Comparative genomics of the Erwinia and Enterobacter olive fly endosymbionts.

The pestivorous tephritid olive fly has long been known as a frequent host of the obligately host-associated bacterial endosymbiont, Erwinia dacicola, as well as other facultative endosymbionts. The genomes of Erwinia dacicola and Enterobacter sp. OLF, isolated from a California olive fly, encode the ability to supplement amino acids and vitamins missing from the olive fruit on which the larvae feed. The Enterobacter sp. OLF genome encodes both uricase and ureases, and the Er. dacicola genome encodes an allantoate transport pathway, suggesting that bird feces or recycling the fly's waste products may be important sources of nitrogen. No homologs to known nitrogenases were identified in either bacterial genome, despite suggestions of their presence from experiments with antibiotic-treated flies. Comparisons between the olive fly endosymbionts and their free-living relatives revealed similar GC composition and genome size. The Er. dacicola genome has fewer genes for amino acid metabolism, cell motility, and carbohydrate transport and metabolism than free-living Erwinia spp. while having more genes for cell division, nucleotide metabolism and replication as well as mobile elements. A 6,696 bp potential lateral gene transfer composed primarily of amino acid synthesis and transport genes was identified that is also observed in Pseudomonas savastanoii pv savastanoii, the causative agent of olive knot disease.

Endophytic Detection in Selected European Herbal Plants.

A total of 181 cultivable endophytic bacterial isolates were collected from stems of 13 species of herbs inhabiting Europe (Poland): Chelidonium majus L., Elymus repens L., Erigeron annuus L., Euphrasia rostkoviana Hayne, Foeniculum vulgare L., Geranium pratense L., Humulus lupulus L., Matricaria chamomilla L., Mentha arvensis L., Papaver rhoeas L., Rosmarinus officinalis L., Solidago gigantea L. and Vinca minor L. The isolates were screened for their antifungal activity and fifty three were found to inhibit fungal growth. Of these, five had strong antifungal properties. These selected isolates were identified as: Pseudomonas azotoformans, P. cedrina, Bacillus subtilis group and Erwinia persicina.

ß-carotene-producing bacteria residing in the intestine provide vitamin A to mouse tissues in vivo.

Vitamin A deficiency (VAD) is an overwhelming public health problem that affects hundreds of millions of people worldwide. A definitive solution to VAD has yet to be identified. Because it is an essential nutrient, vitamin A or its carotenoid precursor ß-carotene can only be obtained from food or supplements. In this study, we wanted to establish whether ß-carotene produced in the mouse intestine by bacteria synthesizing the provitamin A carotenoid could be delivered to various tissues within the body. To achieve this, we took advantage of the Escherichia coli MG1655*, an intestine-adapted spontaneous mutant of E. coli MG1655, and the plasmid pAC-BETA, containing the genes coding for the 4 key enzymes of the ß-carotene biosynthetic pathway (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and lycopene cyclase) from Erwinia herbicola. We engineered the E. coli MG1655* to produce ß-carotene during transformation with pAC-BETA (MG1655*-ßC) and gavaged wild-type and knockout mice for the enzyme ß-carotene 15,15'-oxygenase with this recombinant strain. Various regimens of bacteria administration were tested (single vs. multiple and low vs. high doses). ß-Carotene concentration was measured by HPLC in mouse serum, liver, intestine, and feces. Enumeration of MG1655*-ßC cells in the feces was performed to assess efficiency of intestinal colonization. We demonstrated in vivo that probiotic bacteria could be used to deliver vitamin A to the tissues of a mammalian host. These results have the potential to pave the road for future investigations aimed at identifying alternative, novel approaches to treat VAD.

Identification and genetics of 6-thioguanine secreted by Erwinia species and its interference with the growth of other bacteria.

We identified a compound in culture supernatants of Erwinia species, such as Erwinia amylovora, E. pyrifoliae, E. billingiae, E. tasmaniensis, E. persicina and E. rhapontici absorbing at 340 nm, which was associated before with the yellow pigment produced by E. amylovora on media containing copper ions. The compound was purified from E. tasmaniensis strain Et1/99 supernatants by chromatography on Dowex-1 and Dowex-50 columns and identified by HPLC/MS and NMR analysis as 6-thioguanine (6TG). Its signal at 167 Da matched with the expected molecular mass. By random mutagenesis with miniTn5, we obtained mutants defective in the genes for pyrimidine and purine metabolism. A specific gene cluster with ycf genes described by us before, absent in the corresponding region of Escherichia coli, was identified in the genome sequence of three Erwinia species and named tgs region for thioguanine synthesis. Clones of the tgs gene cluster promoted 6TG synthesis and secretion in E. coli, when the bacteria were grown in minimal medium supplemented with amino acids. 6TG was bacteriostatic for E. coli and Salmonella typhimurium strains, with cell growth resumed after prolonged incubation. Similar results were obtained with P. agglomerans strains. Bacteria from the genus Pectobacterium were barely and Rahnella or Gibbsiella species were not inhibited by 6TG. Adenine and guanine relieved the toxic effect of 6TG on E. coli. Non-producing strains were fully virulent on host plants. 6TG synthesis may help erwinias to interfere with growth of some microorganisms in the environment.

