Expression of two recombinant chloramphenicol acetyltransferase variants in highly reduced genome Escherichia coli strains

Highly reduced E. coli strains, MDS40, MDS41, and MDS42, lacking approximately 15% of the genome, were grown to high cell densities to test their ability to produce a recombinant protein with high yields. These strains lack all transposons and insertion sequences, cryptic prophage and many genes of unknown function. In addition to improving genetic stability, these deletions may reduce the biosynthetic requirements of the cell potentially allowing more efficient production of recombinant protein. Basic growth parameters and the ability of the strains to produce chloramphenicol acetyltransferase (CAT) under high cell density, batch cultivation were assessed. Although growth rate and recombinant protein production of the reduced genome strains are comparable to the parental MG1655 strain, the reduced genome strains were found to accumulate significant amounts of acetate in the medium at the expense of additional biomass. A number of hypotheses were examined to explain the accumulation of acetate, including oxygen limitation, carbon flux imbalance, and metabolic activity of the recombinant protein. Use of a non-catalytic CAT variant identified the recombinant protein activity as the source of this phenomenon; implications for the metabolic efficiency of the reduced genome strains are discussed.     

Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.     

Characterization of a Listeria monocytogenes Scott A Isolate with High Tolerance towards High Hydrostatic Pressure

An isolate of L. monocytogenes Scott A that is tolerant to high hydrostatic pressure (HHP), named AK01, was isolated upon a single pressurization treatment of 400 MPa for 20 min and was further characterized. The survival of exponential- and stationary-phase cells of AK01 in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer was at least 2 log units higher than that of the wild type over a broad range of pressures (150 to 500 MPa), while both strains showed higher HHP tolerance (piezotolerance) in the stationary than in the exponential phase of growth. In semiskim milk, exponential-phase cells of both strains showed lower reductions upon pressurization than in buffer, but again, AK01 was more piezotolerant than the wild type. The piezotolerance of AK01 was retained for at least 40 generations in rich medium, suggesting a stable phenotype. Interestingly, cells of AK01 lacked flagella, were elongated, and showed slightly lower maximum specific growth rates than the wild type at 8, 22, and 30°C. Moreover, the piezotolerant strain AK01 showed increased resistance to heat, acid, and H₂O₂ compared with the wild type. The difference in HHP tolerance between the piezotolerant strain and the wild-type strain could not be attributed to differences in membrane fluidity, since strain AK01 and the wild type had identical in situ lipid melting curves as determined by Fourier transform infrared spectroscopy. The demonstrated occurrence of a piezotolerant isolate of L. monocytogenes underscores the need to further investigate the mechanisms underlying HHP resistance of food-borne microorganisms, which in turn will contribute to the appropriate design of safe, accurate, and feasible HHP treatments.     

Arabinose content of extracellular polysaccharide plays a role in cell aggregation of Azospirillum brasilense

Extracellular polysaccharides play an important role in aggregation and surface colonization of plant-associated bacteria. In this work, we report the time course production and monomer composition of the exopolysaccharide (EPS) produced by wild type strain and several mutants of the plant growth promoting rhizobacterium (PGPR) Azospirillum brasilense. In a fructose synthetic medium, wild type strain Sp7 produced a glucose-rich EPS during exponential phase growth and an arabinose-rich EPS during stationary and death phase growth. D-glucose or L-arabinose did not support cell growth as sole carbon sources. However, glucose and arabinose-rich EPSs, when used as carbon source, supported bacterial growth. Cell aggregation of Sp7 correlated with the synthesis of arabinose-rich EPS. exoB (UDP-glucose 4'-epimerase), exoC (phosphomannomutase) and phbC (poly-beta-hydroxyburyrate synthase) mutant strains, under tested conditions, produced arabinose-rich EPS and exhibited highly cell aggregation capability. A mutant defective in LPS production (dTDP 4-rhamnose reductase; rmlD) produced glucose-rich EPS and did not aggregate. These results support that arabinose content of EPS plays an important role in cell aggregation. Cell aggregation appears to be a time course phenomenon that takes place during reduced metabolic cell activity. Thus, aggregation could constitute a protected model of growth that allows survival in a hostile environment. The occurrence of exoC and rmlD was detected in several species of Azospirillum.     

