Plant health and development are influenced by a broad range of microorganisms inhabiting the root rhizosphere, including beneficial associative bacteria. These bacteria are usually minor components of the rhizosphere microbial community but have been shown to significantly enhance plant growth and yield . This ability to enhance plant growth has been attributed to diverse mechanisms, including biological nitrogen fixation, production of phytohormones, enhancement of nutrient uptake , and bio-control of pathogens and pests . Plant growth-promoting bacteria can colonize roots either on the root surface or as endophytes without eliciting a noticeable plant defense response. Unlike some other bacterium-plant interactions that show strict host specificity, many PGPB can colonize a wide variety of plant species, including agriculturally important members of the Poaceae family, such as rice, maize, wheat, and a variety of bio-energy grasses . PGPB strains have been described for several host plants, including Setaria viridis, a model C4 plant, which is a close relative to a variety of bio-energy grasses. In a previous study, we demonstrated that S. viridis under lab conditions can obtain up to 100% of its nitrogen needs through biological nitrogen fixation mediated by diazotrophic PGPB . Among the strains used were the betaproteobacteria Azoarcus olearius and Herbaspirillum seropedicae, which exhibit strong growth-promoting ability . Azoarcus olearius DQS4T was originally isolated from oil-contaminated soil and, based on its genome sequence, this strain shows high similarity with the well-studied A. olearius strain BH72 . Strain DQS4T can colonize the roots of rice and Setaria to high levels and increase below- and above-ground biomass . H. seropedicae SmR1 is a well-studied endophytic bacterium that colonizes several plants, including maize, wheat, and Setaria. Recently,container size for strawberries in situ metabolomic profiling of S. viridis roots colonized by SmR1 demonstrated that inoculation induced a wide variety of plant metabolic changes, including those affecting nitrogen and phytohormone levels .
Thus, similar to many other, better-studied plant-microbe associations, PGPB inoculation appears to profoundly affect the metabolism of its host. To identify specific PGPB genes that contribute to plant root colonization, we applied transposon mutagenesis sequencing coupled with random barcoding. This technique assesses gene functionality in mutant strains through the generation of reusable libraries of unique and mapped mutant insertions.TnSeq is a powerful and sensitive approach to identify bacterial gene functions that play roles in bacterial fitness under researcher-defined growth conditions . Starting with information on the location and frequency of each mutation in the population, the change in mutational frequency after passage of the population through an environmental challenge leads to the identification of genes that are either essential, important, or detrimental to growth under that condition. For example, TnSeq of Streptococcus pneumoniae identified genes essential for bacterial basal growth, as well as genes involved in transcriptional regulation and carbohydrate transport . In the PGPB Pseudomonas simiae, TnSeq revealed genes involved in carbon metabolism and motility that could enhance or suppress colonization of Arabidopsis roots . In the present work, we applied standard TnSeq and a variant of TnSeq that uses random DNA barcodes to measure strain abundance to construct mutant libraries of strains DQS4T and SmR1, respectively, and to then evaluate genetic contributions to fitness on the host plant Setaria viridis.Gene essentiality is defined as its importance in maintaining competitive fitness under a condition of interest . For this study, two PGPB bacterial species were used, Azoarcus olearius DQS4T and Herbaspirillum seropedicae SmR1, both diazotrophic and capable of promoting plant growth . The mutagenized pool of each strain was inoculated onto Setaria viridis roots or a carbon augmented soil control with no plants.
