A system to study the expression of phytopathogenic genes encoded by Burkholderia glumae.

Rice is often infected by bacterial panicle blight disease caused by Burkholderia glumae. Since most studies have assessed the transcriptome of the plant when it is exposed to bacteria, the gene expression of the phytopathogenic bacteria have not been well elaborated during the infection process or in the host cell. Recently, a few researches were conducted to evaluate the in vivo transcriptome of bacteria during the infective process. Most bacterial cells do not express genes involved in pathogenicity in culture medium making it difficult to investigate gene expression of bacterial cells in plant cells. Here, we sought a simulated patho-system that would allow bacterial cells to express their pathogenic genes. Thus, rice root exudates (RE) and bacterial N-acyl homoserine lactone (AHL) were used and their effects on bacterial gene expression were assessed. Transcription patterns of B. glumae virulence determinants showed that enrichment medium (LB + RE + C8-HSL) could significantly induce virulence factor genes compared with Luria Bertani (LB; control) medium. The data indicate that the artificial environment is similar to the real patho-system, and that this induced maximum relevant gene expression. In this model system, bacterial gene expression changes are traceable in the infection process. Bacterial cells exposed to either an artificial environment or LB + RE + C8-HSL behaved similarly to the natural environment in situ.

The expression of an exogenous ACC deaminase by the endophyte Serratia grimesii BXF1 promotes the early nodulation and growth of common bean.

Ethylene acts as an inhibitor of the nodulation process of leguminous plants. However, some bacteria can decrease deleterious ethylene levels by the action of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase which degrades ACC, the ethylene precursor in all higher plants. Co-inoculation of rhizobia with endophytes enhances the rhizobial symbiotic efficiency with legumes, improving both nodulation and nitrogen fixation. However, not much is understood about the mechanisms employed by these endophytic bacteria. In this regard, the role of ACC deaminase from endophytic strains in assisting rhizobia in this process has yet to be confirmed. In this study, the role of ACC deaminase in an endophyte's ability to increase Rhizobium tropici nodulation of common bean was evaluated. To assess the effect of ACC deaminase in an endophyte's ability to promote rhizobial nodulation, the endophyte Serratia grimesii BXF1, which does not encode ACC deaminase, was transformed with an exogenous acdS gene. The results obtained indicate that the ACC deaminase-overexpressing transformant strain increased common bean growth, and enhanced the nodulation abilities of R. tropici CIAT899, in both cases compared to the wild-type non-transformed strain. Furthermore, plant inoculation with the ACC deaminase-overproducing strain led to an increased level of plant protection against a seed-borne pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we studied the effect of ACC deaminase production by the bacterial endophyte Serratia grimesi BXF1, and its impact on the nodulation process of common bean. The results obtained indicate that ACC deaminase is an asset to the synergetic interaction between rhizobia and the endophyte, positively contributing to the overall legume-rhizobia symbiosis by regulating inhibitory ethylene levels that might otherwise inhibit nodulation and overall plant growth. The use of rhizobia together with an ACC deaminase-producing endophyte is, therefore, an important strategy for the development of new bacterial inoculants with increased performance.

Delay of flower senescence by bacterial endophytes expressing 1-aminocyclopropane-1-carboxylate deaminase.

AIMS: The ability of 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant growth-promoting bacterial (PGPB) endophytes Pseudomonas fluorescens YsS6 and Pseudomonas migulae 8R6, their ACC deaminase minus mutants and the rhizospheric plant growth-promoting bacterium Pseudomonas putida UW4 to delay the senescence of mini carnation cut flowers was assessed. METHODS AND RESULTS: Fresh cut flowers were incubated with either a bacterial cell suspension, the ethylene precursor ACC, the ethylene inhibitor l-α-(aminoethoxyvinyl)-glycine or 0·85% NaCl at room temperature for 11 days. Levels of flower senescence were recorded every other day. To verify the presence of endophytes inside the plant tissues, scanning electron microscopy was performed. Among all treatments, flowers treated with wild-type ACC deaminase-containing endophytic strains exhibited the most significant delay in flower senescence, while flowers treated with the ACC deaminase minus mutants senesced at a rate similar to the control. Flowers treated with Ps. putida UW4 senesced more rapidly than untreated control flowers. CONCLUSION: The only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity so that it may be concluded that this enzyme is directly responsible for the significant delay in flower senescence. Despite containing ACC deaminase activity, Ps. putida UW4 is not taken up by the cut flowers and therefore has no effect on prolonging their shelf life. SIGNIFICANCE AND IMPACT OF THE STUDY: The world-wide cut flower industry currently uses expensive and potentially environmentally dangerous chemical inhibitors of ethylene to prolong the shelf life of cut flowers. The use of PGPB endophytes with ACC deaminase activity has the potential to replace the chemicals that are currently used by the cut flower industry.

