Design rules for efficient transgene expression in plants.

Sustained expression of transgenes in specified developmental patterns is commonly needed in plant biotechnology, but obstructed by transgene silencing. Here, we present a set of gene design rules, tested on the silencing-susceptible beetle luc and bacterial ims genes, expressed in sugarcane. Designs tested independently or in combination included removal of rare codons, removal of RNA instability sequences, blocking of likely endogenous sRNA binding sites and randomization of non-rare codons. Stable transgene expression analyses, on multiple independent lines per construct, showed greatest improvement from the removal of RNA instability sequences, accompanied by greatly reduced transcript degradation evident in northern blot analysis. We provide a set of motifs that readily can be eliminated concurrently with rare codons and undesired structural features such as repeat sequences, using Gene Designer 2.0 software. These design rules yielded 935- and 5-fold increased expression in transgenic callus, relative to the native luc and ims sequences; and gave sustained expression under the control of sugarcane and heterologous promoters over several years in greenhouse and field trials. The rules can be applied easily with codon usage tables from any plant species, providing a simple and effective means to achieve sustained expression of otherwise silencing-prone transgenes in plants.

Diversity of sequences and expression patterns among alleles of a sugarcane loading stem gene.

Modern sugarcane cultivars are highly polyploid and aneuploid hybrids, which are propagated as clones. Their complex genome structure comprises 100-130 chromosomes and 10-13 hom(e)ologous copies of most loci. There is preliminary evidence of very high heterozygosity, with implications for genetic improvement approaches ranging from marker-assisted selection to transgenics. Here, we report that sugarcane cultivar Q200 has at least nine alleles at the Loading Stem Gene (ScLSG) locus. Exon-intron structure is identical and the predicted protein products show at least 92 % identity, across sugarcane alleles and the Sorghum homologue Sb07g027880. There is substantial variation in the 5' UTR and promoter regions including numerous allele-specific nucleotide polymorphisms, insertions and deletions. We developed an allele-specific qRT-PCR method to undertake the first compelling test of allele-specific expression in polyploid sugarcane. Seven alleles distinguished by this method all showed peak expression in the sucrose-loading zone of the stem, but there was apparent variability in expression patterns across other tissues. The ScLSG2 and ScLSG5 alleles appear promising for specificity of expression in stems, relative to leaf, meristem, emerging shoot and root tissues. Within the stem, there was activity in parenchyma, vascular and rind tissues. This expression pattern is of interest in basic research and biotechnology aimed at enhanced sucrose content, engineering value-added products, and manipulation of stem biomass composition.

Sugarcane Loading Stem Gene promoters drive transgene expression preferentially in the stem.

Promoter regions of six sugarcane Loading Stem Gene (ScLSG) alleles were analyzed using bioinformatic and transgenic approaches. Stable transgene expression analyses, on multiple independent lines per construct, revealed differences between ScLSG promoters in absolute levels and in tissue-selectivity of luciferase reporter activity. Four promoters drove peak expression in the sucrose-loading zone and maintained substantial expression throughout mature stems. One drove a pattern of gradual increase along the stem maturation profile. In general, stem: root expression ratio increased with plant age. The ScLSG5 promoter had the fewest light-enhanced and root-expression motifs in bioinformatic analysis, and drove the highest level and specificity of transgene expression in stems. This indicates the potential to further improve the stem specificity of ScLSG promoter sequences by eliminating enhancers of expression in other tissues. An intron in the 5'UTR was important for expression strength. The ScLSG promoters will be useful for research and biotechnology in sugarcane, where the tailored expression of transgenes in stems is important for enhanced accumulation of sugar or value-added products, and for development as a bioenergy feedstock.

Mature-stem expression of a silencing-resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in sugarcane.

