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Applied and Environmental Microbiology, November 2009, p. 6792-6803, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.00902-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mutations That Disrupt Either the pqq or the gdh Gene of Rahnella aquatilis Abolish the Production of an Antibacterial Substance and Result in Reduced Biological Control of Grapevine Crown Gall{triangledown}

Yan Bin Guo,1 Jinyun Li,2* Lei Li,2 Fan Chen,2,3 Wenliang Wu,1 Jianhui Wang,2 and Huimin Wang2*

Department of Ecological Science and Engineering, College of Resources and Environmental Science, China Agricultural University, Beijing 100193, People's Republic of China,1 Department of Plant Pathology, College of Agriculture and Biotechnology, China Agricultural University, and Key Laboratory of Plant Pathology, Ministry of Agriculture, Beijing 100193, People's Republic of China,2 Bureau of Fuzhou Landscape Architecture, Liuyi North Road, Fuzhou 350011, Fujian Province, People's Republic of China3

Received 22 April 2009/ Accepted 29 August 2009

Rahnella aquatilis HX2, a biocontrol agent for grapevine crown gall caused by Agrobacterium vitis, produces an antibacterial substance that inhibits the growth of A. vitis in vitro. In this study, we show that MH15 and MH16, two Tn5-induced mutants of HX2, have lost their abilities to inhibit A. vitis and have reduced biocontrol activities; they grow in logarithmic phase at a rate similar to that of the wild type and have single Tn5 insertions. They are also impaired in producing pyrroloquinoline quinone (PQQ) or glucose dehydrogenase (GDH). Complementation of MH15 and MH16 with cosmid clones of CP465 and CP104 from an HX2 DNA library restored the antibiosis, biocontrol, and PQQ or GDH production phenotypes. A 6.7-kb BamHI fragment from CP465 that fully restored the MH15-affected phenotypes was cloned and sequenced. Sequence analysis of the mutated DNA region resulted in the identification of seven open reading frames (ORFs), six of which share significant homology with PQQ-synthesizing genes in other bacteria, designated pqqA through pqqF. Meanwhile, A 5.5-kb PstI fragment from CP104 fully complemented the MH16 mutant and contained a single ORF highly similar to that of genes coding for GDHs. An in-frame gdh deletion mutant has the same phenotypes as the Tn5 mutant of MH16. Complementation of both deletion and Tn5 gdh mutants restored the affected phenotypes to wild-type levels. Our results suggest that an antibacterial substance plays a role in biocontrol of A. vitis by HX2.

* Corresponding author. Mailing address: Department of Plant Pathology, China Agricultural University, Beijing 100193, People's Republic of China. Phone: 86-10-62733018. Fax: 86-10-62733532. E-mail for Jinyun Li: lijinyun{at}cau.edu.cn. E-mail for Huimin Wang: wanghm69{at}cau.edu.cn

{triangledown} Published ahead of print on 4 September 2009.

Applied and Environmental Microbiology, November 2009, p. 6792-6803, Vol. 75, No. 21
0099-2240/09/$08.00+0     doi:10.1128/AEM.00902-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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