Carol Boyd

Cloning and characterization of a xylanase gene from corn strains of Erwinia chrysanthemi.

The gene encoding a 42-kDa endoxylanase was cloned from Erwinia chrysanthemi strain D1. Sequencing of this gene, called xynA, showed that it encoded a primary protein product of 413 amino acids with an unusual and long (31 amino acid) leader peptide that was cleaved during secretion to the bacterial periplasm. This protein is distinct from xylanases in glycohydrolase families 10 and 11 and, instead, appears to be intermediate between families 5 and 30. The xynA gene is located downstream from a gene with high homology to ATP-dependent RNA helicases and the Escherichia coli recD gene. Large amounts of the mature xylanase were produced by E. coli cells carrying a T7 expression plasmid construct and the protein was isolated from the bacterial periplasmic fraction by chromatography on a CM Bio-gel column. Marker exchange mutagenesis of the xynA gene eliminated the ability of strain D1 to produce detectable extracellular xylanase activity but did not affect virulence on corn leaves.

Cloning and characterization of the bgxA gene from Erwinia chrysanthemi D1 which encodes a β-glucosidase/xylosidase enzyme

A β-glucosidase/xylosidase gene from Erwinia chrysanthemi strain D1 was cloned and sequenced. This gene, named bgxA, encodes a ca. 71 kDa protein product which, following removal of the leader peptide, resulted in a ca. 69 kDa mature protein that accumulated in the periplasmic space of E. chrysanthemi strain D1 and Escherichia coli cells expressing the cloned gene. The protein exhibited both β-glucosidase and β-xylosidase activities but gave no detectable activity on xylan or carboxymethyl cellulose. The enzyme was classified as a type 3 glycosyl hydrolase, but was unusual in having a truncated B region at the carboxyl-terminus. Several E. chrysanthemi strains isolated from corn produced the glucosidase/xylosidase activity but not those isolated from dicot plants. However, bgxA marker exchange mutants of strain D1 were not detectably altered in virulence on corn leaves.

Comparison of avrD alleles from Pseudomonas syringae pv. glycinea

Avirulence gene D alleles resided on indigenous plasmids in races 0, 2, 3, 4, 5, and 6 of Pseudomonas syringae pv. glycinea (Psg), but the allele in race 1 appeared to be chromosomal. These were all nonfunctional avirulence genes because they neither induced the avirulence phenotype on Rpg4 soybean cultivars nor directed the production of syringolide elicitors when expressed in Escherichia coli cells. The predicted proteins encoded by the seven Psg avrD genes were very similar to that of a functional class II allele from P. syringae pv. phaseolicola G50 race 2, but contained mutations collectively affecting only nine amino acid positions. Despite these relatively small amino acid differences and the location of avrD from each isolate on a 5.6-kb HindIII restriction fragment, the flanking regions varied considerably among the Psg isolates. The presence of avrD alleles with few alterations but different locational contexts in all tested Psg races argues that they provide an important selected function in the bacteria but have been modified to escape defense surveillance in Rpg4 soybean plants.

Two different classes of avrD alleles occur in pathovars of Pseudomonas syringae

Considerable variation was observed in the occurrence of avirulence gene D (avrD) in different isolates and pathovars of Pseudomonas syringae. Three functional alleles of avrD were cloned and characterized from P. s. pv. phaseolicola and P. s. pv. lachrymans. These avrD genes occurred on indigenous plasmids in both pathovars, like the allele originally cloned from P. s. pv. tomato. P. s. pv. lachrymans was unique in that it carried two different alleles on plasmids of different sizes. These alleles were cloned on 5.6- or 3.8-kb HindIII fragments that are conserved in several other P. syringae pathovars. Surprisingly, the two avrD alleles from P. s. pv. lachrymans were the most divergent of those compared, with only 85% amino acid identity. Allele 1 from P. s. pv. lachrymans was 95% identical to avrD from P. s. pv. tomato but less similar to the other three avrD genes. These two alleles were accordingly called homology class I. The avrD gene from P. s. pv. phaseolicola and allele 2 from P. s. pv. lachrymans were 97 and 98% identical, respectively, at the amino acid level with the nonfunctional P. s. pv. glycinea allele. These three alleles were therefore grouped into homology class II. Comparison of all the avrD alleles permitted the identification of four amino acid substitutions unique to the P. s. pv. glycinea allele at positions 19, 245, 280, and 304.

Functional implications of the three-dimensional structures of pectate lyases

Abstract The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, PelC and PelE, have been refined to a resolution of 2.2. A superposition of the two structures has been used to correct the multiple sequence alignment of the extracellular pectate lyase superfamily. The corrected alignment has revealed two clusters of ‘potentially catalytic’ invariant amino acids, compatible with two active sites. Site-directed mutagenesis studies have confirmed that the pectinolytic active site includes the region around the Ca2+ binding site. Furthermore, mutagenesis studies suggest catalytic roles for individual amino acids. Although PelC and PelE are structurally similar in the overall fold of the polypeptide backbone, there are significant differences in the size and conformation of the polypeptide backbone, there are significant differences in the size and conformation of the loops that comprise the pectinolytic active site. The differences in the surface charges between PelC and PelE in the groove extending from the Ca2+ site suggest that the optimal oligosaccharide substrates are different for each enzyme.

Avirulence gene avrPphC from Pseudomonas syringae pv. phaseolicola 3121: a plasmid-borne homologue of avrC closely linked to an avrD allele.

Cosmid clone pPsp01 from race 1 Pseudomonas syringae pv. phaseolicola isolate 3121 conferred a unique pattern of soybean cultivar reactions when expressed in P. s. pv. glycinea R4. The avirulence phenotype was shown to result from the presence in clone pPsp01 of an avrD allele as well as an additional avirulence gene located approximately 5-kb upstream. The new gene, called avrPphC, shows high identity to and is phenotypically identical to avrC, previously cloned from P. s. pv. glycinea race 0. avrD and avrPphC occur on an approximately 120-kb indigenous plasmid in P. s. pv. phaseolicola 3121. Although commonly observed in Xanthomonas campestris, this is the first noted occurrence of multiple avirulence genes on a single plasmid in Pseudomonas syringae. Unlike avrD, however, avrPphC does not appear to occur widely in pathovars of Pseudomonas syringae.

TMV protein synthesis is not translationally regulated by heat shock.

SummaryTobacco mosaic virus (TMV) protein synthesis in tobacco leaf tissue was not translationally regulated under conditions of heat shock as were most of the other proteins that were produced at 25°C. Upon shift from 25°C to 37–40°C, most host protein synthesis was inhibited followed by initiation of synthesis of heat shock proteins. In contrast, TMV protein synthesis continued after the temperature shift. This phenomenon allowed the enhancement of detection of TMV protein synthesis in tobacco leaves. The most prominent proteins labeled were viral when tissue was labeled during the first hr following the shift to 40°C, a period after heat shock repression of host protein synthesis, but before the onset of most heat shock protein synthesis. Another method to predominately label viral proteins was to incubate infected leaves for periods at 35°C which induced repression of preexisting host protein synthesis without inducing synthesis of heat shock proteins.

Characterization of the prtA and prtB genes of Erwinia chrysanthemi EC 16

Two tandem metalloprotease-encoding structural genes, prtA and prtB, were sequenced from Erwinia chrysanthemi EC16. These were highly homologous to previously reported genes from the same bacteria, as well as to three other metalloprotease-encoding genes from enteric bacteria. The three tandem prt structural genes from strain EC16 were closely linked to a cluster of genes previously found to be essential for extracellular secretion of the metalloproteases.