Mesorhizobium ciceri LMS-1 expressing an exogenous 1-aminocyclopropane-1-carboxylate (ACC) deaminase increases its nodulation abilities and chickpea plant resistance to soil constraints.

AIMS: Our goal was to understand the symbiotic behaviour of a Mesorhizobium strain expressing an exogenous 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which was used as an inoculant of chickpea (Cicer arietinum) plants growing in soil. METHODS AND RESULTS: Mesorhizobium ciceri LMS-1 (pRKACC) was tested for its plant growth promotion abilities on two chickpea cultivars (ELMO and CHK3226) growing in nonsterilized soil that displayed biotic and abiotic constraints to plant growth. When compared to its wild-type form, the M. ciceri LMS-1 (pRKACC) strain showed an increased nodulation performance of c. 125 and 180% and increased nodule weight of c. 45 and 147% in chickpea cultivars ELMO and CHK3226, respectively. Mesorhizobium ciceri LMS-1 (pRKACC) was also able to augment the total biomass of both chickpea plant cultivars by c. 45% and to reduce chickpea root rot disease susceptibility. CONCLUSIONS: The results obtained indicate that the production of ACC deaminase under free living conditions by Mesorhizobium strains increases the nodulation, plant growth abilities and biocontrol potential of these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study regarding the use of a transformed rhizobial strain expressing an exogenous ACC deaminase in different plant cultivars growing in soil. Hence, obtaining Mesorhizobium strains with high ACC deaminase activity is a matter of extreme importance for the development of inoculants for field applications.

Interactions between Pseudomonas putida UW4 and Gigaspora rosea BEG9 and their consequences for the growth of cucumber under salt-stress conditions.

AIMS: After the determination of the toxic but nonlethal concentration of NaCl for cucumber, we examined the interaction between an ACC (1-aminocyclopropane-1-carboxylate) deaminase producing bacterial strain and an arbuscular mycorrhizal fungus (AMF) and their effects on cucumber growth under salinity. METHODS AND RESULTS: In the first experiment, cucumber seedlings were exposed to 0.1, 50, 100 or 200 mmol l(-1) NaCl, and plant biomass and leaf area were measured. While seeds exposed to 200 mmol l(-1) NaCl did not germinate, plant growth and leaf size were reduced by 50 or 100 mmol l(-1) salt. The latter salt cancentration caused plant death in 1 month. In the second experiment, seeds were inoculated with the ACC deaminase-producing strain Pseudomonas putida UW4 (AcdS(+)), its mutant unable to produce the enzyme (AcdS(-)), or the AMF Gigaspora rosea BEG9, individually or in combination and exposed to 75 mmol l(-1) salt. Plant morphometric and root architectural parameters, mycorrhizal and bacterial colonization and the influence of each micro-organism on the photosynthetic efficiency were evaluated. The AcdS(+) strain or the AMF, inoculated alone, increased plant growth, affected root architecture and improved photosynthetic activity. Mycorrhizal colonization was inhibited by each bacterial strain. CONCLUSIONS: Salinity negatively affects cucumber growth and health, but root colonization by ACC deaminase-producing bacteria or arbuscular mycorrhizal fungi can improve plant tolerance to such stressful condition. SIGNIFICANCE AND IMPACT OF THE STUDY: Arbuscular mycorrhizal fungus and bacterial ACC deaminase may ameliorate plant growth under stressful conditions. It was previously shown that, under optimal growth conditions, Ps. putida UW4 AcdS(+) increases root colonization by Gi. rosea resulting in synergistic effects on cucumber growth. These results suggest that while in optimal conditions ACC deaminase is mainly involved in the bacteria/fungus interactions, while under stressful conditions this enzyme plays a role in plant/bacterium interactions. This finding is relevant from an ecological and an applicative point of view.