Metabolic modeling for multi-objective optimization of ethanol production in a Synechocystis mutant

Cyanobacteria have potential to produce drop-in bio-fuels such as ethanol via photoautotrophic metabolism. Although model cyanobacterial strains have been engineered to produce such products, systematic metabolic engineering studies to identify optimal strains for the same have not been performed. In this work, we identify optimal ethanol producing mutants corresponding to appropriate gene deletions that result in a suitable redirection in the carbon flux. In particular, we systematically simulate exhaustive single and double gene deletions considering a genome scale metabolic model of a mutant strain of the unicellular cyanobacterium Synechocystis species strain PCC 6803. Various optimization based metabolic modeling techniques, such as flux balance analysis (FBA), method of minimization of metabolic adjustment (MOMA) and regulatory on/off minimization (ROOM) were used for this analysis. For single gene deletion MOMA simulations, the Pareto front with biomass and ethanol fluxes as the two objectives to be maximized was obtained and analyzed. Points on the Pareto front represent maximal utilization of resources constrained by substrate uptake thereby representing an optimal trade-off between the two fluxes. Pareto analysis was also performed for double gene deletion MOMA and single and double gene deletion ROOM simulations. Based on these analyses, two mutants, with combined gene deletions in ethanol and purine metabolism pathways, were identified as promising candidates for ethanol production. The relevant genes were adk, pta and ackA. An ethanol productivity of approximately 0.15 mmol/(gDW h) was predicted for these mutants which appears to be reasonable based on experimentally reported values in literature for other strains.     

Carbon-Flux Distribution within Streptomyces coelicolor Metabolism: A Comparison between the Actinorhodin-Producing Strain M145 and Its Non-Producing Derivative M1146

Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and ¹³C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.     

一株阴环炭团菌(香灰菌)转化子形态变异的成因

遗传转化方法是基因功能研究的重要方法之一,但在转化过程常出现转化子变异的现象,影响目标基因功能的判断。本文以阴环炭团菌菌落形态特征异常的拟基因TJAS01-V10012900敲除菌株12900-5为研究对象,通过PCR手段验证基因敲除的真实性,基因组重测序分析各转化子在基因敲除区域以及基因组其他区域的差异,转录组测序比较12900-5与其他转化子在基因表达层面的差异。比较基因组发现,12900-5与12900-6菌株在潮霉素抗性基因插入位置与片段一致,且不存在多拷贝现象。12900-5菌株有两个特异SNP位于不同的基因,一个与细胞周期S期有关,另一个与囊泡转运有关,两者均与菌丝生长有关。由此推测,转化子12900-5形态特征异常很有可能与这两个非靶标位点突变有关。转录组分析结果发现12900-5菌株在氨基酸代谢通路、翻译过程等多个代谢与信号途径基因的表达量明显下降。本研究例证了转化过程存在的非靶标变异现象,但导致这种变异的原因仍需进一步研究。     

Genome Shuffling of Clostridium acetobutylicum CICC 8012 for Improved Production of Acetone–Butanol–Ethanol (ABE)

Genome shuffling was applied to increase ABE production of the strict anaerobe C. acetobutylicum CICC 8012. By using physical and chemical mutagenesis, strains with superior streptomycin sulfate, 2-deoxy-D-glucose and butanol tolerance levels were isolated. These strains were used for genome shuffling. The best performing strain F2-GA was screened after two rounds of genome shuffling. With 55 g glucose/l as carbon source, F2-GA produced 22.21 g ABE/l in 72 h and ABE yield reached 0.42 g/g which was about 34.53 % improvement compared to the wild type. Fermentation parameters and gene expression of several key enzymes in ABE metabolic pathways were varied significantly between F2-GA and the wild type. These results demonstrated the potential use of genome shuffling to microbial breeding which were difficult to deal with traditional methods.     

Mapmi gene contributes to stress tolerance and virulence of the entomopathogenic fungus, Metarhizium acridum

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate (Fru-6-P) and mannose 6-phosphate (Man-6-P), providing a link between glycolysis and the mannose metabolic pathway. In this study, we identified pmi gene (Mapmi) from the entomopathogenic fungus, Metarhizium acridum, and analyzed its functions using RNA interference (RNAi). Amending the growth medium with cell stress chemicals significantly reduced growth, conidial production and percent germination in Mapmi-RNAi mutant strain, compared to the wild-type strain. Growth of RNAi mutant was lower than the wild type strain with glucose or fructose as sole carbon source. RNAi mutant exhibited a normal growth phenotype with mannose at low concentrations, while trace or high concentration of mannose was more negatively impacted the growth of RNAi mutant than the wild type strain. Infection with Mapmi-RNAi mutant against Locusta migratoria manilensis (Meyen) led to a significantly reduced virulence compared to infection with the wild-type strain. These results suggest that Mapmi plays essential roles in stress tolerance and pathogenicity of M. acridum.     

