaminase producers. The shortlisted ACC deaminase producers were checked for their ability to promote growth of Cajanus cajan, and mitigate stress under laboratory conditions followed by validation of their potency in naturally saline field conditions. Physiological stress markers were assessed to evaluate the impact of salinity in plants treated with ACC deaminase producer, compared to controls. Further, the contribution of ACC deaminase in stress mitigation was demonstrated by using a chemical inhibitor for ethylene biosynthesis. This study presents a polyphasic approach, transitioning from the rhizospheric soil to the laboratory to validation in the field, and puts forth a promising eco-friendly alternative for sustainable agriculture.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01031-0.     

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.     

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.     

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.     

Tolerance of transgenic canola expressing 1-aminocyclopropane-1-carboxylic acid deaminase to growth inhibition by nickel.

Plant growth-promoting bacteria are useful to phytoremediation strategies in that they confer advantages to plants in contaminated soil. When plant growth-promoting bacteria contain the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, the bacterial cell acts as a sink for ACC, the immediate biosynthetic precursor of the plant growth regulator ethylene thereby lowering plant ethylene levels and decreasing the negative effects of various environmental stresses. In an effort to gain the advantages provided by bacterial ACC deaminase in the phytoremediation of metals from the environment two transgenic canola lines with the gene for this enzyme were generated and tested. In these transgenic canola plants, expression of the ACC deaminase gene is driven by either tandem constitutive cauliflower mosaic virus (CaMV) 35S promoters or the root specific rolD promoter from Agrobacterium rhizogenes. Following the growth of transgenic and non-transformed canola in nickel contaminated soil, it was observed that the rolD plants demonstrate significantly increased tolerance to nickel compared to the non-transformed control plants.     

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.     

旱地小麦根际细菌中产生1-氨基环丙烷-1-羧酸(ACC)脱氨酶菌株的分离和鉴定

采用富集定向筛选法,从旱地小麦的根际土壤中分离到2株产生1-氨基环丙烷-1-羧酸(ACC)脱氨酶的菌株AS和CS。经测定菌株AS和CS的ACC脱氨酶的比活力分别为0.018 6 U/mg和0.016 7 U/mg蛋白。根据培养特征观察和生理生化指标测定结果,结合16S rDNA碱基序列测定和系统发育同源性分析,确定菌株AS和菌株CS分别属于霍氏肠杆菌(Enterobacter hormaechei)和变形斑沙雷氏菌(Serratia proteamaculans)。     

Cloning and Characterization of a Plasmid Encoded ACC Deaminase from an Indigenous Pseudomonas fluorescens FY32

In addition to the characterized mechanisms responsible for many direct effects of plant growth promoting bacteria (PGPB) on plants, it has been suggested that a number of PGPB contain the enzyme ACC deaminase that catalyzes degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, into α-ketobutyrate and ammonia. As part of an effort to obtain an ACC deaminase encoding gene from a collection of soil samples, only one bacterial isolate, Pseudomonas fluorescens FY32 was capable of growing on ACC as a sole source of nitrogen. The ACC deaminase gene was amplified from the above isolate by polymerase chain reaction (PCR) giving an expected DNA fragment, 1017 bp. Sequence analysis of the fragment showed that it was highly homologous (94% and 98% identities at nucleotide and amino acid levels, respectively) to the previously characterized acdS gene from Pseudomonas sp. 6G5. Furthermore, fusion of the ACC deaminase ORF with lacZ gene resulted in the expression of active enzyme in Escherichia coli. In addition, further analyses revealed that the acdS gene was plasmid-encoded so that a large plasmid (pFY32) with almost 50 kb in size was identified from this bacterium. Furthermore, transfer of pFY32 into E. coli DH5α proved its ACC deaminase activity. This result was in accordance with previous reports suggesting horizontal transfer of the acdS gene. However, it needs more investigation to identify whether this pFY32 plasmid has undergone lateral gene transfer during the evolutionary process.     

Nitrogen fixation in annual and perennial legume-grass mixtures across a fertility gradient

Background and aims

The selection of legume species and species mixtures influences agroecosystem nitrogen (N) and carbon cycling. We utilized a fertility gradient to investigate the effects of plant species interactions on biological N fixation of an annual and perennial legume in response to shifting soil resource availability.

Methods

Legume N fixation of annual field pea (Pisum sativum) and perennial red clover (Trifolium pratense) grown in monoculture and mixtures with oats (Avena sativa) or orchardgrass (Dactylis glomerata) was estimated using the ¹⁵N natural abundance method across 15 farm fields and we measured six soil N pools ranging from labile to more recalcitrant.

Results

Evidence of complementary and facilitative species interactions was stronger for the perennial red clover-orchardgrass mixture than for the annual field pea-oat mixture (N Land Equivalency Ratios were 1.6 and 1.2, respectively). We estimated that the transfer of fixed N from red clover to orchardgrass increased aboveground N fixation estimates by 15% from 33 to 38?kg?N ha^?1. Despite a more than 2-fold range in soil organic matter levels and more than 3-fold range in labile soil N pools across field sites, the N fertility gradient was not a strong predictor of N fixation. While grass N assimilation was positively correlated with soil N pools, we found only weak, inverse correlations between legume N fixation and soil N availability. In grass-legume mixtures, soil N availability indirectly influenced N fixation through plant competition.

Conclusions

These results suggest that increasing diversity of cropping systems, particularly through the incorporation of perennial mixtures into rotations, could improve overall agroecosystem N cycling efficiency.     