Evidence for horizontal transfer of 1-aminocyclopropane-1-carboxylate deaminase genes.

PCR was used to rapidly identify and isolate 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes from bacteria. The Shimodaira-Hasegawa test was used to assess whether phylogenetically anomalous gene placements suggestive of horizontal gene transfer (HGT) were significantly favored over vertical transmission. The best maximum likelihood (ML) ACC deaminase tree was significantly more likely than four alternative ML trees, suggesting HGT.

Burkholderia phytofirmans sp. nov., a novel plant-associated bacterium with plant-beneficial properties.

A Gram-negative, non-sporulating, rod-shaped, motile bacterium, with a single polar flagellum, designated strain PsJN(T), was isolated from surface-sterilized onion roots. This isolate proved to be a highly effective plant-beneficial bacterium, and was able to establish rhizosphere and endophytic populations associated with various plants. Seven related strains were recovered from Dutch soils. Based on 16S rRNA gene sequence data, strain PsJN(T) and the Dutch strains were identified as representing a member of the genus Burkholderia, as they were closely related to Burkholderia fungorum (98.7 %) and Burkholderia phenazinium (98.5 %). Analysis of whole-cell protein profiles and DNA-DNA hybridization experiments confirmed that all eight strains belonged to a single species. Strain PsJN(T) had a DNA G+C content of 61.0 mol%. Only low levels of DNA-DNA hybridization to closely related species were found. Qualitative and quantitative differences in fatty acid composition between strain PsJN(T) and closely related species were identified. The predominant fatty acids in strain PsJN(T) were 16 : 0, 18 : 1omega7c and summed feature 3 (comprising 16 : 1omega7c and/or iso-15 : 0 2-OH). Isolate PsJN(T) showed high 1-aminocyclopropane-1-carboxylate deaminase activity and is therefore able to lower the ethylene level in a developing or stressed plant. Production of the quorum-sensing signal compound 3-hydroxy-C8-homoserine lactone was detected. Based on the results of this polyphasic taxonomic study, strain PsJN(T) and the seven Dutch isolates are considered to represent a single, novel species, for which the name Burkholderia phytofirmans sp. nov. is proposed. The type strain is strain PsJN(T) (=LMG 22146(T) = CCUG 49060(T)).

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.

Relationship between antifreeze protein and freezing resistance in Pseudomonas putida GR12-2.

Following transposon Tn5 mutagenesis of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2, mutants that have different freeze-resistant properties were selected. Five of the freeze-sensitive mutants, i.e. FSM-5, -6, -14, -29, and -41, secreted a lower amount of antifreeze protein-(AFP) into the culture broth compared with the wild-type. Among of these five mutants, the three mutants (FSM-6, FSM-14, and FSM-41) that have the lowest level of freezing resistance (4.0-6.0% survival) also produce AFP at low levels (0.5-0.9 microg/mL) compared with the wild-type (4.8 microg/ml). The antifreeze and ice-nucleating activities of the AFP from these three mutant strains were similar to those of wild-type. Furthermore, the decreased freezing resistance from three mutants could be partially restored by adding purified AFP to mutant cell suspensions. Freezing resistance of three mutants was found to increase in proportion to the addition of AFP up to a concentration of 50 microg/mL. We conclude that accumulation of AFP is one component of the mechanism for freezing resistance in bacteria.

Involvement of gacS and rpoS in enhancement of the plant growth-promoting capabilities of Enterobacter cloacae CAL2 and UW4.