Increased glycopeptide production after overexpression of shikimate pathway genes being part of the balhimycin biosynthetic gene cluster

Amycolatopsis balhimycina produces the vancomycin-analogue balhimycin. The strain therefore serves as a model strain for glycopeptide antibiotic production. Previous characterisation of the balhimycin biosynthetic cluster had shown that the border sequences contained both, a putative 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (dahp), and a prephenate dehydrogenase (pdh) gene. In a metabolic engineering approach for increasing the precursor supply for balhimycin production, the dahp and pdh genes from the biosynthetic cluster were overexpressed both individually and together and the resulting strains were subjected to quantitative physiological characterisation. The constructed strains expressing an additional copy of the dahp gene and the strain carrying an extra copy of both dahp and pdh showed improved specific glycopeptide productivities by approximately a factor three, whereas the pdh overexpression strain showed a production profile similar to the wild type strain. In addition to the overexpression strains, corresponding deletion mutants, Δdahp and Δpdh, were constructed and characterised. Deletion of dahp resulted in significant reduction in balhimycin production whereas the Δpdh strain had production levels similar to the parent strain. Based on these results the relation between primary and secondary metabolism with regards to Dahp and Pdh is discussed.     

Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122

The synthesis of human superoxide dismutase (SOD) in batch cultures of a Saccharomyces cerevisiae strain using a glucose-limited minimal medium was studied through metabolic flux analysis. A stoichiometric model was built, which included 78 reactions, according to metabolic pathways operative in these strains during respirofermentative and oxidative metabolism. It allowed calculation of the distribution of metabolic fluxes during diauxic growth on glucose and ethanol. Fermentation profiles and metabolic fluxes were analyzed at different phases of diauxic growth for the recombinant strain (P+) and for its wild type (P-). The synthesis of SOD by the strain P+ resulted in a decrease in specific growth rate of 34 and 54% (growth on glucose and ethanol respectively) in comparison to the wild type. Both strains exhibited similar flux of glucose consumption and ethanol synthesis but important differences in carbon distribution with biomass/substrate yields and ATP production 50% higher in P-. A higher contribution of fermentative metabolism, with 64% of the energy produced at the phosphorylation level, was observed during SOD production. The flux of precursors to amino acids and nucleotides was higher in the recombinant strain, in agreement with the higher total RNA and protein levels. Lower specific growth rates in strain P+ appear to be related to the decrease in the rate of synthesis of nonrecombinant protein, as well as a decrease in the activities of the pentose phosphate (PP) pathway and TCA cycle. A very different way of entry into the stationary phase was observed for each strain: in the wild-type strain most metabolic fluxes decreased and fluxes related to energy reserve synthesis increased, while in the P+ strain the flux of 22 reactions (including PP pathway and amino acids biosynthesis) related to SOD production increased their fluxes. Changes in SOD production rates at different physiological states appear to be related to the differences in building blocks availability between respirofermentative and oxidative metabolism. Using the present expression system, ideal conditions for SOD synthesis are represented by either active growth during respirofermentative metabolism or transition from a growing to a nongrowing state. An increase in SOD flux could be achieved using an expression system nonassociated to growth and potentially eliminating part of the metabolic burden.     