Disparate role of rhizobial ACC deaminase in root-nodule symbioses

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase converts ACC, a precursor of the plant hormone ethylene, into ammonia and ??-ketobutyrate. ACC deaminase is widespread among the rhizobia in which it might play a crucial role in protecting rhizobia against inhibitory effects of ethylene synthesized by the host plant in response to the nodulation process. The beneficial action of this enzyme was demonstrated in several rhizobia such as Mesorhizobium loti and Rhizobium leguminosarum where knock-out mutants of the ACC deaminase gene showed nodulation defects. The genome of the slow-growing rhizobial species Bradyrhizobium japonicum also carries an annotated gene for a putative ACC deaminase (blr0241). Here, we tested the possible importance of this enzyme in B. japonicum by constructing an insertion mutant of blr0241 and studying its phenotype. First, the activity of ACC deaminase itself was measured. Unlike the B. japonicum wild type, the blr0241 mutant did not show any enzymatic activity. By contrast, the mutant was not impaired in its ability to nodulate soybean, cowpea, siratro, and mungbean. Likewise, symbiotic nitrogen fixation activity remained unaffected. Furthermore, a co-inoculation assay with the B. japonicum wild type and the blr0241 mutant for soybean and siratro nodulation revealed that the mutant was not affected in its competitiveness for nodulation and nodule occupation. The results show that the role previously ascribed to ACC deaminase in the rhizobia cannot be generalized, and species-specific differences may exist.     

The interconversion of ACC deaminase and <Emphasis Type="SmallCaps">d</Emphasis>-cysteine desulfhydrase by directed mutagenesis

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. These enzymes cleave ACC, the immediate precursor of ethylene in plants, into ammonia and alpha-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. In the work reported here, it was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather it utilizes D: -cysteine as a substrate, and in fact encodes a D: -cysteine desulfhydrase. Kinetic characterization of the tomato enzyme indicates that it is similar to other, previously characterized, D: -cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering only two amino acid residues within the predicted active site served to change the enzyme from D: -cysteine desulfhydrase to ACC deaminase. Conversely, by altering two amino acid residues at the same positions within the active site of ACC deaminase from Pseudomonas putida UW4 the enzyme was converted into D: -cysteine desulfhydrase. Therefore, it is possible that a change in these two residues may have occurred in an ancestral protein to result in two different enzymatic activities.     

Bacterial biosynthesis of 1-aminocyclopropane-1-caboxylate (ACC) deaminase,a useful trait to elongation and endophytic colonization of the roots of rice under constant flooded conditions

This study was conducted to investigate the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Pseudomonas fluorescens strain REN₁ and its ability to reduce ethylene levels produced during stress, endophytically colonize and promote the elongation of the roots of rice seedlings under gnotobiotic conditions. We isolated 80 bacteria from inside roots of rice plants grown in the farmers’ fields in Guilan, Iran. All of the isolates were characterized for plant growth promoting (PGP) traits. In addition, the colonization assay of these isolates on rice seedlings was carried out to screen for competent endophytes. The best bacterial isolate, based on ACC deaminase production, was identified and used for further study. 16S rDNA sequence analysis revealed that the endophyte was closely related to Pseudomonas fluorescens. The results of this study showed ACC deaminase containing P. fluorescens REN1 increased in vitro root elongation and endophytically colonized the root of rice seedlings significantly, as compared to control under constant flooded conditions. The trait of low amount of indole-3-acetic acid (IAA) production (<15 μg mL⁻¹) and the high production of ACC deaminase by bacteria may be main factors in colonizing rice seedling roots compared to other PGP traits (siderophore production and phosphate solubilization) in this study. Endophytic IAA and ACC deaminase-producing bacteria may be preferential selections by rice seedlings. Therefore, it may be suggested that the utilization of ACC as a nutrient gives the isolates advantages in more endophytic colonization and increase of root length of rice seedlings.     

A Method for Multiplex Gene Synthesis Employing Error Correction Based on Expression

Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in E. coli. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.     

Synthesis and degradation of 1-aminocyclopropane-1-carboxylic acid by Penicillium citrinum

1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M(r) 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the Km for S-adenosyl-L-methionine of 1.74 mM and kcat of 0.56 s-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M(r) 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a Km for ACC of 4.8 mM and kcat of 3.52 s-1. The presence of 7 mM Cu2+ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.     

Regulation of Expression of the prb-1b / ACC Deaminase Gene by UV-8 in Transgenic Tomatoes

Transgenic tomato plants with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4 under the control of a pathogenesis-related promoter (prb-1b) from tobacco were challenged by abiotic stresses to determine the expression patterns ofthe transgene. No ACC deaminase RNA or protein was detected by RT-PCR and in western blots prepared from leaf proteins of transgenic plants after wounding or treatment with α-amino butyric acid, xylanase, ethephon, salicylic acid, jasmonic acid, ethylene, or ethylene plus jasmonic acid. However, expression of the ACC deaminase transgene was observed in leaves and roots oftransformed tomato lines exposed to UV light. The UV response required a minimum of 48 h of exposure and was specific to UV-8 light.     

In vitro selection of GTP-binding proteins by block shuffling of estrogen-receptor fragments

To what extent has alternative splicing contributed to the evolution of protein-function diversity? We previously constructed a pool of block-deletion mutants of the human estrogen receptor α ligand binding domain by random multi-recombinant PCR. Here we performed iterative in vitro selection of GTP-binding proteins by using the library of mRNA-displayed proteins and GTP-affinity chromatography combined with quantitative real-time PCR. We obtained a novel GTP-binding protein with moderate affinity and substrate-specificity. The results of our in vitro simulation imply that alternative splicing may have contributed substantially to the diversification of protein function during evolution.