Root-associated bacterial cells were recovered, transposon DNA was sequenced, and resulting reads were mapped to each bacterial genome. The relative incidence of a specific mutation was then determined and used to calculate fitness values. By high throughput sequence analysis of insertion mutants, we covered about 3,692 genes of DQS4T and 3,878 genes of SmR1 distributed throughout either strain’s genome. Thus, regardless of method, very good coverage of each genome was achieved. In the case of DQS4T transposon mutagenesis, 89.2% of 4,135 total genes were mapped to a gene harboring at least 1 insertion event. For the SmR1 library, 81.1% of the 4781 genes had identified mutations. We assume that genes without an insertion in the library likely identify those essential for bacterial growth in culture. Our screening identified 89 and 130 gene mutations that significantly affected the ability of strains DQS4T and SmR1 to colonize S. viridis roots, respectively. Given that our interest was to identify genes that exclusively affected root colonization, we excluded from further consideration any genes that also affected fitness under soil conditions . The genes affecting root colonization were categorized based on the phenotypes as either enhanced fitness value, mutations that increased root association ; or decreased fitness value, mutations that impaired root association . For the complete list of gene mutations affecting fitness under each condition, see Table S2. Unsurprisingly, many of the genes identified lacked a clear functional annotation, especially for A. olearius DQS4T . Even though each strain showed a distinct pattern of mutations that affected fitness, a few common gene functions were identified impairing root colonization in both strains. The most common COG categories important for SmR1 root colonization were those involved in amino acid transport and metabolism, followed by energy production and conversion and coenzyme transport and metabolism. In the case of DQS4Tthe most common COGs were cell wall/membrane/envelope bio-genesis and signal transduction, followed by translation, ribosomal structure and bio-genesis, and inorganic ion transport and metabolism .
Although we cannot rule out that the different mutagenesis methods could contribute to the different fitness profiles, the overall mutational coverage of the genomes of both strains suggest that such effects are likely minor. Hence, the results seem to suggest that any given PGPB strain will have different major requirements for root colonization reflecting the specific metabolic needs of the bacterium.Gene mutations that positively impacted the ability of bacterial strains to colonize roots were defined as those with a fitness score $ 1. We identified 8 and 14 genes in this category in A. olearius and H. seropedicae, respectively . This list included the Azoarcus gene predicted to encode a pyrroloquinoline quinone -dependent alcohol dehydrogenase , which is involved in the oxidoreductase process of ADH. Based on sequence alignment with Azoarcus olearius BH72,container size for blueberries the DQS_RS19730 gene encodes an ExaA5 protein that was previously reported to be induced upon exposure to ethanol as a carbon source . In our study, we found that exaA5 plays an important role in root colonization. Disruption of this gene enhanced root colonization of S. viridis roots . However, the mutant was less competitive in root colonization than the wild type at 1 and 3 days after inoculation but seemed to recover to wild-type levels by 5 days after inoculation . An insertion mutation in the gene encoding a diguanylate cyclase significantly increased bacterial root colonization 3 and 5 days postinoculation and showed the same trend in competition with the wild type . Diguanylate cyclases with a GGDEF activesite motif produce cyclic di-GMP and play a major role in the transition between motile and sessile bacterial lifestyles . We also identified an iron regulator outer membrane protein, TonB , presumably required for Fe31uptake . This protein was reported to interact with ExbA and ExbB, forming a complex that regulates iron acquisition . In SmR1, 6 of the 14 gene mutations that increased root colonization are predicted to play a role in flagellar biosynthesis. FliF, FliG, FlhB, and FliQ are structural components of the flagellar basal body . MotA is complexed with MotB and functions as a proton channel for torque generation . Our testing showed that the absence of flagellar genes in the SmR1 strain conveyed an advantage for root colonization in S. viridis 10 days after inoculation. We also identified a poly-3-hydroxybutyrate -related gene, a poly-3-hydroxyalkanoate depolymerase PhaZ1 , that enhanced colonization ability when mutated . PhaZ1 is a PHB depolymerization enzyme required for the granule mobilization . Furthermore, mutations in the genes encoding transcriptional regulators and ABC transporters also resulted in increased root colonization by strain SmR1 .Mutant strains corresponding to 81 DQS4T and 114 SmR1 genes were identified as significantly reducing root colonization . Therefore, these gene functions are normally required for efficient colonization by wild-type bacteria. Within this group of mutations, we identified two genes present in both DQS4T and SmR1: cheY , which is related to chemotaxis, and ampD , which is involved in peptidogly can degradation. Chemotaxis is directly involved in modulating the movement of the flagellum in response to attractants, helping bacterial orientation and active motion for survival toward favorable conditions .