Robust metabolic responses to varied carbon sources in natural and laboratory strains of Saccharomyces cerevisiae

Understanding factors that regulate the metabolism and growth of an organism is of fundamental biologic interest. This study compared the influence of two different carbon substrates, dextrose and galactose, on the metabolic and growth rates of the yeast Saccharomyces cerevisiae. Yeast metabolic and growth rates varied widely depending on the metabolic substrate supplied. The metabolic and growth rates of a yeast strain maintained under long-term laboratory conditions was compared to strain isolated from natural condition when grown on different substrates. Previous studies had determined that there are numerous genetic differences between these two strains. However, the overall metabolic and growth rates of a wild isolate of yeast was very similar to that of a strain that had been maintained under laboratory conditions for many decades. This indicates that, at in least this case, metabolism and growth appear to be well buffered against genetic differences. Metabolic rate and cell number did not co-vary in a simple linear manner. When grown in either dextrose or galactose, both strains showed a growth pattern in which the number of cells continued to increase well after the metabolic rate began a sharp decline. Previous studied have reported that O₂ consumption in S. cerevisiae grown in reduced dextrose levels were elevated compared to higher levels. Low dextrose levels have been proposed to induce caloric restriction and increase life span in yeast. However, there was no evidence that reduced levels of dextrose increased metabolic rates, measured by either O₂ consumption or CO₂ production, in the strains used in this study.     

Disruption of the pdhB Pyruvate Dehydrogenase Gene Affects Colony Morphology,In Vitro Growth and Cell Invasiveness of Mycoplasma agalactiae

The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae.     

iTRAQ analysis reveals the effect of gabD and sucA gene knockouts on lysine metabolism and crystal protein formation in Bacillus thuringiensis

Lysine metabolism plays an important role in the formation of the insecticidal crystal proteins of Bacillus thuringiensis (Bt). The genes lam, gabD and sucA encode three key enzymes of the lysine metabolic pathway in Bt4.0718. The lam gene mainly affects the cell growth at stable period, negligibly affected sporulation and insecticidal crystal protein (ICP) production. While, the deletion mutant strains of the gabD and sucA genes showed that the growth, sporulation and crystal protein formation were inhibited, cells became slender, and insecticidal activity was significantly reduced. iTRAQ proteomics and qRT-PCR used to analyse the differentially expressed protein (DEP) between the two mutant strains and the wild type strain. The functions of DEPs were visualized and statistically classified, which affect bacterial growth and metabolism by regulating biological metabolism pathways: the major carbon metabolism pathways, amino acid metabolism, oxidative phosphorylation pathways, nucleic acid metabolism, fatty acid synthesis and peptidoglycan synthesis. The gabD and sucA genes in lysine metabolic pathway are closely related to the sporulation and crystal proteins formation. The effects of DEPs and functional genes on basic cellular metabolic pathways were studied to provide new strategies for the construction of highly virulent insecticidal strains, the targeted transformation of functional genes.     

Rational design and construction of an efficient E. coli for production of diapolycopendioic acid

We applied elementary mode analysis to a recombinant metabolic network of carotenoid-producing E. coli in order to identify multiple-gene knockouts for an enhanced synthesis of the carotenoids diapolycopendial (DPL) and diapolycopendioic acid (DPA). Based on the model, all inefficient carotenoid biosynthesis pathways were eliminated in a strain containing a combination of eight gene deletions. To validate the model prediction, the designed strain was constructed and tested for its performance. The designed mutant produces the carotenoids at significantly increased yields and rates as compared to the wild-type. The consistency between model prediction and experimental results demonstrates that elementary mode analysis is useful as a guiding tool also for the rational strain design of more complex pathways for secondary metabolite production.     

Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism

The transformation of trinitrotoluene (TNT) by several mutant strains of Clostridium acetobutylicum has been examined to analyze the maximal rate of initial transformation, determine the effects of metabolic mutations of the host on transformation rate, and to assess the cell metabolic changes brought about during TNT transformation. Little difference in the maximal rate of TNT degradation in early acid phase cultures was found between the parental ATCC 824 strain and strains altered in the acid forming pathways (phosphotransacetylase, or butyrate kinase) or in a high-solvent-producing strain (mutant B). This result is in agreement with the previous findings of a similar degradation rate in a degenerate strain (M5) that had lost the ability to produce solvent. A series of antisense constructs were made that reduced the expression of hydA, encoding the Fe-hydrogenase, or hydE and hydF, genes encoding hydrogenase maturating proteins. While the antisense hydA strain had only ～30 % of the activity of wild type, the antisense hydE strain exhibited a TNT degradation rate around 70 % that of the parent. Overexpression of hydA modestly increased the TNT degradation rate in acid phase cells, suggesting the amount of reductant flowing into hydrogenase rather than the hydrogenase level itself was a limiting factor in many situations. The redox potential, hydrogen evolution, and organic acid metabolites produced during rapid TNT transformation in early log phase cultures were measured. The redox potential of the acid-producing culture decreased from ?370 to ?200 mV immediately after addition of TNT and the hydrogen evolution rate decreased, lowering the hydrogen to carbon dioxide ratio from 1.4 to around 1.1 for 15 min. During the time of TNT transformation, the treated acidogenic cells produced less acetate and more butyrate. The results show that during TNT transformation, the cells shift metabolism away from hydrogen formation to reduction of TNT and the resulting effects on cell redox cofactors generate a higher proportion of butyrate.     

