Dual two-component regulatory systems are involved in aromatic compound degradation in a polychlorinated-biphenyl degrader, Rhodococcus jostii RHA1.

A Gram-positive polychlorinated-biphenyl (PCB) degrader, Rhodococcus jostii RHA1, degrades PCBs by cometabolism with biphenyl. A two-component BphS1T1 system encoded by bphS1 and bphT1 (formerly bphS and bphT) is responsible for the transcription induction of the five gene clusters, bphAaAbAcAdC1B1, etbAa1Ab1CbphD1, etbAa2Ab2AcD2, etbAdbphB2, and etbD1, which constitute multiple enzyme systems for biphenyl/PCB degradation. The bphS2 and bphT2 genes, which encode BphS2 and BphT2, virtually identical to BphS1 (92%) and BphT1 (97%), respectively, were characterized. BphS2T2 induced the activation of the bphAa promoter in a host, Rhodococcus erythropolis IAM1399, in the presence of a variety of aromatics, including benzene, toluene, ethylbenzene, xylenes, isopropylbenzene, and chlorinated benzenes, as effectively as BphS1T1. The substrate spectrum of BphS2T2 was the same as that of BphS1T1, except for biphenyl, which is a substrate only for BphS1T1. BphS2T2 activated transcription from the five promoters of biphenyl/PCB degradation enzyme gene clusters as effectively as BphS1T1. The targeted disruptions of the bphS1, bphS2, bphT1, and bphT2 genes indicated that all these genes are involved in the growth of RHA1 on aromatic compounds. The hybrid system with bphS1 and bphT2 and that with bphS2 and bphT1 were constructed, and both systems conducted induced activation of the bphAa promoter, indicating cross-communication. These results indicated that RHA1 employs not only multiple enzyme systems, but also dual regulatory systems for biphenyl/PCB degradation. Comparison of the sequences, including bphS2T2, with the bphS1T1-containing sequences and the corresponding sequences in other rhodococcal degraders suggests that bphS2T2 might have originated from bphS1T1.

Proteomics approach for identifying osmotic-stress-related proteins in soybean roots.

Osmotic stress can endanger the survival of plants. To investigate the mechanisms by which plants respond to osmotic stress, protein profiles from soybean plants treated with polyethylene glycol (PEG) were monitored by a proteomics approach. Treatment with 10% aqueous PEG reduced the lengths of roots and hypocotyls of soybean seedlings. Proteins from soybean roots were separated by two-dimensional polyacrylamide gel electrophoresis, and 415 proteins were detected by Coomassie brilliant blue staining. Thirty-seven proteins changed by PEG treatment were analyzed using Edman sequencing and peptide-mass fingerprinting method and this group included proteins involved in disease/defense. Seven proteins were selected for further experiments using the results of cluster analysis and statistical analysis of the abundance change. A comparison with the effects of other abiotic stresses showed that caffeoyl-CoA-O-methyltransferase and 20S proteasome alpha subunit A were decreased and increased by abiotic stresses, respectively. Expression analyses of these transcripts were also changed by PEG treatment. Caffeoyl-CoA-O-methyltransferase and 20S proteasome alpha subunit A may control the sensitivity of several regulatory genes specific to short exposure to osmotic stress.

Enhanced soybean infection by the legume "super-virulent" Agrobacterium tumefaciens strain KAT23.

Agrobacterium tumefaciens KAT23 harbors a nopaline-type Ti plasmid and is "super-virulent" to soybean (Glycine max) and other leguminous plants. The right and left border sequences of the essential cis-element for T-DNA transfer were removed in order to utilize the high infectivity of this strain in an Agrobacterium-mediated soybean transformation system. The resulting strain, named Soy2, showed no oncogenic activity. After inoculation with disarmed Soy2 harboring binary vector pIG121-Hm and pCAMBIA-WR, soybean epicotyls exhibited high beta-glucuronidase activities, with efficiencies higher than EHA105, an A. tumefaciens strain widely used in making transgenic plants.

Cytosolic ascorbate peroxidase 2 (cAPX 2) is involved in the soybean response to flooding.

Proteomic analyses of soybean seedlings responding to flooding were conducted to identify key proteins involved. The seeds were germinated on a spongy matrix for two days, and then subjected to flooding for three days. After flooding, the total number of roots, the length of the main root, the lengths of the lateral and adventitious roots, and the fresh weight of the underground tissues of flooded soybean seedlings were significantly suppressed compared with nontreated plants. To identify the early flooding-responsive proteins, the seedling roots were used for preparing cytosolic and membrane fractions. After two-dimensional polyacrylamide gel electrophoresis and silver staining, 208 proteins were detected, and the levels of 44 were different from those of the control. The expression pattern of 10 proteins among the 44 from six different soybean cultivars confirmed that the 10 were flooding-responsive proteins. One of the 10 proteins was dominantly down-regulated under flooding conditions and was identified as cytosolic ascorbate peroxidase 2 (cAPX 2). Northern-hybridization showed that the abundance of cAPX 2 transcript decreased significantly after flooding, as did the enzymatic activity of APX. These results suggest that cAPX 2 is involved in flooding stress responses in young soybean seedlings.

Characterization and host range determination of soybean super virulent Agrobacterium tumefaciens KAT23.

Agrobacterium tumefaciens KAT23 isolated from peach root causes crown gall disease in a number of grain legume plants, including the common bean (Phaseolus vulgaris) and soybean (Glycine max). KAT23 caused tumor formation in each of these plants more effectively than strain C58. Biotype determination suggested that this strain is biotype II. KAT23 was able to utilize nopaline as a carbon source. Partial sequence analysis indicated that KAT23 harbors a nopaline-type Ti plasmid, designated pTiKAT23, which was highly homologous with other nopaline-type Ti plasmids (pTiC58 and pTiSAKURA). KAT23 transferred not only the T-DNA of the Ti plasmid but also introduced T-DNA of the binary vector efficiently. The common bean inoculated with KAT23 (pIGFP121-Hm) showed crown galls, and some plants showed beta-glucuronidase (GUS) and sGFP (S65T) gene expression. This virulent ability of KAT23 indicates its potential application to legumes, especially to soybean transformation.