Isomaltulose (IM) is a natural isomer of sucrose. It is widely approved as a food with properties including slower digestion, lower glycaemic index and low cariogenicity, which can benefit consumers. Availability is currently limited by the cost of fermentative conversion from sucrose. Transgenic sugarcane plants with developmentally-controlled expression of a silencing-resistant gene encoding a vacuole-targeted IM synthase were tested under field conditions typical of commercial sugarcane cultivation. High yields of IM were obtained, up to 483 mm or 81% of total sugars in whole-cane juice from plants aged 13 months. Using promoters from sugarcane to drive expression preferentially in the sugarcane stem, IM levels were consistent between stalks and stools within a transgenic line and across consecutive vegetative field generations of tested high-isomer lines. Germination and early growth of plants from setts were unaffected by IM accumulation, up to the tested level around 500 mm in flanking stem internodes. These are the highest yields ever achieved of value-added materials through plant metabolic engineering. The sugarcane stem promoters are promising for strategies to achieve even higher IM levels and for other applications in sugarcane molecular improvement. Silencing-resistant transgenes are critical to deliver the potential of these promoters in practical sugarcane improvement. At the IM levels now achieved in field-grown sugarcane, direct production of IM in plants is feasible at a cost approaching that of sucrose, which should make the benefits of IM affordable on a much wider scale.

Metabolic engineering of sugars and simple sugar derivatives in plants.

Carbon captured through photosynthesis is transported, and sometimes stored in plants, as sugar. All organic compounds in plants trace to carbon from sugars, so sugar metabolism is highly regulated and integrated with development. Sugars stored by plants are important to humans as foods and as renewable feedstocks for industrial conversion to biofuels and biomaterials. For some purposes, sugars have advantages over polymers including starches, cellulose or storage lipids. This review considers progress and prospects in plant metabolic engineering for increased yield of endogenous sugars and for direct production of higher-value sugars and simple sugar derivatives. Opportunities are examined for enhancing export of sugars from leaves. Focus then turns to manipulation of sugar metabolism in sugar-storing sink organs such as fruits, sugarcane culms and sugarbeet tubers. Results from manipulation of suspected 'limiting' enzymes indicate a need for clearer understanding of flux control mechanisms, to achieve enhanced levels of endogenous sugars in crops that are highly selected for this trait. Outcomes from in planta conversion to novel sugars and derivatives range from severe interference with plant development to field demonstration of crops accumulating higher-value sugars at high yields. The differences depend on underlying biological factors including the effects of the novel products on endogenous metabolism, and on biotechnological fine-tuning including developmental expression and compartmentation patterns. Ultimately, osmotic activity may limit the accumulation of sugars to yields below those achievable using polymers; but results indicate the potential for increases above current commercial sugar yields, through metabolic engineering underpinned by improved understanding of plant sugar metabolism.

Comparison of Agrobacterium and particle bombardment using whole plasmid or minimal cassette for production of high-expressing, low-copy transgenic plants.

Transgene integration complexity in the recipient genome can be an important determinant of transgene expression and field performance in transgenic crops. We provide the first direct comparison of Agrobacterium-mediated transformation (AMT) and particle bombardment using whole plasmid (WP) and excised minimal cassettes (MC), for transformation efficiency, transgene integration complexity and transgene expression in plants. To enable direct comparison, a selectable marker and a luciferase reporter gene were linked in identical configurations in plasmids suitable for AMT or direct gene transfer into sugarcane. Transformation efficiencies were similar between WP and MC when equal molar DNA quantities were delivered. When the MC concentration was reduced from 66 to 6.6 ng per shot, transformation efficiency dropped fourfold, to a level equivalent with AMT in amenable genotype Q117. The highest proportion of transformants combining low copy number (estimated below two integrated copies by qPCR) with expression of the non-selected reporter gene was obtained using AMT (55 %) or MC at low DNA concentration (30 %). In sugarcane, both of these methods yielded high-expressing, single-copy transgenic plant lines at a workable efficiency for practical plant improvement; but AMT is currently limited to a few amenable genotypes. These methods are best coupled with rapid early screens for desired molecular characteristics of transformants, e.g. PCR screens for low copy number and/or transcription of the gene of practical interest.

Microbial sucrose isomerases: producing organisms, genes and enzymes.