Surrogate biochemistry: use of Escherichia coli to identify plant cDNAs that impact metabolic engineering of carotenoid accumulation.

Carotenoids synthesized in plants but not animals are essential for human nutrition. Therefore, ongoing efforts to metabolically engineer plants for improved carotenoid content benefit from the identification of genes that affect carotenoid accumulation, possibly highlighting potential challenges when pyramiding traits represented by multiple biosynthetic pathways. We employed a heterologous bacterial system to screen for maize cDNAs encoding products that alter carotenoid accumulation either positively or negatively. Genes encoding carotenoid biosynthetic enzymes from the bacterium Erwinia uredovora were introduced into Escherichia coli cells that were subsequently transfected with a maize endosperm cDNA expression library; and these doubly transformed cells were then screened for altered carotenoid accumulation. DNA sequencing and characterization of one cDNA class conferring increased carotenoid content led to the identification of maize cDNAs encoding isopentenyl diphosphate isomerase. A cDNA that caused a reduced carotenoid content in E. coli was also identified. Based on DNA sequence analysis, DNA hybridization, and further functional testing, this latter cDNA was found to encode the small subunit of ADP-glucose pyrophosphorylase, a rate-controlling enzyme in starch biosynthesis that has been of interest for enhancing plant starch content.

Biological sensor for sucrose availability: relative sensitivities of various reporter genes.

A set of three sucrose-regulated transcriptional fusions was constructed. Fusions p61RYTIR, p61RYlac, and p61RYice contain the scrR sucrose repressor gene and the promoterless gfp, lacZ, and inaZ reporter genes, respectively, fused to the scrY promoter from Salmonella enterica serovar Typhimurium. Cells of Erwinia herbicola containing these fusions are induced only in media amended with sucrose, fructose, or sorbose. While a large variation in sucrose-dependent reporter gene activity was observed in cells harboring all gene fusions, fusions to the inaZ reporter gene yielded a much wider range of activity and were responsive to lower levels of sucrose than either lacZ or gfp. The lacZ reporter gene was found to be more efficient than gfp, requiring approximately 300-fold fewer cells for a detectable response over all concentrations of sucrose. Similarly, inaZ was found to be more efficient than lacZ, requiring 30-fold fewer cells at 1.45 microM sucrose and 6,100-fold fewer cells at 29 mM sucrose for a quantifiable response. The fluorescence of individual cells containing p61RYTIR was quantified following epifluorescence microscopy in order to relate the fluorescence exhibited by populations of cells in batch cultures with that of individual cells in such cultures. While the mean fluorescence intensity of a population of individual cells increased with increasing concentrations of sucrose, a wide range of fluorescence intensity was seen among individual cells. For most cultures the distribution of fluorescence intensity among individual cells was log-normally distributed, but cells grown in intermediate concentrations of sucrose exhibited two distinct populations of cells, one having relatively low fluorescence and another with much higher fluorescence. When cells were inoculated onto bean leaves, whole-cell ice nucleation and gfp-based biological sensors for sucrose each indicated that the average concentration of sucrose on moist leaf surfaces was about 20 microM. Importantly, the variation in green fluorescent protein fluorescence of biosensor cells on leaves suggested that large spatial variations in sugar availability occur on leaves.

Cloning and characterization of the gene cluster for palatinose metabolism from the phytopathogenic bacterium Erwinia rhapontici.

