Jeffrey M. Werneke

Development of broad-host-range vectors for expression of cloned genes in Pseudomonas

The cloning and expression of genes in Pseudomonas have been difficult, until now, due to the absence of vector systems that contain multiple restriction sites downstream from promoter sequences that are functional in Pseudomonas. We report here the construction of several broad-host-range vectors that can be utilized in either Pseudomonas or Escherichia coli and that rely on easily selectable antibiotic resistance markers with multiple cloning sites. These vectors were constructed by inserting the entire pUC13 sequence into derivatives of the RSF1010 wide-host-range plasmid. From this construction, other derivatives were obtained, specifically a lacZ::KmR fusion gene which provides an easily selectable marker in both E. coli and Pseudomonas. These vectors have been used to express the Pseudomonas putida cytochrome P450 monoxygenase gene in a P450-deficient P. putida strain. Thus, these vectors allow for the cloning, expression and selection of Pseudomonas genes in Pseudomonas by complementation.

Rubisco Activase: A New Enzyme in the Regulation of Photosynthesis

Rubisco has been one of the most studied enzymes in the photosynthetic process, since its discovery in 1956 (1). However our biochemical knowledge of the mechanism of the enzyme in vitro has been inadequate to account for its ability to function in vivo as measured by the photosynthetic rate of a leaf relative to its rubisco content. It was not until the middle 1970’s that sufficient activity could be achieved in vitro to account for in vivo rates of photosynthesis (2,3). However, as soon as this problem was apparently resolved, another became more evident as research progressed. Namely, discrepancies exist between measurements of enzyme capacity in vivo and our knowledge of how the capacity of the enzyme for catalysis can be maintained in vitro (4–9). With the discovery and initial characterization of rubisco activase, many of these discrepancies have been resolved and a more detailed understanding of rubisco functioning should be possible in the near future.

Rubisco Activase; Purification, Subunit Composition and Species Distribution

The activity or RuBP carboxylase/oxygenase (rubisco) in leaves increases upon illumination (1,2). Somerville et al. (3) provided evidence that light activation of rubisco is a prerequisite for catalytic competence in vivo by isolating a rubisco light activation mutant of Arabidopsis, designed rca, in which rubisco appeared to be poorly activated in vivo. Electrophoretic analysis of chloroplast proteins from mutant and wild-type Arabidopsis demonstrated that two polypeptides were missing in the mutant (4). A causal relationship between the missing polypeptides in the mutant and the inability to activate rubisco in vivo was established by genetic analysis (4). Using a reconstituted assay for rubisco activation, Salvucci et al (4) demonstrated that rubisco activation required a protein present in chloroplast extracts of spinach and Arabidopsis wild type but absent in extracts of the rca mutant. Based on these data it was proposed that rubisco activation is catalyzed in vivo by a specific stromal protein, rubisco activase (4,5). This paper reports the purification of rubisco activase from spinach, its subunit composition, and the identification of this protein in several plant species.