National Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, China department of Microbiology, China Agricultural University, Beijing 100094, China
A positive clone of a 2.6 kb Sail fragment was isolated by screening the Azospirillum brasilenseYxi62 genomic library by using a probe of PCR product of conserved central region of nifA gene. Sequencing of the 2.6 kb Sail fragment revealed three ORFs. One ORF (from 713 bp to 2227 bp) shares 59% identity with FlbD of Caulobacter crescentus, which is a global transcriptional regulator of many cj54-dependent flagellar genes. Two incomplete ORFs were found in the flanking regions of flbD. The upstream one shows homology with motA in many bacteria, e.g. Rhodospirillum centenum. MotA is a membrane protein that enables flagellar motor rotation. The downstream incomplete ORF is homologous to flhA which encodes another membrane protein suggested to be involved in regulation or secretion of some flagellar proteins. The overall homology suggests this 2.6 kb fragment is a portion of a flagellar gene cluster in A. brasilense.
The flbD mutant designated as A. brasilenseYVl was obtained by inserting a 1.2 kb kanamycin resistance cassette (Kan1) at the Aatll site offlbD. This mutant strain can neither swim in liquid medium (observed under light microscopy), nor swarm on semisolid plates. Electron microscopy showed that YF2 (the flbD mutant) grown in semisolid medium lost both polar and lateral flagella (Fla'Laf).
A flbD complementation plasmid pFV5 was constructed by inserting the 2.6 kb flbD fragment into the Xhol site of plasmid pYKlOO. Plasmid pFV5 was introduced into Yu62 (wild-type) and YF2 (flbD"mutant), respectively. Motility was restored after introducing pFV5 into flbD mutant YF2, with a larger swarming circle than wild type Yu62.
Further electron microscopic studies indicated that the complemented mutant strain YF2/pFV5 possesses a single polar flagellum and 2-3 lateral flagella (fewer than wild type Yu62), whereas the wild type strain Yu62 carrying pFV5 has multiple polar flagella but loses lateral flagella. It appears that different flagellation gave rise to differences in motility of the above strains. In summary, the mutation and complementation analysis clearly indicated that flbD is essential for both polar and lateral flagellar biosynthesis in A. brasilense.
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