Sucrose isomerase (SI) activity is used industrially for the conversion of sucrose into isomers, particularly isomaltulose or trehalulose, which have properties advantageous over sucrose for some food uses. All of the known microbial SIs are TIM barrel proteins that convert sucrose without need for any cofactors, with varying kinetics and product specificities. The current analysis was undertaken to bridge key gaps between the information in patents and scientific publications about the microbes and enzymes useful for sucrose isomer production. This analysis shows that microbial SIs can be considered in 5 structural classes with corresponding functional distinctions that broadly align with the taxonomic differences between producing organisms. The most widely used bacterial strain for industrial production of isomaltulose, widely referred to as "Protaminobacter rubrum" CBS 574.77, is identified as Serratia plymuthica. The strain producing the most structurally divergent SI, with a high product specificity for trehalulose, widely referred to as "Pseudomonas mesoacidophila" MX-45, is identified as Rhizobium sp. Each tested SI-producer is shown to have a single SI gene and enzyme, so the properties reported previously for the isolated proteins can reasonably be associated with the products of the genes subsequently cloned from the same isolates and SI classes. Some natural isolates with potent SI activity do not catabolize the isomer under usual production conditions. The results indicate that their industrial potential may be further enhanced by selection for variants that do not catabolize the sucrose substrate.

Field performance of transgenic sugarcane expressing isomaltulose synthase.

Transgenic sugarcane plants expressing a vacuole-targeted isomaltulose (IM) synthase in seven recipient genotypes (elite cultivars) were evaluated over 3 years at a field site typical of commercial cane growing conditions in the Burdekin district of Australia. IM concentration typically increased with internode maturity and comprised up to 217 mm (33% of total sugars) in whole-cane juice. There was generally a comparable decrease in sucrose concentration, with no overall decrease in total sugars. Sugarcane is vegetatively propagated from stem cuttings known as setts. Culture-derived plants were slower to establish and generally gave shorter and thinner stalks at harvest than those grown from field-sourced setts in the initial field generations. However, after several cycles of field propagation, selections were obtained with cane yields similar to the recipient genotypes. There was no apparent adverse effect of IM accumulation on vigour assessed by stalk height and diameter or other visual indicators including germination of setts and establishment of stools. There was some inconsistency in IM levels in juice, between samplings of the vegetatively propagated transgenic lines. Until the causes are resolved, it is prudent to selectively propagate from stalks with higher IM levels in the initial vegetative field generations. Pol/Brix ratio allowed rapid identification of lines with high IM levels, using common sugar industry instruments. Sucrose isomerase activity was low in these transgenic lines, and the results indicate strong potential to develop sugarcane for commercial-scale production of IM if higher activity can be engineered in appropriate developmental patterns.

Isomaltulose is actively metabolized in plant cells.

Isomaltulose is a structural isomer of sucrose (Suc). It has been widely used as a nonmetabolized sugar in physiological studies aimed at better understanding the regulatory roles and transport of sugars in plants. It is increasingly used as a nutritional human food, with some beneficial properties including low glycemic index and acariogenicity. Cloning of genes for Suc isomerases opened the way for direct commercial production in plants. The understanding that plants lack catabolic capabilities for isomaltulose indicated a possibility of enhanced yields relative to Suc. However, this understanding was based primarily on the treatment of intact cells with exogenous isomaltulose. Here, we show that sugarcane (Saccharum interspecific hybrids), like other tested plants, does not readily import or catabolize extracellular isomaltulose. However, among intracellular enzymes, cytosolic Suc synthase (in the breakage direction) and vacuolar soluble acid invertase (SAI) substantially catabolize isomaltulose. From kinetic studies, the specificity constant of SAI for isomaltulose is about 10% of that for Suc. Activity varied against other Suc isomers, with V(max) for leucrose about 6-fold that for Suc. SAI activities from other plant species varied substantially in substrate specificity against Suc and its isomers. Therefore, in physiological studies, the blanket notion of Suc isomers including isomaltulose as nonmetabolized sugars must be discarded. For example, lysis of a few cells may result in the substantial hydrolysis of exogenous isomaltulose, with profound downstream signal effects. In plant biotechnology, different V(max) and V(max)/K(m) ratios for Suc isomers may yet be exploited, in combination with appropriate developmental expression and compartmentation, for enhanced sugar yields.

Transgenic expression of trehalulose synthase results in high concentrations of the sucrose isomer trehalulose in mature stems of field-grown sugarcane.