Erwinia rhapontici is able to convert sucrose into isomaltulose (palatinose, 6-O-alpha-D-glucopyranosyl-D-fructose) and trehalulose (1-O-alpha-D-glucopyranosyl-D-fructose) by the activity of a sucrose isomerase. These sucrose isomers cannot be metabolized by plant cells and most other organisms and therefore are possibly advantageous for the pathogen. This view is supported by the observation that in vitro yeast invertase activity can be inhibited by palatinose, thus preventing sucrose consumption. Due to the lack of genetic information, the role of sucrose isomers in pathogenicity has not been evaluated. Here we describe for the first time the cloning and characterization of the palatinose (pal) genes from Erwinia rhapontici. To this end, a 15-kb chromosomal DNA fragment containing nine complete open reading frames (ORFs) was cloned. The pal gene products of Erwinia rhapontici were shown to be homologous to proteins involved in uptake and metabolism of various sugars from other microorganisms. The palE, palF, palG, palH, palK, palQ, and palZ genes were oriented divergently with respect to the palR and palI genes, and sequence analysis suggested that the first set of genes constitutes an operon. Northern blot analysis of RNA extracted from bacteria grown under various conditions implies that the expression of the palI gene and the palEFGHKQZ genes is oppositely regulated at the transcriptional level. Genes involved in palatinose uptake and metabolism are down regulated by sucrose and activated by palatinose. Palatinose activation is inhibited by sucrose. Functional expression of palI and palQ in Escherichia coli revealed sucrose isomerase and palatinase activity, respectively.

Pectate lyase PelI of Erwinia chrysanthemi 3937 belongs to a new family.

Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pel4, pelB, pelC, pelD, and pelE genes and a set of secondary pectate lyases, two of which, pelL and pelZ, have been already identified. We cloned the pelI gene, encoding a ninth pectate lyase of E. chrysanthemi 3937. The pelI reading frame is 1,035 bases long, corresponding to a protein of 344 amino acids including a typical amino-terminal signal sequence of 19 amino acids. The purified mature PelI protein has an isoelectric point of about 9 and an apparent molecular mass of 34 kDa. PelI has a preference for partially methyl esterified pectin and presents an endo-cleaving activity with an alkaline pH optimum and an absolute requirement for Ca2+ ions. PelI is an extracellular protein secreted by the Out secretory pathway of E. chrysanthemi. The PelI protein is very active in the maceration of plant tissues. A pelI mutant displayed reduced pathogenicity on chicory leaves, but its virulence did not appear to be affected on potato tubers or Saintpaulia ionantha plants. The pelI gene constitutes an independent transcriptional unit. As shown for the other pel genes, the transcription of pelI is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, temperature, nitrogen starvation, and catabolite repression. Regulation of pelI expression appeared to be dependent on the three repressors of pectinase synthesis, KdgR, PecS, and PecT, and on the global activator of sugar catabolism, cyclic AMP receptor protein. A functional KdgR binding site was identified close to the putative pelI promoter. Analysis of the amino acid sequence of PelI revealed high homology with a pectate lyase from Erwinia carotovora subsp. carotovora (65% identity) and low homology with pectate lyases of the phytopathogenic fungus Nectria haematococca (Fusarium solani). This finding indicates that PelI belongs to pectate lyase class III. Using immunoblotting experiments, we detected PelI homologs in various strains of E. chrysanthemi and E. carotovora subsp. carotovora but not in E. carotovora subsp. atroseptica.

Expression in Escherichia coli and properties of the carotene ketolase from Haematococcus pluvialis.

High level expression of the functional beta-carotene ketolase gene bkt from Haematococcus pluvialis occurred in Escherichia coli transformants producing beta-carotene or zeaxanthin as a result of the presence of additional carotenoid genes from Erwinia uredovora. Requirement of molecular oxygen for the insertion of the keto group was demonstrated. The final product of this two-step ketolase reaction from beta-carotene is canthaxanthin (4,4'-diketo-beta-carotene) with the 4-monoketo derivative echinenone as an intermediate. A reaction sequence for the formation of astaxanthin from beta-carotene was established based on kinetic data on astaxanthin formation in E. coli transformants carrying the hydroxylase gene crtZ from Erwinia along with bkt. We conclude that the carotenoids zeaxanthin and adonixanthin which accumulate in addition to astaxanthin in this transformant are products of side reactions rather than direct precursors of astaxanthin. The possible mechanisms for the formation of the keto derivatives are discussed.

Synergism between Erwinia pectate lyase isoenzymes that depolymerize both pectate and pectin.