The plant growth-promoting bacteria Enterobacter cloacae CAL2 and UW4 were genetically transformed with a multicopy plasmid containing an rpoS or gacS gene from Pseudomonas fluorescens. The transformed strains were compared with the nontransformed strains for growth, indoleacetic acid (IAA) production, antibiotic production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, siderophore production, cell morphology, and the ability to promote canola root elongation. All transformed strains had a longer lag phase, were slower in reaching stationary phase, and attained a higher cell density than the nontransformed strains. Transformation resulted in cells that were significantly shorter than the nontransformed cells. The transformed strains also produced significantly more IAA than the nontransformed strains. Introduction of rpoS or gacS from Pseudomonas fluorescens was associated with a reduction in the production of both antibiotics, 2,4-diacetylphloroglucinol and mono-acetylphloroglucinol, produced by Enterobacter cloacae CAL2. With Enterobacter cloacae CAL2, plasmid-borne rpoS, but not gacS, increased the level of ACC deaminase activity, while introduction of rpoS in Enterobacter cloacae UW4 caused a decrease in ACC deaminase activity. Neither gacS nor rpoS significantly affected the level of siderophores synthesized by either bacterial strain. Overproduction of either GacA or RpoS in Enterobacter cloacae CAL2 resulted in a significant increase in the root lengths of canola seedlings when seeds were treated with the bacteria, and overproduction of RpoS caused an increase in canola shoot as well as root lengths.

Expression of the ACC Deaminase Gene fromEnterobacter cloacae UW4 in Azospirillum brasilense.

The ACC deaminase structural gene (acdS) from Enterobacter cloacae UW4 was cloned in the broad host range plasmid pRK415 under the control of the lac promoter and transferred into Azospirillum brasilense Cd and Sp245. A. brasilenseCd and Sp245 transformants showed high ACC deaminase activity, similar to that observed in Enterobacter cloacae UW4. The expression of ACC deaminase improved the existing growth promoting activity of Azospirillum. The roots of tomato and canola seedlings were significantly longer in plants inoculated with A. brasilense Cd transformants than those in plants inoculated with the nontransformed strains of the same bacterium. In the case of wheat seedlings, inoculation with A. brasilense Cd transformants did not promote root growth. The difference in plant response (canola and tomato versus wheat) is attributed to the greater sensitivity of canola and tomato plants to ethylene as compared to wheat plants.

Transcriptional regulation of the Enterobacter cloacae UW4 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene (acdS).

Based on DNA sequence analysis and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, the region of DNA immediately upstream of the Enterobacter cloacae UW4 ACC deaminase gene (acdS) contains several features that appear to be involved in its transcriptional regulation. In the present study, the 5' upstream region of acdS was cloned into the promoter-probe vector, pQF70, which carries the promoterless luciferase gene (luxAB), and luciferase expression was monitored. The data obtained from studying the expression of the luciferase gene showed that (i) a leucine responsive regulatory protein (LRP)-like protein encoded within the upstream region is located on the opposite strand from acdS under the control of a promoter stronger than the one responsible for acdS transcription, (ii) luciferase gene expression required both ACC and the LRP-like protein, (iii) luciferase expression was increased three-fold under anaerobic conditions, consistent with the involvement of a fumarate-nitrate reduction (FNR)-like regulatory protein box within the upstream region, and (iv) the addition of leucine to the growth medium decreased luciferase activity in the presence of ACC and increased luciferase activity in the absence of ACC, consistent with leucine acting as a regulator of the expression of the LRP-like protein.

Levels of ACC and related compounds in exudate and extracts of canola seeds treated with ACC deaminase-containing plant growth-promoting bacteria.

It was previously proposed that plant growth-promoting bacteria that possess 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase could utilize ACC that is present in the exudate of germinating canola seeds. The uptake and cleavage of ACC by these bacteria would lower the level of ACC, and thus ethylene within the plant, and reduce the extent of its inhibition on root elongation. To test part of the above mentioned model, ACC levels were monitored in canola seed tissues and exudate during germination. Lower amounts of ACC were present in the exudate and tissues of seeds treated with the plant growth-promoting bacterium Enterobacter cloacae CAL3, than in control seeds treated with MgSO4. The ACC-related compounds, alpha- and gamma-aminobutyric acids, both known to stimulate ethylene production, were also measured in the canola seed exudate and tissues. Approximately the same levels of alpha-aminobutyric acid were present in the exudates of the bacterium-treated seeds and the control seeds, but the amount of alpha-aminobutyric acid was lower in the tissues of the bacterium-treated seeds than in the control seeds. Smaller quantities of gamma-aminobutyric acid were seen in both the exudate and tissues of the E. cloacae CAL3-treated seeds than in the control seeds.

