SummaryAbscisic acid (ABA) is a plant hormone involved in seed development and responses to various environmental stresses. Oxidation of abscisic aldehyde is the last step of ABA biosynthesis and is catalysed by aldehyde oxidase (EC 1.2.3.1). We have reported the occurrence of three isoforms of aldehyde oxidase, AOa, AOb and AOg, in Arabidopsis thaliana seedlings, but none oxidized abscisic aldehyde. Here we report a new isoform, AOd, found in rosette leaf extracts, which ef®ciently oxidizes abscisic aldehyde. AOd was speci®cally recognized by antibodies raised against a recombinant peptide encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3 and AAO4). Functionally expressed AAO3 protein in the yeast Pichia pastoris showed a substrate preference very similar to that of rosette AOd. These results indicate that AOd is encoded by AAO3. AOd produced in P. pastoris exhibited a very low K m value for abscisic aldehyde (0.51 mM), and the oxidation product was determined by gas chromatography±mass spectrometry to be ABA. Northern analysis showed that AAO3 mRNA is highly expressed in rosette leaves. When the rosette leaves were detached and exposed to dehydration, AAO3 mRNA expression increased rapidly within 3 h of the treatment. These results suggest that AOd, the AAO3 gene product, acts as an abscisic aldehyde oxidase in Arabidopsis rosette leaves.

A new developmental gene, fruA, of Myxococcus xanthus was cloned using a one-step cloning vector, TnV. DNA sequencing of the wild-type allele of the fruA gene indicated that the fruA gene encodes a protein of 229 amino acid residues with a calculated molecular weight of 24672. The deduced amino acid sequence of FruA protein showed similarity to those of many bacterial regulatory proteins carrying a DNA-binding helix-turn-helix motif. The transcription-initiation site of the fruA gene was determined by a primer-extension experiment. Development of M. xanthus cells with a disrupted fruA gene stopped at the stage of mound formation. Although cells were able to aggregate to form mounds, myxospores were not formed. By Northern and Western blot analysis, it was found that the fruA expression was not detected during vegetative growth but initiated at around 6 h and reached the highest level at 12 h after the onset of development. Expression of the fruA gene was dependent on the expression of asg, bsg, csg, dsg, and esg genes, indicating that a series of intercellular signalling is necessary for the expression of the fruA gene. The effects of the fruA mutation on beta-galactosidase expression of various developmentally regulated genes fused with the lacZ gene were analysed; three developmental lacZ fusions (omega 4469, omega 4273 and omega 4500) were either poorly induced or not induced at all, while three other lacZ fusions (omega 4408, omega 4521 and omega 4455) expressed at the early stage of development were normally induced but were unable to be repressed at a later stage of development as in the wild-type strain. Interestingly, in the fruA mutant, tps (the gene for protein S) was not activated. From these results together with analysis of the amino acid sequence of FruA, we propose that FruA is a putative transcription factor required for the development of M.xanthus.

The entire nucleotide sequence of the pil region of the IncI1 plasmid R64 was determined. Analysis of the sequence indicated that 14 genes, designated pilI through pilV, are involved in the formation of the R64 thin pilus. Protein products of eight pil genes were identified by the maxicell procedure. The pilN product was shown to be a lipoprotein by an experiment using globomycin. A computer search revealed that several R64 pil genes have amino acid sequence homology with proteins involved in type IV pilus biogenesis, protein secretion, and transformation competence. The pilS and pilV products were suggested to be prepilins for the R64 thin pilus, and the pilU product appears to be a prepilin peptidase. These results suggest that the R64 thin pilus belongs to the type IV family, specifically group IVB, of pili. The requirement of the pilR and pilU genes for R64 liquid mating was demonstrated by constructing their frameshift mutations. Comparison of three type IVB pilus biogenesis systems, the pil system of R64, the toxin-coregulated pilus (tcp) system of Vibrio cholerae, and the bundle-forming pilus (bfp) system of enteropathogenic Escherichia coli, suggests that they have evolved from a common ancestral gene system.Type IV pili are rod-like surface appendages produced by gram-negative bacteria such as Pseudomonas aeruginosa, Neisseria gonorrhoeae, Moraxella bovis, Myxococcus xanthus, and Vibrio cholerae, as well as enteropathogenic and enterotoxigenic Escherichia coli (for reviews, see references 27, 28, and 35). They are flexible, with a diameter of 6 to 7 nm and a length of up to 20 m. They are produced at the polar position of the bacterial cell. Many type IV pili play important roles in the attachment of bacterial pathogens to membranes of eukaryotic host cells, as do the other pili (13, 35). Type IV pili are also associated with the twitching motility of various bacteria and with the social motility of myxobacteria (40).Type IV pili are composed of pilin subunits (35). Pilin molecules from various bacteria have amino acid sequence homology (see Fig. 4E). The type IV pilin family is usually divided into two groups. Group A consists of pilins from P. aeruginosa, N. gonorrhoeae, M. bovis, and so on. They are closely related in amino acid sequence and are produced from prepilin molecules through the cleavage of 6-to 7-amino-acid signal peptides. The N-terminal amino acid of type IVA mature pilins is phenylalanine and is N methylated. Group B pilins, including toxin-coregulated pilus (tcp) in V. cholerae (26) and bundleforming pilus (bfp) in enteropathogenic E. coli (33,34), are substantively different from type IVA pilins. Their signal peptides are longer than those of type IVA pilins. The N-terminal amino acid of type IVB mature pilins is methionine or leucine.The C-terminal amino acid (glycine) of signal peptides and the 5th amino acid (glutamic acid) of mature pilins are completely conserved among type IVA and type IVB prepilins and related proteins (see Fig. 4E). A long hydrophobic segment is present at th...

SummaryThe entire nucleotide sequence of the transfer region of IncI1 plasmid R64 was determined together with previously reported sequences. Twenty-two transfer genes, traE±Y and nuc, were newly identified in the present study. The protein products of 17 genes were detected by maxicell experiments or by the T7 RNA polymerase expression system. Mutagenesis experiments indicated that 16 genes were indispensable for R64 transfer both in liquid and on surfaces. In summary, the R64 transfer region located within an < 54 kb DNA segment was shown to encode the most complex transfer system so far studied. It contains at least 49 genes and may produce 58 different proteins as a result of shufflon DNA rearrangement and overlapping genes. Among the 49 genes, 23 tra, trb and nik genes have been shown to be indispensable for R64 conjugal transfer in liquid and on surfaces. Twelve additional pil genes are required only for liquid matings. The amino acid sequences of 10 R64 tra/trb products share similarity with those of the icm/dot products of Legionella pneumophila that are responsible for its virulence, suggesting that the R64 transfer and L. pneumophila icm/dot systems have evolved from a common ancestral genetic system.

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