The molecular genetics of indole-3-acetic (IAA) synthesis and regulation in Azospirillum brasilense was investigated in this study. Tn5 mutagenesis was performed and five mutants with decreased IAA production were isolated. Five Tn5-inserted genes from these mutants were cloned and sequenced. Four genes were reported for the first time to be involved in IAA production, namely, atrA, ftsA, omaA and aldA that code for GntR-family transcriptional regulator, iron-binding protein component of ABC-type Fe(3+) transport system, outer membrane protein, and aldehyde dehydrogenase, respectively. In addition, two genes atrB and atrC, with predicted proteins that showed high homology to aminotransferases, were cloned from the downstream of atrA in this bacterium. Studies also showed that complementation of atrA, ftsA and omaA were able to restore the IAA production of the corresponding IAA(-) mutants. Comparison of Fe(3+) concentrations in culture supernatants of the wild-type strain, the ftsA mutant and the complemented strain revealed that the iron-uptake ability of the ftsA mutant was highly reduced. This result also points to the necessity of iron as a metal ion in IAA synthesis. Statistical analysis showed no significant difference in the IAA accumulated in cells between the omaA mutant and the wild-type strain, suggesting the omaA might not affect IAA secretion but be involved in IAA production in other unknown ways.

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A 324 bp of nifH fragment was PCR amplified from Paenibacillus massiliensis T7 using the universal degenerate primers. The PCR-amplified nifH fragment was labeled with DIG and then used as a probe in Southern blot analysis. Southern blot result showed that there were two positive signals, indicating that there might be two copies of nifH in P. massiliensis T7. A total of 10254 bp DNA sequence containing purD and nifBHDKENX was obtained by five rounds of inverse-PCR amplification. The predicted proteins of nifBHDKENX had high homology with those from other nitrogen-fixing bacteria. Only one putative sigma54-dependent promoter sequence was detected upstream of the nifB gene and nifBHDKENX were likely to be organized in one operon. Assays of 3-galactosidase activity of P. massiliensis T7PB carrying a nifB-lacZ fusion under different concentrations of NH4+ and O2 showed that the expression of nifB-lacZ was strongly inhibited by O2.

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