Sugarcane plants were developed that produce the sucrose isomers trehalulose and isomaltulose through expression of a vacuole-targeted trehalulose synthase modified from the gene in 'Pseudomonas mesoacidophila MX-45' and controlled by the maize ubiquitin (Ubi-1) promoter. Trehalulose concentration in juice increased with internode maturity, reaching about 600 mM, with near-complete conversion of sucrose in the most mature internodes. Plants remained vigorous, and trehalulose production in selected lines was retained over multiple vegetative generations under glasshouse and field conditions.

Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars.

Amenability to tissue culture stages required for gene transfer, selection and plant regeneration are the main determinants of genetic transformation efficiency via particle bombardment into sugarcane. The technique is moving from the experimental phase, where it is sufficient to work in a few amenable genotypes, to practical application in a diverse and changing set of elite cultivars. Therefore, we investigated the response to callus initiation, proliferation, regeneration and selection steps required for microprojectile-mediated transformation, in a diverse set of Australian sugarcane cultivars. 12 of 16 tested cultivars were sufficiently amenable to existing routine tissue-culture conditions for practical genetic transformation. Three cultivars required adjustments to 2,4-D levels during callus proliferation, geneticin concentration during selection, and/or light intensity during regeneration. One cultivar gave an extreme necrotic response in leaf spindle explants and produced no callus tissue under the tested culture conditions. It was helpful to obtain spindle explants for tissue culture from plants with good water supply for growth, especially for genotypes that were harder to culture. It was generally possible to obtain several independent transgenic plants per bombardment, with time in callus culture limited to 11-15 weeks. A caution with this efficient transformation system is that separate shoots arose from different primary transformed cells in more than half of tested calli after selection for geneticin resistance. The results across this diverse cultivar set are likely to be a useful guide to key variables for rapid optimisation of tissue culture conditions for efficient genetic transformation of other sugarcane cultivars.

Genetic transformation of sweet sorghum.

Sweet sorghum has substantial potential as a biofuel feedstock, with advantages in some environments over alternatives such as sugarcane or maize. Gene technologies are likely to be important to achieve yields sufficient for food, fuel and fibre production from available global croplands, but sorghum has proven difficult to transform. Tissue culture recalcitrance and poor reproducibility of transformation protocols remain major challenges for grain sorghum, and there has been no reported success for sweet sorghum. Here we describe a repeatable transformation system for sweet sorghum, based on (1) optimized tissue culture conditions for embryogenic callus production with >90% regenerability in 12-week-old calli, and (2) an effective selection regimen for hygromycin resistance conferred by a Ubi-hpt transgene following particle bombardment. Using this method, we have produced sixteen independent transgenic lines from multiple batches at an overall efficiency of 0.09% transformants per excised immature embryo. Co-expression frequency of a non-selected luciferase reporter was 62.5%. Transgene integration and expression were confirmed in T(0) and T(1) plants by Southern analysis and luciferase assays. This success using the major international sweet sorghum cultivar Ramada provides a foundation for molecular improvement of sweet sorghum through the use of transgenes. Factors likely to be important for success with other sweet sorghum cultivars are identified.

Functional analysis of genes for benzoate metabolism in the albicidin biosynthetic region of Xanthomonas albilineans.

Albicidins are potent DNA-gyrase-inhibiting antibiotics and phytotoxins synthesised by Xanthomonas albilineans. Functions have been deduced for some clustered biosynthetic genes, including a PKS-NRPS megasynthase, methyltransferases and regulatory genes, and resistance genes including a transporter and a gyrase-binding protein. More puzzling is the presence in this cluster of apparent aromatic metabolism genes. Here, we describe functional analysis of several such genes and propose a model for their role. An apparent benzoate CoA ligase (xabE) proved essential for albicidin production and pathogenicity. A neighbouring operon includes genes for p-aminobenzoate (PABA) metabolism. A PABA synthase fusion (pabAB) restored prototrophy in pabA and pabB mutants of Escherichia coli, proving functionality. Inactivation of pabAB increased susceptibility to sulphanilamide but did not block albicidin production. X. albilineans contains a remote pabB gene which evidently supplies enough PABA for albicidin biosynthesis in culture. Additional capacity from pabAB may be advantageous in more demanding environments such as infected plants. Downstream from pabAB are a known resistance gene (albG) and ubiC which encodes a p-hydroxybenzoate (PHBA) synthase. PHBA protects X. albilineans from inhibition by PABA. Therefore, coordinated expression may protect X. albilineans against toxicity of both the PABA intermediate and the albicidin product, under conditions that induce high-level antibiotic biosynthesis.

