Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids.

Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.

Catharanthus roseus, an Experimental Host Plant for the Citrus Strain of Xylella fastidiosa.

We verified by pathogenicity tests that the herbaceous plant Catharanthus roseus (Madagascar periwinkle) can be used as an experimental host for the strain of Xylella fastidiosa that causes citrus variegated chlorosis (CVC). Plants were mechanically inoculated with CVC strain 9a5c, the genome of which was recently sequenced. Plants were inoculated with the virulent 8th passage (9a5c-8) and the 51st passage (9a5c-51). Leaf deformation and stunting were seen 2 months after inoculation on 18 of 21 plants with 9a5c-8 and 8 of 21 plants with 9a5c-51. The plants were infected with X. fastidiosa as shown by polymerase chain reaction. The bacterium could be reisolated from all plants tested, showing that CVC-X. fastidiosa multiplied and moved systemically in C. roseus plants causing dysfunction in plant growth. The disease symptoms evolved within 4 months post-inoculation to a severe leaf chlorosis in all inoculated plants. The localization of X. fastidiosa in the xylem was verified by immunofluorescence. Genes coding for proteins with homologies to plant sterol-C-methyltransferase, a transketolase-like protein, subunit III of photosystem I, and a desiccation protectant protein were found to be differentially expressed in symptomatic C. roseus plants as a response to infection with X. fastidiosa in comparison to healthy plants. A tentative correlation between the pattern of expression of these C. roseus genes with the mechanism of pathogenicity of X. fastidiosa is discussed.

Structural interactions responsible for the assembly of the troponin complex on the muscle thin filament.

Skeletal muscle contraction is regulated by a complex of five polypeptides which are stably associated with the actin filament. This complex consists of two proteins: troponin with three subunits (TnC; TnI and TnT) and tropomyosin (a dimer of two chains). Using deletion mutants of TnC, TnI and TnT we determined that each of these polypeptides can be divided into at least two domains. One domain is responsible for the regulatory properties of the protein. Its interaction with the other components of the system change upon calcium binding to TnC. A second domain present in each of these proteins is responsible for the stable association of the complex to the actin filament. The interactions among this second set of domains is not influenced by calcium binding to TnC. The structural interactions are: 1) interactions between the C-domain of TnC with the N-domain of TnI; 2) interactions of the N-domain of TnI with the C-terminal domain of TnT and 3) interactions between the N-domain of TnT (T1) and actin/tropomyosin.

Functional alpha-tropomyosin produced in Escherichia coli. A dipeptide extension can substitute the amino-terminal acetyl group.

Unlike the muscle protein, alpha-tropomyosin expressed in Escherichia coli does not bind actin, does not exhibit head-to-tail polymerization, and does not inhibit actomyosin ATPase activity in the absence of troponin. The only chemical difference between recombinant and muscle tropomyosins is that the first methionine is not acetylated in the recombinant protein (Hitchcock-De-Gregori, S.E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). We expressed three fusion tropomyosins in E. coli with 2, 3, and 17 amino acids fused to its amino terminus. All three fusions restored actin binding, head-to-tail polymerization, and the capacity to inhibit the actomyosin ATPase to these unacetylated tropomyosins. Unlike larger fusions, the small fusions of 2 and 3 amino acids do not interfere with regulatory function. Therefore the presence of a fused dipeptide at the amino terminus of unacetylated tropomyosin is sufficient to replace the function of the N-acetyl group present in muscle tropomyosin. A structural interpretation for the function of the acetyl group, based on our results and the coiled coil structure of tropomyosin, is presented.