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.

Molecular cloning of the phosphoenolpyruvate carboxylase gene of Anabaena variabilis.

Purified Anabaena variabilis chromosomal DNA was partially digested with restriction endonuclease Sau3A and ligated into the BamHI site of plasmid pBR322. Escherichia coli 342-167, a mutant with a decreased level of phosphoenolpyruvate carboxylase (PEPCase) activity was transformed with plasmids from the A. variabilis genomic library. A transformant that grew on minimal media in the absence of glutamate was isolated and its plasmid, pTRH1, was shown to encode the A. variabilis PEPCase. E. coli HB101 cells transformed with plasmid pTRH1 have approx. 50 times the normal amount of PEPCase activity and also overproduce a protein with the apparent Mr (99,000) of the A. variabilis PEPCase.

The identification and partial characterization of a plasmid containing the gene for the membrane-associated hydrogenase from E. coli.

Escherichia coli DNA was digested with restriction endonuclease PstI and ligated into the PstI site of plasmid pBR322. Recombinant plasmids that were constructed in this manner were used to transform E. coli H61, a mutant with a decreased level of hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for the membrane-associated E. coli hydrogenase in plasmid pBL101. In E. coli minicells, the pBL101 DNA directed the synthesis of a protein of a size corresponding to that of the precursor of the E. coli membrane-associated hydrogenase, which appears to contain an uncleaved leader peptide. A restriction map of the cloned DNA was determined for 14 endonucleases.

Metabolic load and heterologous gene expression.

The expression of a foreign protein(s) in a recombinant host cell or organism often utilizes a significant amount of the host cell's resources, removing those resources away from host cell metabolism and placing a metabolic load (metabolic drain, metabolic burden) on the host. As a consequence of the imposed metabolic load, the biochemistry and physiology of the host may be dramatically altered. The numerous physiological changes that may occur often lowers the amount of the target foreign protein that is produced and eventually recovered from the recombinant organism. In this review the physiological changes to host cells, the causes of the phenomenon of metabolic load, and several strategies to avoid some of the problems associated with metabolic load are elaborated and discussed.

Biotechnological uses of cyanobacteria.

Cyanobacteria (blue-green algae) are O(2)-evolving photosynthesizing prokaryotes that have an extensive history of use as a human food source and as a fertilizer in rice fields. They have also been recognized as an excellent source of vitamins and proteins and as such are found in health food stores in North America and elsewhere. Cyanobacteria have a great deal of potential as a source of fine chemicals, as a biofertilizer and as a source of renewable fuel. This potential is being realized as data from research in the areas of the physiology and chemistry of these organisms are gathered and as the knowledge of cyanobacterial genetics and genetic engineering increases. We review, here, the present (and possible future) uses of cyanobacteria and assess the state of the art with respect to the genetic manipulation of cyanobacteria.

Isolation, characterization and manipulation of cellulase genes.

The complete hydrolysis of cellulose requires a number of different enzymes including endoglucanase, exoglucanase and beta-glucosidase. These enzymes function in concert as part of a 'cellulase'complex called a cellulosome. In order (i) to develop a better understanding of the biochemical nature of the cellulase complex as well as the genetic regulation of its integral components and (ii) to utilize cellulases either as purified enzymes or as part of an engineered organism for a variety of purposes, researchers have, as a first step, used recombinant DNA technology to isolate the genes for these enzymes from a variety of organisms. This review provides some perspective on the current status of the isolation, characterization and manipulation of cellulase genes and specifically discusses (i) strategies for the isolation of endoglucanase, exoglucanase and beta-glucosidase genes; (ii) DNA sequence characterization of the cellulase genes and their accompanying regulatory elements; (iii) the expression of cellulase genes in heterologous host organisms and (iv) some of the proposed uses for isolated cellulase genes.

Genetic manipulation of plant growth-promoting bacteria to enhance biocontrol of phytopathogens.

Plant growth-promoting bacteria (PGPB) control the damage to plants from phytopathogens by a number of different mechanisms including: outcompeting the phytopathogen, physical displacement of the phytopathogen, secretion of siderophores to prevent pathogens in the immediate vicinity from proliferating, synthesis of antibiotics, synthesis of a variety of small molecules that can inhibit phytopathogen growth, production of enzymes that inhibit the phytopathogen and stimulation of the systemic resistance of the plant. Biocontrol PGPB may be improved by genetically engineering them to overexpress one or more of these traits so that strains with several different anti-phytopathogen traits which can act synergistically are created. In engineering these strains it is essential to ensure that the normal functioning of the bacterium is not impaired, i.e., that there is no problem with metabolic load.