Biological activity and colonization pattern of the bioluminescence-labeled plant growth-promoting bacterium Kluyvera ascorbata SUD165/26.

Kluyvera ascorbata SUD165/26 is a spontaneous siderophore-overproducing mutant of K. ascorbata SUD165, which was previously isolated from nickel-contaminated soil and shown to significantly enhance plant growth in soil contaminated with high levels of heavy metals. To develop a better understanding of the functioning of K. ascorbata SUD165/26 in the environment, and to trace its distribution in the rhizosphere, isolates of this bacterium were labeled with either green fluorescent protein or luciferase. When the plant growth-promoting activities of the labeled strains were assayed and compared with the activities of the unlabeled strain, none of the monitored parameters had changed to any significant extent. When the spatial colonization patterns of the labeled bacteria on canola roots were determined after seed application, it was observed that the bacterium was tightly attached to the surface of both roots and seeds, and formed aggregates. The majority of the bacterial population inhabited the upper two thirds of the roots, with no bacteria detected around the root tips.

Synergism between Phyllobacterium sp. (N(2)-fixer) and Bacillus licheniformis (P-solubilizer), both from a semiarid mangrove rhizosphere.

Mangrove seedlings were treated with a mixture of two bacterial species, the slow-growing, N(2)-fixing bacterium Phyllobacterium sp. and the fast-growing, phosphate-solubilizing bacterium Bacillus licheniformis, both isolated from the rhizosphere from black, white, and red mangroves of a semiarid zone. Nitrogen fixation and phosphate solubilization increased when the mixture was used compared to the effects observed when adding individual cultures, notwithstanding that there was no increase in bacterial multiplication under these conditions. Inoculation of black mangrove seedlings in artificial seawater showed the mixture performed somewhat better than inoculation of the individual bacterium; more leaves were developed and higher levels of (15)N were incorporated into the leaves, although the total nitrogen level decreased. This study demonstrates that interactions between individual components of the rhizosphere of mangroves should be considered when evaluating these bacteria as plant growth promoters.

Determination of 1-aminocycopropane-1-carboxylic acid (ACC) to assess the effects of ACC deaminase-containing bacteria on roots of canola seedlings.

Previously, it was proposed that plant growth-promoting bacteria that possess the enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, can reduce the amount of ethylene produced by a plant and thereby promote root elongation. To test this model, canola seeds were imbibed in the presence of the chemical ethylene inhibitor, 2-aminoethoxyvinyl glycine (AVG), various strains of plant growth-promoting bacteria, and a psychrophilic bacterium containing an ACC deaminase gene on a broad host range plasmid. The extent of root elongation and levels of ACC, the immediate precursor of ethylene, were measured in the canola seedling roots. A modification of the Waters AccQ.Tag Amino Acid Analysis Method was used to quantify ACC in the root extracts. It was found that, in the presence of the ethylene inhibitor, AVG, or any one of several ACC deaminase-containing strains of bacteria, the growth of canola seedling roots was enhanced and the ACC levels in these roots were lowered.

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.

Reduced symptoms of Verticillium wilt in transgenic tomato expressing a bacterial ACC deaminase.

Summary Ethylene evolved during compatible or susceptible disease interactions may hasten and/or worsen disease symptom development; if so, the prevention of disease-response ethylene should reduce disease symptoms. We have examined the effects of reduced ethylene synthesis on Verticillium wilt (causal organism, Verticillium dahliae) of tomato by transforming tomato with ACC deaminase, which cleaves ACC, the immediate biosynthetic precursor of ethylene in plants. Three promoters were used to express ACC deaminase in the plant: (i) CaMV 35S (constitutive expression); (ii) rolD (limits expression specifically to the site of Verticillium infection, i.e. the roots); and (iii) prb-1b (limits expression to certain environmental cues, e.g. disease infection). Significant reductions in the symptoms of Verticillium wilt were obtained for rolD- and prb-1b-, but not for 35S-transformants. The pathogen was detected in stem sections of plants with reduced symptoms, suggesting that reduced ethylene synthesis results in increased disease tolerance. The effective control of formerly recalcitrant diseases such as Verticillium wilt may thus be obtained by preventing disease-related ethylene production via the tissue-specific expression of ACC deaminase.