In addition, four other genes encoding transmembrane chemoreceptors, also known as methyl-accepting chemotaxis proteins , were identified in H. seropedicae. The protein CheA undergoes autophosphorylation induced by MCPs, and CheR and CheW acts to modulate the phosphorylation state of CheY . Mutations in these genes impaired the capacity of H. seropedicae to colonize roots, consistent with a previous report .Analysis of the DQS4T TnSeq data identified a gene predicted to encode a two-component sensor histidine kinase containing a HAMP domain involved in signal conversion between the transmembrane-sensing and kinase activity control . One assumes that this histidine kinase is reacting to an unknown factor in the rhizosphere that normally enhances root association in wild-type cells. We also observed genes involved in cell wall formation, such as murI , a glutamate racemase responsible for the conversion of L-glutamate in D-glutamate, and murA , a UDP-N-acetylglucosamine 1-carboxyvinyltransferase responsible for converting UDP-N-acetylglucosamine to UDP-N-acetylglucosamine enolpyruvate. A previous study of H. seropedicae reported the importance of the mur genes for maize root association and also showed that their expression was repressed in the presence of naringenin. In addition, a N-acetyl-anhydromuranmyl-L-alanine amidase AmpD, likely involved in the degradation of peptidoglycan by hydrolysis of muropeptides that can serve as signals for induction of b-lactamase , was identified as important for root colonization in both strains. We also identified a putative transcriptional regulator of catechol 2,3-dioxygenase, a flavonoidrelated compound that might be involved in the degradation of aromatic compounds important for plant-bacterial interactions . Among such compounds are flavonoids that can serve as chemoattractants for rhizobia in legumes . Flavonoids can also mediate PGPB colonization, as reported for H. seropedicae colonization of Arabidopsis and the colonization of rice by Serratia spp. . Interestingly, mutations in genes involved in PHB metabolism—including PhaP1 , a phasin family protein that affects PHB production, and PhbA1 , an acetyl-CoA acetyltransferase— decreased root colonization. The importance of PHB metabolism in bacteria during plant root colonization was reported previously in studies of Setaria-Herbaspirillum and legumerhizobium symbiosis .To more definitively test the importance of individual genes identified in our screen, we selected candidate genes and created insertion mutations in 15 separate genes. These genes were selected to cover a diversity of putative functions representing operons containing multiple genes, as well as single genes, including those associated with both positive and negative fitness scores . Using a gnotobiotic system, a competitive colonization assay was performed in which colonization of S. viridis roots by the individual mutants was measured, as well as their ability to compete for colonization when coinoculated with the corresponding wild-type strain. Since root colonization is a dynamic process and might change over time, we evaluated competition at three different time points. For A. olearius DQS4T , we selected six genes where mutations reduced colonization, specifically a transcriptional regulator, ybeZ, ketoacyl ACP synthase , peaF, pcm, and purU . Mutants defective in genes that resulted in reduced colonization in competition with the wild-type strain showed delayed colonization of Setaria roots. Mutation of the transcriptional regulator gene represents the most extreme case where colonization was completely blocked when inoculated singly onto Setaria roots . This gene encodes an AphS protein known to be involved in the phenol degradation pathway, where phenol is converted into catechol via catechol 2,3-dioxygenase . Analysis of A. olearius DQS4T and BH72 genomes showed two that is produced by the wild type that can compensate for its loss in the mutant. As for the other mutants that impaired A. olearius DQS4T colonization ybeZ, ketoacyl ACP synthase , peaF, pcm, and purU, all showed a significant reduction in colonization that was most pronounced 3 and 5 days after inoculation both when inoculated individually or in competition with the wild-type strain .