Environmental implications of recombinant DNA technology.

Applications of recombinant DNA technology are discussed as a backdrop for evaluation of the environmental impacts of this technology. Some of applications include using traditional biological techniques for specific purposes, including nitrogen fixation, microbial pesticides, and waste treatment. In these instances the final product lies along a continuum, beginning with an organism marginally performing its function, and ending with one that is highly specialised and very efficient in what it does. One may move along this continuum toward the 'perfect' microorganism by using traditional methodologies of mutagenesis and selection, recombinant DNA technology, or a combination of the two.

Molecular laser biotechnology.

Laser technology has developed to the point where it is possible to utilize lasers as a sophisticated but accessible tool in understanding and manipulating gene functioning. This review emphasizes some of the systems that employ lasers in the new and growing field of molecular laser biotechnology. Here the main emphasis is on the manipulation and understanding of bacterial and plant systems.

Development of DNA-mediated transformation systems for plants.

The genetic engineering of plants by DNA-mediated gene transfer requires that efficient transformation systems be developed. Considerable progress has been made in manipulating the Ti plasmid of Agrobacterium tumefaciens as a vehicle for delivery of foreign genes into protoplasts of dicotyle-donous plants. Part of the Ti plasmid, the T-DNA, can be incorporated into the genome of the host cell; the T-DNA can carry a foreign DNA sequence which co-integrates with it; under normal conditions, the tumorigenic-causing portion of the T-DNA can be inactivated so that transformed protoplasts can be regenerated and T-DNA with an inserted foreign gene can be stably maintained during regeneration, meiosis and gamete formation. A foreign gene has yet to be expressed in regenerated plants although a T-DNA gene for opine synthesis can function in regenerates. Developing a more ubiquitous transformation system for monocotyledons is further from fruition. Based on transformation systems for simple eukaryotic organisms, it is reasonable to expect that a DNA vector which is capable of amplifying a novel plant gene and which contains both a drug resistance marker to facilitate the selection of transformed plant protoplasts and a species-specific autonomously replicating sequence to ensure the stable maintenance of the input gene in the recipient cell can be constructed.

Increased ability of transgenic plants expressing the bacterial enzyme ACC deaminase to accumulate Cd, Co, Cu, Ni, Pb, and Zn.

Transgenic tomato plants Lycopersicon esculentum (Solanaceae) cv. Heinz 902 expressing the bacterial gene 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, under the transcriptional control of either two tandem 35S cauliflower mosaic virus promoters (constitutive expression), the rolD promoter from Agrobacterium rhizogenes (root specific expression) or the pathogenesis related PRB-1b promoter from tobacco, were compared to non-transgenic tomato plants in their ability to grow in the presence of Cd, Co, Cu, Mg, Ni, Pb, or Zn and to accumulate these metals. Parameters that were examined include metal concentration and ACC deaminase activity in both plant shoots and roots; root and shoot development; and leaf chlorophyll content. In general, transgenic tomato plants expressing ACC deaminase, especially those controlled by the PRB-1b promoter, acquired a greater amount of metal within the plant tissues, and were less subject to the deleterious effects of the metals on plant growth than were non-transgenic plants.

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.

Enzymes that regulate ethylene levels--1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, ACC synthase and ACC oxidase.

The plant enzymes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase, catalyze essential steps in the biosynthesis of the phytohormone ethylene; the microbial enzyme ACC deaminase catalyses the hydrolytic cleavage of ACC, the immediate precursor of ethylene, and is therefore an inhibitor of ethylene biosynthesis. In this manuscript, the biochemical properties and mechanisms of these three enzymes and the genes that encode them are examined and compared. Despite the fact that ACC oxidase and ACC deaminase both act on the same substrate, i.e., ACC, these two enzymes and the mechanisms that they employ are quite different. Conversely, although ACC synthase catalyses the synthesis of ACC and ACC deaminase catalyses its hydrolysis, these enzymes share a number of important physical and biochemical properties.