Phytopathogenic Erwinia bacteria cause tissue maceration by secretion of pectinolytic enzymes such as pectate lyase (PL). Sequencing of overlapping genomic fragments from Erwinia carotovora subsp. atroseptica established the organization of a 7.5 kbp region encoding PL isoenzymes. Two intergenic regions of 656 and 645 bp separate three enzyme coding regions of 1125 bp exhibiting approximately 80% positional identity. The promoters of each of the three genes contain a segment with high homology to the binding sequence of the E. chrysanthemi KdgR transcription repressor, implying similar mechanisms of gene regulation in the two bacterial species. Separate expression of the pel genes in the Escherichia coli-pT7-7 system and purification of their products yielded PLs at 7-33 mg (I culture)-1 with greater than 95% purity. Availability of the recombinant enzymes allowed determination of the kinetic differences amongst the PL isoforms, PL1, PL2 and PL3. The results show that PL is not strictly confined to depolymerization of pectate since each isoenzyme more readily degrades 31% esterified pectin. Addition of isoenzyme combinations revealed no synergism with respect to degradation of pectate or 31% esterified pectin. However, addition of enzyme combinations containing PL3 enhanced the activity towards 68% esterified pectin, against which individual PL activities were low, by up to 64%. These data suggest that the combination of PL isoenzymes extends the range of pectic substrates which the bacterium can degrade.

Expression of the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene together with the Erwinia pectate lyase and polygalacturonase genes in Saccharomyces cerevisiae.

Recombinant Saccharomyces cerevisiae strains capable of simultaneous secretion of bacterial glucanase and pectinase enzymes have been developed. The Butyrivibrio fibrrisolvens endo-beta-1,4-glucanase gene (end1), the Erwinia chrysanthemi pectate lyase gene (pelE) and E. carotovora polygalacturonase gene (peh1) were each inserted between a yeast expression-secretion cassette and yeast gene terminator, and cloned into yeast-centromeric shuttle vectors. Transcription initiation signals present in the expression-secretion cassette were derived from the yeast alcohol dehydrogenase gene promoter (ADC1P), whereas the transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of glucanase and pectinases was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S). These YCplac111-based constructs, designated END1, PEL5, AND PEH1, respectively, were transformed into S. cerevisiae. The END1, PEL5 and PEH1 constructs were co-expressed in laboratory strains of S. cerevisiae as well as in wine and distillers' yeasts. DNA-RNA hybridization analysis showed the presence of END1, PEL5 and PEH1 transcripts. Carboxymethylcellulose and polypectate agarose assays revealed the production of biologically active endo-beta-1,4-glucanase, pectate lyase and polygalacturonase by the S. cerevisiae transformants. Interestingly, although the same expression-secretion cassette was used in all three constructs, time-course assays indicated that the pectinases were secreted before the glucanase. It is tempting to speculate that the bulkiness of the END1-encoded protein and the five alternating repeats of Pro-Asp-Pro-Thr(Gln)-Pro-Val-Asp within the glucanase moiety could be involved in the delayed secretion of the glucanase.

A Chlorella virus gene promoter functions as a strong promoter both in plants and bacteria.

An upstream region from an algal virus methyltransferase gene was tested for promoter function in transgenic plants, electroporated monocot protoplasts and bacteria. Fusion of the 851 bp upstream region to a reporter gene significantly enhanced the reporter gene expression in transgenic Arabidopsis and potato plants and in electroporated maize and Sorghum cells relative to the cauliflower mosaic virus 35S promoter. The promoter was also functional in several Escherichia coli strains and in three species of phytopathogenic bacteria, Erwinia, Pseudomonas, and Xanthomonas. These findings indicate that the upstream region is a strong promoter uniquely functional in both eukaryotes and prokaryotes and capable of using both eukaryotic RNA polymerase II and bacterial RNA polymerases.

Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica.

Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed.

A pleiotropic reduced virulence (Rvi-) mutant of Erwinia carotovora subspecies atroseptica is defective in flagella assembly proteins that are conserved in plant and animal bacterial pathogens.

Erwinia carotovora subsp. atroseptica was mutagenized and assayed for virulence in planta. Those mutants which exhibited reduced virulence (Rvi-) were assayed for growth rate, auxotrophy and extracellular enzyme secretion and seven mutants were found to be wild type for all of these phenotypes. When screened for other phenotypes, two were found to be non-motile. One mutant was complemented for motility by a heterologous gene library. A 2.7kb XmaIII-ClaI complementing fragment was sequenced and the gene products were found to have similarity to flagella biosynthesis gene products from several bacteria. Further similarity was found to a pathogenicity protein from the plant pathogen Xanthomonas campestris pv. glycines and to the Spa pathogenicity proteins of the human pathogen Shigella flexneri, which are involved in the surface presentation of antigens. These studies highlight the emergence of common themes in the molecular strategies employed by both plant and animal bacterial pathogens for the targeting of proteins involved in the elaboration of disease.

Environmental conditions affect transcription of the pectinase genes of Erwinia chrysanthemi 3937.