Evaluation and application of a luciferase fusion system for rapid in vivo analysis of RNAi targets and constructs in plants.

The practical use of RNA-mediated approaches including antisense RNA, ribozymes and siRNAs for specific inhibition of gene expression is limited by lack of simple quantitative methods to rapidly test efficacy in vivo. There have been indications that cotransfer of target::reporter gene fusions with constructs designed against the target sequence, followed by quantification of transient reporter gene activity might be effective. Here, we report detailed testing of the approach in plants, using diverse target::luciferase fusions and antisense or ribozyme constructs. We used quantitative transient luciferase activity (Luc) assays to test antisense constructs against beta-glucuronidase, PR glucanase, vacuolar invertase and cucumber mosaic virus, as well as ribozymes against watermelon mosaic virus and tobacco anionic peroxidase. For constructs previously tested in transgenic plants, the results correspond well with those from the transient expression assay. Target susceptibility was generally not strongly influenced by luciferase fusion, and the assay was not highly dependent on target sequence length. Some sequences reduced Luc activity below the level for reliable quantification, but suitable alternative fusions were readily produced. Transcriptional and translation fusions were effective for 5' target::luc constructs. Translational fusions were more reliable for luc::target 3' constructs. With minimal preliminary work to prepare suitable target::luciferase fusions, the approach appears generally applicable for rapid in vivo validation of effectiveness and specificity of constructs designed for RNA-mediated down-regulation of plant genes.

Efficient silencing of reporter transgenes coupled to known functional promoters in sugarcane, a highly polyploid crop species.

Sugarcane is a crop of great interest for engineering of sustainable biomaterials and biofuel production. Isolated sugarcane promoters have generally not maintained the expected patterns of reporter transgene expression. This could arise from defective promoters on redundant alleles in the highly polyploid genome, or from efficient transgene silencing. To resolve this question we undertook detailed analysis of a sugarcane gene that combines a simple pattern in genomic Southern hybridization analysis with potentially useful, sink-specific, expression. Sequence analysis indicates that this gene encodes a member of the SHAQYF subfamily of MYB transcription factors. At least eight alleles were revealed by PCR analysis in sugarcane cultivar Q117 and a similar level of heterozygosity was seen in BAC clones from cultivar Q200. Eight distinct promoter sequences were isolated from Q117, of which at least three are associated with expressed alleles. All of the isolated promoter variants were tested for ability to drive reporter gene expression in sugarcane. Most were functional soon after transfer, but none drove reporter activity in mature stems of regenerated plants. These results show that the ineffectiveness of previously tested sugarcane promoters is not simply due to the isolation of non-functional promoter copies from the polyploid genome. If the unpredictable onset of silencing observed in most other plant species is associated with developmental polyploidy, approaches that avoid efficient transgene silencing in polyploid sugarcane are likely to have much wider utility in molecular improvement.

DNA gyrase from the albicidin producer Xanthomonas albilineans has multiple-antibiotic-resistance and unusual enzymatic properties.

