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A trans-acting protein interacting with a specific sequence motif proximal to the transcriptional start site of the L-asparaginase promoter has been observed previously (E. Vincze, J. M. Reeves, E. Lamping, K. J. F. Farnden, and P. H. S. Reynolds, Plant Mol. Biol. 26:303-311, 1994). Gel retardation experiments in which protein extracts of Mesorhizobium loti and developing nodules were used suggested a bacterial origin for the repressor binding protein (rep2037). Nodulation tests were performed by using different [Fix.sup.-] Tn5 mutants of M. loti. Analyses of these mutants revealed a correlation between the presence of Mesorhizobium in the nodule-like structures and the ability of nodule protein extracts to bind the repressor binding domain (RBD). Through the use of mutated RBD sequences, the RBD sequence was identified as CTAAAAT. The repressor protein was isolated from M. loti NZP2037 by multiple chromatographic procedures and affinity separation by using concatemers of RBD attached to magnetic beads. Sequencing of the recovered protein resulted in identification of the repressor protein as the sarcosine oxidase [alpha] subunit. This was confirmed by expression of the gene encoding the M. loti [alpha] subunit of sarcosine oxidase in Escherichia coli. When the expressed peptide was bound to RBD, the gel retardation result was identical to the result obtained with rep2037 from M. loti strain NZP2037.

Summary: The common polysaccharide antigen (CPA) of the lipopolysaccharide (LPS) from Pseudomonas syringae is highly variable, but the genetic basis for this is poorly understood. We have characterized the CPA locus from P. syringae pv. actinidiae (Psa). This locus has genes for l‐ and d‐rhamnose biosynthesis and an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an o‐methyltransferase. This operon is predicted to have a role in the transport, elongation and termination of the CPA oligosaccharide and is referred to as the TET operon. Two alleles of the TET operon were present in different biovars (BV) of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from BV1 and replacement with that from BV3, demonstrated the link between the genetic locus and the biochemical properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a range of P. syringae and P. viridiflava isolates displayed a phylogenetic history incongruent with core gene phylogeny but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility. [ABSTRACT FROM AUTHOR]

The plant growth-repressing DELLA proteins (DELLAs) are known to represent a convergence point in integration of multiple developmental and environmental signals in planta, one of which is hormone gibberellic acid (GA). Binding of the liganded GA receptor (GID1/GA) to the N-terminal domain of DELLAs is required for GA-induced degradation of DELLAs via the ubiquitin-proteasome pathway, thus derepressing plant growth. However, the conformational changes of DELLAs upon binding to GID1/GA, which are the key to understanding the precise mechanism of GID1/GA-mediated degradation of DELLAs, remain unclear. Using biophysical, biochemical, and bioinformatics approaches, we demonstrated for the first time that the unbound N-terminal domains of DELLAs are intrinsically unstructured proteins under physiological conditions. Within the intrinsically disordered N-terminal domain of DELLAs, we have identified several molecular recognition features, sequences known to undergo disorder-to-order transitions upon binding to interacting proteins in intrinsically unstructured proteins. In accordance with the molecular recognition feature analyses, we have observed the binding-induced folding of N-terminal domains of DELLAs upon interaction with AtGID1/GA. Our results also indicate that DELLA proteins can be divided into two subgroups in terms of their molecular compactness and their interactions with monoclonal antibodies. [ABSTRACT FROM AUTHOR]

The identification of clones expressing high levels of recombinant protein in Pichia pastoris is usually dependant upon SDS-PAGE, Western blotting, or bioactivity-based assays that are labour and time-consuming. We describe a rapid method that images green fluorescence protein (GFP) of individual P. pastoris clones transformed with vectors that express the proteins as GFP C- terminal fusion. In this report we have used the system to monitor expression of three proteins from Venturia inaequalis. Culture plates containing individual colonies were imaged on a Fuji LAS-3000 system and the intensity of fluorescence of GFP [Mean Gray Value (MGV)] of each colony recorded. Two common variables, the time course of expression and induction temperature were also optimised using this method. The results show that colonies with high levels of GFP fluorescence can be successfully used to identify, at an early stage, colonies expressing high levels of recombinant proteins. This correlation can be used to monitor the conditions for optimization of the expression and accumulation of extracellular recombinant protein in medium and to identify fractions containing GFP-tagged recombinant proteins during protein purification.