To depolymerize plant pectin, the phytopathogenic enterobacterium Erwinia chrysanthemi produces a series of enzymes which include a pectin-methyl-esterase encoded by the pem gene and five isoenzymes of pectate lyases encoded by the five genes pelA, pelB, pelC, pelD, and pelE. We have constructed transcriptional fusions between the pectinase gene promoters and the uidA gene, encoding beta-glucuronidase, to study the regulation of these E. chrysanthemi pectinase genes individually. The transcription of the pectinase genes is dependent on many environmental conditions. All the fusions were induced by pectic catabolic products and responded, to different degrees, to growth phase, catabolite repression, temperature, and nitrogen starvation. Transcription of pelA, pelD, and pelE was also increased in anaerobic growth conditions. High osmolarity of the culture medium increased expression of pelE but decreased that of pelD; the other pectinase genes were not affected. The level of expression of each gene was different. Transcription of pelA was very low under all growth conditions. The expression of the pelB, pelC, and pem genes was intermediate. The pelE gene had a high basal level of expression. Expression of pelD was generally the most affected by changes in culture conditions and showed a low basal level but very high induced levels. These differences in the expression of the pectinase genes of E. chrysanthemi 3937 presumably reflect their role during infection of plants, because the degradation of pectic polymers of the plant cell walls is the main determinant of tissue maceration caused by soft rot erwiniae.

Physical and chemical control of released microorganisms at field sites.

An important consideration in the environmental release of a genetically engineered microorganism is the capability for reduction or elimination of microorganism populations once their function is completed or if adverse environmental effects are observed. In this study the decontamination treatments of burning and biocide application, alone and in combination with tilling, were evaluated for their ability to reduce populations of bacteria released on the phylloplane. Field plots of bush beans (Phaseolus vulgaris), sprayed with the bacterium Erwinia herbicola, received the following treatments: control; control + till; burn; burn + till; Kocide (cupric hydroxide); Kocide + till; Agri-Strep (streptomycin sulfate); and Agri-Strep + till. Leaves and soil from the plots were sampled -1, 1, 5, 8, 12, 15, 19, and 27 days after application of the decontamination treatments. Burning produced a significant reduction in the number of E. herbicola, whereas tilling, alone or in combination with the biocide treatments, stimulated a significant increase in E. herbicola populations, which persisted for several weeks. The individual treatments of the biocides, Kocide and Agri-Strep, produced a rate of decline in E. herbicola populations that did not significantly differ from that of the control treatment.

Molecular cloning of the structural gene for exopolygalacturonate lyase from Erwinia chrysanthemi EC16 and characterization of the enzyme product.

The ability of Erwinia chrysanthemi to cause soft-rot diseases involving tissue maceration in many plants has been linked to the production of endo-pectate lyase E. chrysanthemi EC16 mutant UM1005, however, contains deletions in the pel genes that encode the known endopectate lyases, yet still macerates plant tissues. In an attempt to identify the remaining macerating factor(s), a gene library of UM1005 was constructed in Escherichia coli and screened for pectolytic activity. A clone (pPNL5) was identified in this library that contained the structural gene for an exopolygalacturonate lyase (ExoPL). The gene for ExoPL was localized on a 3.3-kb EcoRV fragment which contained an open reading frame for a 79,500-Da polypeptide. ExoPL was purified to apparent homogeneity from Escherichia coli DH5 alpha (pPNL5) and found to have an apparent molecular weight of 76,000 with an isoelectric point of 8.6. Purified ExoPL had optimal activity between pH 7.5 and 8.0 and could utilize pectate, citrus pectin, and highly methyl-esterified Link pectin as substrates. A PL- ExoPL- mutant of EC16 was constructed that exhibited reduced growth on pectate, but retained pathogenicity on chrysanthemum equivalent to that of UM1005. The results indicate that ExoPL does not contribute to the residual macerating activity of UM1005.

Cloning of genes encoding extracellular metalloproteases from Erwinia chrysanthemi EC16.

A 14-kilobase BamHI-EcoRI DNA fragment cloned from Erwinia chrysanthemi EC16 contained a gene encoding a metalloprotease inhibitor as well as three tandem prt genes encoding metalloproteases. The prt genes were separated from the inhibitor gene by a ca. 4-kilobase region that was necessary for extracellular secretion of the proteases. When individually subcloned downstream from vector promoters, the three prt genes each led to substantial extracellular secretion of the proteases by Escherichia coli cells, provided that the 4-kilobase required region was supplied in cis or trans. One of the protease structural genes, prtC, was sequenced and had high homology to a metalloprotease gene previously described from Serratia species as well as to the prtB gene of E. chrysanthemi B374. Marker exchange mutants of E. chrysanthemi EC16 defective in production of one or all of the extracellular proteases were not impaired in virulence on plant tissue.