A chromosomal DNA fragment from Erwinia amylovora was identified that complemented a deletion mutant in the gut(srl) operon of Escherichia coli. The E. amylovora srl operon on the cloned fragment was localized by transposon mutagenesis. A DNA fragment including the srl genes of E. amylovora was sequenced and found to contain six open reading frames (ORFs). These ORFs were highly homologous to genes of the gut operon of E. coli. No large gene was found that encoded a protein equivalent to GutA of E. coli; instead two ORFs with extensive similarity to GutA were identified in the E. amylovora srl operon. All transposon insertions were mapped by PCR analysis, and several insertions in a plasmid bearing the srl operon were unable to complement a mutation in the E. coli gutD gene. All E. amylovora srl mutants could be complemented by introducing the sorbitol operon from E. coli. The direction of transcription was confirmed by analysis of lacZ fusions. Expression of the srl operon in E. amylovora was high in the presence of sorbitol in the medium and was repressed by glucose. Mutants with a sorbitol deficiency were still virulent on slices of immature pears, but were unable to cause significant fire blight symptoms on apple shoots. Since sorbitol is used for carbohydrate transport in host plants of E. amylovora, this sugar alcohol may be an important factor in determining host specificity for the fire blight pathogen.

Galactose metabolism mutants of Erwinia amylovora were created by transposon insertions and characterized for their growth properties and interaction with plant tissue. The nucleotide sequence of the galE gene was determined. The gene, which encodes UDP-galactose 4-epimerase, shows homology to the galE genes of Escherichia coli, Neisseria gonorrhoeae, Rhizobium meliloti, and other gram-negative bacteria. Cloned DNA with the galE and with the gaiT and galK genes did not share borders, as judged by the lack of common fragments in hybridization with chromosomal DNA. These genes are thus located separately on the bacterial chromosome. In contrast to the gal operon of E. coli, the galE gene of E. amylovora is constitutively expressed, independently of the presence of galactose in the medium. The function of the galE gene but not of the galT or galK gene is required for bacterial virulence on pear fruits and seedlings. In the absence of galactose, the galE mutant was deficient in amylovoran synthesis. Subsequently, the galE mutant cells elicited host defense reactions, and they were not stained by fluorescein isothiocyanate-labelled lectin, which efficiently binds to amylovoran capsules ofE. amylovora. The mutation affected the side chains of bacterial lipopolysaccharide, but an intact 0 antigen was not required for virulence. This was shown with another mutant, which could be complemented for virulence but not for side chain synthesis of lipopolysaccharide.The gram-negative bacterium Erwinia amylovora is the causal agent of fire blight, a necrotic disease of rosaceous plants (49). To escape plant defense mechanisms and to cause symptoms, E. amylovora requires the capsular exopolysaccharide amylovoran (4, 20, 44). Amylovoran synthesis mutants are nonpathogenic (6, 47). A chromosomal cluster of ams genes necessary for amylovoran synthesis has been characterized (7). Cloned E. amylovora genes were able to complement mutants in the cps genes of the maize pathogen Erwinia stewartii, which are involved in exopolysaccharide (EPS) synthesis. The galE gene is adjacent to the cps gene cluster in E. stewartii (14). The dominant sugar in amylovoran is galactose (45). Consequently, disruption of the galactose metabolism affects the capsule synthesis and virulence of E. amylovora, and growth in the presence of galactose restores the ability to produce EPS (8). The genetics of gal genes in E. amylovora have not been resolved.Enterobacteria utilize galactose by the LeLoir pathway (1). The first reaction in this pathway is catalyzed by galactokinase (EC 2.7.1.6), which phosphorylates free galactose to galactose-1-phosphate. In the next steps, galactose-1-phosphate uridylyltransferase (EC 2.7.7.10) transfers the UDP residue from UDP-glucose to galactose-1-phosphate, and UDP-galactose 4-epimerase (EC 5.1.3.2) catalyzes the reversible conversions of UDP-galactose and UDP-glucose. In Escherichia coli, the structural genes galK, galT, and galE are organized in an operon which is induced by galactose (1). Two adjacent promoters are regulat...

The suicide plasmid pfdA31-Tn5 was constructed to mutagenize Erwinia amylovora and Escherichia coli strains by electorporation. This vector carries the bacteriophage fd replication origin, a beta-lactamase gene and the transposon Tn5. For propagation the plasmid depends on host cells producing fd gene-2 protein. Electroporation of E.amylovora or E.coli cells with plasmid pfdA31-Tn5 yielded more than 10(4) transposition events per micrograms DNA. We have produced and characterized transposon mutants of E.amylovora affecting either galactose metabolism or the synthesis of the phytotoxin (L)-2,5-dihydrophenylalanine. A Tn5-insertion in a gene, involved in exopolysaccharide synthesis of E.amylovora strain Ea7/74, was subcloned into vector pfdA31 and used to mutagenize E.amylovora strain Ea1/79 by site-directed recombination.

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