Many rhizobacteria isolated from plant rhizosphere produce various phytohormones in the form of secondary metabolites, the most common of which is Indole-3-acetic acid (IAA). Here, we detail analytical protocols of IAA detection and quantification, in vitro and in situ, as recently applied to Klebsiella SGM 81, a rhizobacterium isolated from the rhizosphere of Dianthus caryophyllus (a commercially important flower across the globe). Specifically, we describe a detailed protocol for a colorimetric assay using the Salkowski reagent method, which can be used to screen for the presence of Indole compounds. To further detect and quantify IAA, a highly accurate analytical approach of LC-MS/MS is used. To detect the presence of IAA around the root system of Dianthus caryophyllus, in situ staining of plant roots is done using Salkowski reagent. Bio-protocol is an online peer-reviewed protocol journal. Its mission is to make life science research more efficient and reproducible by curating and hosting high quality, free access protocols.

The bacterial AAA+ enhancer-binding proteins (EBPs) HrpR and HrpS (HrpRS) of Pseudomonas syringae (Ps) activate σ⁵⁴-dependent transcription at the hrpL promoter; triggering type-three secretion system-mediated pathogenicity. In contrast with singly acting EBPs, the evolution of the strictly co-operative HrpRS pair raises questions of potential benefits and mechanistic differences this transcription control system offers. Here, we show distinct properties of HrpR and HrpS variants, indicating functional specialization of these non-redundant, tandemly arranged paralogues. Activities of HrpR, HrpS and their control proteins HrpV and HrpG from Ps pv. tomato DC3000 in vitro establish that HrpRS forms a transcriptionally active hetero-hexamer, that there is a direct negative regulatory role for HrpV through specific binding to HrpS and that HrpG suppresses HrpV. The distinct HrpR and HrpS functionalities suggest how partial paralogue degeneration has potentially led to a novel control mechanism for EBPs and indicate subunit-specific roles for EBPs in σ⁵⁴-RNA polymerase activation.

Nitrogen regulation in Escherichia coli is a model system for gene regulation in bacteria. Growth on glutamine as a sole nitrogen source is assumed to be nitrogen limiting, inferred from slow growth and strong NtrB/NtrC-dependent gene activation. However, we show that under these conditions, the intracellular glutamine concentration is not limiting but 5.6-fold higher than in ammonium-replete conditions; in addition, α-ketoglutarate concentrations are elevated. We address this glutamine paradox from a systems perspective. We show that the dominant role of NtrC is to regulate glnA transcription and its own expression, indicating that the glutamine paradox is not due to NtrC-independent gene regulation. The absolute intracellular NtrC and GS concentrations reveal molecular control parameters, where NtrC-specific activities were highest in nitrogen-starved cells, while under glutamine growth, NtrC showed intermediate specific activity. We propose an in vivo model in which α-ketoglutarate can derepress nitrogen regulation despite nitrogen sufficiency. IMPORTANCE Nitrogen is the most important nutrient for cell growth after carbon, and its metabolism is coordinated at the metabolic, transcriptional, and protein levels. We show that growth on glutamine as a sole nitrogen source, commonly assumed to be nitrogen limiting and used as such as a model system for nitrogen limitation, is in fact nitrogen replete. Our integrative quantitative analysis of key molecules involved in nitrogen assimilation and regulation reveal that glutamine is not necessarily the dominant molecule signaling nitrogen sufficiency and that α-ketoglutarate may play a more important role in signaling nitrogen status. NtrB/NtrC integrates α-ketoglutarate and glutamine signaling—sensed by the UTase ( glnD ) and PII ( glnB ), respectively—and regulates the nitrogen response through self-regulated expression and phosphorylation-dependent activation of the nitrogen ( ntr ) regulon. Our findings support α-ketoglutarate acting as a global regulatory metabolite.

Transcriptional initiation invariably involves the transition from a closed RNA polymerase (RNAP) promoter complex to a transcriptional competent open complex. Activators of the bacterialσ⁵⁴-RNAP are AAA+ proteins that couple ATP hydrolysis to restructure the σ⁵⁴-RNAP promoter complex. Structures of the σ⁵⁴ activator PspF AAA+ domain (PspF_1–275) bound to σ⁵⁴ show two loop structures proximal to σ⁵⁴ as follows: the σ⁵⁴ contacting the GAFTGA loop 1 structure and loop 2 that classifies σ⁵⁴ activators as pre-sensor 1 β-hairpin AAA+ proteins. We report activities for PspF_1–275 mutated in the AAA+ conserved sensor I threonine/asparagine motif (PspF_1–275^T148A, PspF_1–275^N149A, and PspF_1–275^N149S) within the second region of homology. We show that sensor I asparagine plays a direct role in ATP hydrolysis. However, low hydrolysis rates are sufficient for functional output in vitro. In contrast, PspF_1–275^T148A has severe defects at the distinct step of σ⁵⁴ promoter restructuring. This defect is not because of the failure of PspF_1–275^T148A to stably engage with the closed σ⁵⁴ promoter, indicating (i) an important role in ATP hydrolysis-associated motions during energy coupling for remodeling and (ii) distinguishing PspF_1–275^T148A from PspF_1–275 variants involved in signaling to the GAFTGA loop 1, which fail to stably engage with the promoter. Activities of loop 2 PspF_1–275 variants are similar to those of PspF_1–275^T148A suggesting a functional signaling link between Thr¹⁴⁸ and loop 2. In PspF_1–275 this link relies on the conserved nucleotide state-dependent interaction between the Walker B residue Glu¹⁰⁸ and Thr¹⁴⁸. We propose that hydrolysis is relayed via Thr¹⁴⁸ to loop 2 creating motions that provide mechanical force to the GAFTGA loop 1 that contacts σ⁵⁴.

The plant pathogen Pseudomonas syringae pv. tomato (DC3000) causes virulence by delivering effector proteins into host plant cells through its type three secretion system (T3SS). In response to the plant environment DC3000 expresses hypersensitive response and pathogenicity genes (hrp). Pathogenesis depends on the ability of the pathogen to manipulate the plant metabolism and to inhibit plant immunity, which depends to a large degree on the plant’s capacity to recognise both pathogen and microbial determinants (PAMP/MAMP-triggered immunity). We have developed and employed MS-based shotgun and targeted proteomics to (i) elucidate the extracellular and secretome composition of DC3000 and (ii) evaluate temporal features of the assembly of the T3SS and the secretion process together with its dependence of pH. The proteomic screen, under hrp inducing in vitro conditions, of extracellular and cytoplasmatic fractions indicated the segregated presence of not only T3SS implicated proteins such as HopK1, HrpK1, HrpA1 and Avrpto1, but also of proteins not usually associated with the T3SS or with pathogenicity. Using multiple reaction monitoring MS (MRM-MS) to quantify HrpA1 and Avrpto1, we found that HrpA1 is rapidly expressed, at a strict pH-dependent rate and is post-translationally processed extracellularly. These features appear to not interfere with rapid Avrpto1 expression and secretion but may suggest some temporal post-translational regulatory mechanism of the T3SS assembly. The high specificity and sensitivity of the MRM-MS approach should provide a powerful tool to measure secretion and translocation in infected tissues.