In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates

Here, we present systems metabolic engineering driven by in-silico modeling to tailor Pseudomonas putida for synthesis of medium chain length PHAs on glucose. Using physiological properties of the parent wild type as constraints, elementary flux mode analysis of a large-scale model of the metabolism of P. putida was used to predict genetic targets for strain engineering. Among a set of priority ranked targets, glucose dehydrogenase (encoded by gcd) was predicted as most promising deletion target. The mutant P. putida Δgcd, generated on basis of the computational design, exhibited 100% increased PHA accumulation as compared to the parent wild type, maintained a high specific growth rate and exhibited an almost unaffected gene expression profile, which excluded detrimental side effects of the modification. A second mutant strain, P. putida Δpgl, that lacked 6-phosphogluconolactonase, exhibited a substantially decreased PHA synthesis, as was also predicted by the model. The production potential of P. putida Δgcd was assessed in batch bioreactors. The novel strain showed an increase of the PHA yield (+80%), the PHA titer (+100%) and cellular PHA content (+50%) and revealed almost unaffected growth and diminished by-product formation. It was thus found superior in all relevant criteria towards industrial production. Beyond the contribution to more efficient PHA production processes at reduced costs that might replace petrochemical plastics in the future, the study illustrates the power of computational prediction to tailor microbial strains for enhanced biosynthesis of added-value compounds.     

Characterizing the Metabolism of Dehalococcoides with a Constraint-Based Model

Dehalococcoides strains respire a wide variety of chloro-organic compounds and are important for the bioremediation of toxic, persistent, carcinogenic, and ubiquitous ground water pollutants. In order to better understand metabolism and optimize their application, we have developed a pan-genome-scale metabolic network and constraint-based metabolic model of Dehalococcoides. The pan-genome was constructed from publicly available complete genome sequences of Dehalococcoides sp. strain CBDB1, strain 195, strain BAV1, and strain VS. We found that Dehalococcoides pan-genome consisted of 1118 core genes (shared by all), 457 dispensable genes (shared by some), and 486 unique genes (found in only one genome). The model included 549 metabolic genes that encoded 356 proteins catalyzing 497 gene-associated model reactions. Of these 497 reactions, 477 were associated with core metabolic genes, 18 with dispensable genes, and 2 with unique genes. This study, in addition to analyzing the metabolism of an environmentally important phylogenetic group on a pan-genome scale, provides valuable insights into Dehalococcoides metabolic limitations, low growth yields, and energy conservation. The model also provides a framework to anchor and compare disparate experimental data, as well as to give insights on the physiological impact of “incomplete” pathways, such as the TCA-cycle, CO₂ fixation, and cobalamin biosynthesis pathways. The model, referred to as iAI549, highlights the specialized and highly conserved nature of Dehalococcoides metabolism, and suggests that evolution of Dehalococcoides species is driven by the electron acceptor availability.     

茎瘤固氮根瘤菌环二鸟苷酸代谢相关基因的功能鉴定

【目的】考察茎瘤固氮根瘤菌ORS571中c-di-GMP合成酶AZC-2412的编码基因缺失的突变表型,初步探究其功能机理。【方法】本实验构建基于cre-loxp重组酶系统的根瘤菌基因敲除系统,以及采用三亲接合技术构建突变株。测定野生型和突变株的生长速率、趋化能力、胞外多糖产量、生物膜形成等表型。【结果】突变株与野生型生长速率几乎相同。与野生型相比突变株由于细胞内c-di-GMP水平降低,胞外多糖、生物膜产量等均有所下降。【结论】实验表明,环二鸟苷酸合成酶AZC-2412缺失,使得c-di-GMP水平降低,对胞外多糖生成、细菌的运动能力、生物膜的形成、细胞絮凝、与植物的互作等均有调控作用。