# Bacterial Indole-3-Acetic Acid Influences Soil Nitrogen Acquisition in Barley and Chickpea
## 2 Results  
### Characterization for Biological Nitrogen Fixation and Phytolormone Production in Klebsiella Strains  
The strains _K.q._ SGM81, _K.o._ M5a1, and _K.p._ SSN1 were based on the prediction of BNF and IAA production activities from their whole genome sequence data (details described in Section 4). The presence of _nif_ gene cluster and _ipdC_ gene pathway in all three genomes indicate the BNF and IAA production activities in _K.p._ SSN1, _K.q._ SGM81, and _K.o._ M5a1 strains.  
First, we screened all the _Klebsiella_ strains (_K.p._ SSN1, _K.q._ SGM81, _K.o._ M5a1, M5a1_nifH_) for their nitrogenase activity and IAA production ability in batch culture. The results of acetylene reduction assay (ARA) showed that the wild type strains exhibit considerable nitrogenase activity, indicating the active expression of _nifH_ gene. As shown in Figure 1, _K.q._ SGM81 showed maximum, i.e., 396.5 nmol, C\({}_{2}\)H\({}_{4}\) h\({}^{-1}\) mg protein\({}^{-1}\) of nitrogenase activity, followed by _K.p._ SSN1 (297.0 nmol C\({}_{2}\)H\({}_{4}\) h\({}^{-1}\) mg protein\({}^{-1}\)). Nitrogenase activity of _K.o._ M5a1 was recorded as 293.5 nmol C\({}_{2}\)H\({}_{4}\) h\({}^{-1}\) mg protein\({}^{-1}\). As expected, no nitrogenase activity was detected in M5a1_nifH_.  
Next, we quantified the tryptophan-induced indole-3-acetic acid using the [PERSON] method. The results of this assay are presented in Figure 1. The highest IAA production was seen in _K.q._ SGM81 (85.75 \(\mu\)g mL\({}^{-1}\)) followed by _K.p._ SSN1. _K.o._ M5a1 and its _nifH_ mutant did not produce any IAA, suggesting a non-functional _ipdC_ gene. A Clustal O multiple sequence alignment of _ipdC_ sequences of the three strains (Figure S1) was carried out, showing pairwise percent identities 85.17%, 85.35%, and 99.64% similarity in protein sequence between _K.o._ M5a1-_K.q._ SGM81, between _K.o._ M5a1-_K.p._ SSN1, and between _K.q._ SGM81-_K.p._ SSN1, respectively. However, the amino acid sequence alignment of _ipdC_ of M5a1, SGM81, and SSN1 (Figure S1) shows 100% identity of known highly conserved residues involved in Mg\({}^{2+}\) binding (red) and thiamine pyrophosphate binding (blue highlighted) with the functional _ipdC_ of _Enterobacter cloacae_, for which the structure and function has been detailed. Therefore, there is no obvious structure/function rational from the _K.o._ M5a1 _ipdC_ coding sequence to explain the lack of _K.o._ M5a1 to produce IAA. Next, we inspected the promoter regions of _K.q._ SGM81 and _K.o._ M5a1. Recall that _ipdC_ transcription was proposed to be 54-dependent in _Enterobacter cloacae_ and is s54-dependent diazotrophic _Azospirillum brasilense_[18]. We show that _K.q._ SGM81 has two near perfect s54 promoter sites at and around consensus the \(-24\) and \(-12\) sites from the transcriptional start with the required distance between them, whereas _K.o._ M5a1 shows no good s54 promoter sites (Figure S2). These finding suggest that the _K.o._ M5a1 ipdC s54 promoter is likely to have been corrupted. This seems feasible, given that _K.o._ M5a1 has been a decade-long laboratory strain.