The sugarcane pathogen Xanthomonas albilineans produces a family of antibiotics and phytotoxins termed albicidins, which inhibit plant and bacterial DNA gyrase supercoiling activity, with a 50% inhibitory concentration (50 nM) comparable to those of coumarins and quinolones. Here we show that X. albilineans has an unusual, antibiotic-resistant DNA gyrase. The X. albilineans gyrA and gyrB genes are not clustered with previously described albicidin biosynthesis and self-protection genes. The GyrA and GyrB products differ from Escherichia coli homologues through several insertions and through changes in several amino acid residues implicated in quinolone and coumarin resistance. Reconstituted X. albilineans DNA gyrase showed 20- to 25-fold-higher resistance than E. coli DNA gyrase to albicidin and ciprofloxacin and 8-fold-higher resistance to novobiocin in the supercoiling assay. The X. albilineans DNA gyrase is unusual in showing a high degree of distributive supercoiling and little DNA relaxation activity. X. albilineans GyrA (XaA) forms a functional gyrase heterotetramer with E. coli GyrB (EcB) and can account for albicidin and quinolone resistance and low levels of relaxation activity. XaB probably contributes to both coumarin resistance and the distributive supercoiling pattern. Although XaB shows fewer apparent changes relative to EcB, the EcA.XaB hybrid relaxed DNA in the presence or absence of ATP and was unable to supercoil. A fuller understanding of structural differences between albicidin-sensitive and -resistant gyrases may provide new clues into features of the enzyme amenable to interference by novel antibiotics.

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species.

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

Doubled sugar content in sugarcane plants modified to produce a sucrose isomer.

Sucrose is the feedstock for more than half of the world's fuel ethanol production and a major human food. It is harvested primarily from sugarcane and beet. Despite attempts through conventional and molecular breeding, the stored sugar concentration in elite sugarcane cultivars has not been increased for several decades. Recently, genes have been cloned for bacterial isomerase enzymes that convert sucrose into sugars which are not metabolized by plants, but which are digested by humans, with health benefits over sucrose. We hypothesized that an appropriate sucrose isomerase (SI) expression pattern might simultaneously provide a valuable source of beneficial sugars and overcome the sugar yield ceiling in plants. The introduction of an SI gene tailored for vacuolar compartmentation resulted in sugarcane lines with remarkable increases in total stored sugar levels. The high-value sugar isomaltulose was accumulated in storage tissues without any decrease in stored sucrose concentration, resulting in up to doubled total sugar concentrations in harvested juice. The lines with enhanced sugar accumulation also showed increased photosynthesis, sucrose transport and sink strength. This remarkable step above the former ceiling in stored sugar concentration provides a new perspective into plant source-sink relationships, and has substantial potential for enhanced food and biofuel production.

The phytotoxin albicidin is a novel inhibitor of DNA gyrase.

Xanthomonas albilineans produces a family of polyketide-peptide compounds called albicidins which are highly potent antibiotics and phytotoxins as a result of their inhibition of prokaryotic DNA replication. Here we show that albicidin is a potent inhibitor of the supercoiling activity of bacterial and plant DNA gyrases, with 50% inhibitory concentrations (40 to 50 nM) less than those of most coumarins and quinolones. Albicidin blocks the religation of the cleaved DNA intermediate during the gyrase catalytic sequence and also inhibits the relaxation of supercoiled DNA by gyrase and topoisomerase IV. Unlike the coumarins, albicidin does not inhibit the ATPase activity of gyrase. In contrast to the quinolones, the albicidin concentration required to stabilize the gyrase cleavage complex increases 100-fold in the absence of ATP. The slow peptide poisons microcin B17 and CcdB also access ATP-dependent conformations of gyrase to block religation, but in contrast to albicidin, they do not inhibit supercoiling under routine assay conditions. Some mutations in gyrA, known to confer high-level resistance to quinolones or CcdB, confer low-level resistance or hypersensitivity to albicidin in Escherichia coli. Within the albicidin biosynthesis region in X. albilineans is a gene encoding a pentapeptide repeat protein designated AlbG that binds to E. coli DNA gyrase and that confers a sixfold increase in the level of resistance to albicidin in vitro and in vivo. These results demonstrate that DNA gyrase is the molecular target of albicidin and that X. albilineans encodes a gyrase-interacting protein for self-protection. The novel features of the gyrase-albicidin interaction indicate the potential for the development of new antibacterial drugs.

Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene.

Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35 degrees C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50 degrees C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35 degrees C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the Km was 39.9 mM, the Vmax was 638 U mg(-1), and the Kcat/Km was 1.79 x 10(4) M(-1) s(-1), compared to a Km of 76.0 mM, a Vmax of 423 U mg(-1), and a Kcat/Km of 0.62 x 10(4) M(-1) s(-1) for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.