NprR belongs to the RNPP family of quorum-sensing receptors, a group of intracellular regulators activated directly by signaling oligopeptides in Gram-positive bacteria. In Bacillus thuringiensis (Bt), nprR is located in a transcriptional cassette with nprRB that codes for the precursor of the signaling peptide NprRB. NprR is a transcriptional regulator activated by binding of reimported NprRB; however, several reports suggest that NprR also participates in sporulation but the mechanism is unknown. Our in silico results, based on the structural similarity between NprR from Bt and Spo0F-binding Rap proteins from Bacillus subtilis, suggested that NprR could bind Spo0F to modulate the sporulation phosphorelay in Bt. Deletion of nprR-nprRB cassette from Bt caused a delay in sporulation and defective trigger of the Spo0A∼P-activated genes spoIIA and spoIIIG. The DNA-binding domain of NprR was not necessary for this second function, since truncated NprRΔHTH together with nprRB gene was able to restore the sporulation wild type phenotype in the ΔnprR-nprRB mutant. Fluorescence assays showed direct binding between NprR and Spo0F, supporting that NprR is a bifunctional protein. To understand how the NprR activation by NprRB could result in two different functions, we studied the molecular recognition mechanism between the signaling peptide and the receptor. Using synthetic variants of NprRB, we found that SSKPDIVG displayed the highest affinity (Kd = 7.19 nM) toward the recombinant NprR and demonstrated that recognition involves conformational selection. We propose that the peptide concentration in the cell controls the oligomerization state of the NprR-NprRB complex for switching between its two functions.
Quorum-sensing (QS) is a bacterial mechanism for regulation of gene expression in response to cell density. In Gram-positive bacteria, oligopeptides are the signaling molecules to elicit QS. The RNPP protein family (Rap, NprR, PlcR, and PrgX) are intracellular QS receptors that bind directly to their specific signaling peptide for regulating the transcription of several genes. NprR is the activator of a neutral protease in Bacillus subtilis, and it has been recently related to sporulation, cry genes transcription and extracellular protease activity in strains from the B. cereus group. In the B. thuringiensis genome, downstream nprR, a gene encoding a putative QS signaling propeptide (nprRB) was found. We hypothesized that the nprR and nprRB co-evolved because of their coordinated function in the B. cereus group. A phylogenetic tree of nucleotide sequences of nprR revealed six pherotypes, each corresponding to one putative mature NprRB sequence. The nprR tree does not match the current taxonomic grouping of the B. cereus group or the phylogenetic arrangement obtained when using MLST markers from the same strains. SKPDI and other synthetic peptides encoded in the nprRB gene from B. thuringiensis serovar thuringiensis strain 8741 had effect on temporal regulation of sporulation and expression of a cry1Aa'Z transcriptional fusion, but those peptides that stimulated earlier detection of spores decreased cry1Aa expression suggesting that NprR may either activate or repress the transcription of different genes.
The NprR protein and NprRB signaling peptide comprise a bifunctional quorum–sensing system from the Bacillus cereus group that is involved in transcriptional activation through DNA‐binding and in sporulation initiation by binding to Spo0F. We characterized in vitro the direct interactions established by NprR that may be relevant for performing its two functions. Apo‐NprR interacted with Spo0F, but not with the target DNA. The NprRB signaling peptide SSKPDIVG that binds strongly to Apo‐NprR, failed to bind and disrupt the NprR‐Spo0F complex. Finally, the NprR‐NprRB complex bound both to Spo0F and the target DNA with similar affinity. Based on our findings, we propose that rather than a switch triggered by NprRB, the NprR/NprRB ratio and the availability of Spo0F binding sites define the function of NprR.
BACKGROUND: Ethanologenic Escherichia coli KO11 was modified to channel carbon flux from glucose through the Entner-Doudoroff (ED-P) and pentose phosphate (PP-P) pathways by using a phosphoglucose isomerase (pgi) knockout in the glycolytic pathway. This strain grows very slowly under non-aerated conditions with minimal media supplemented with 4% glucose. To improve the capacity to grow, KO11 pgi was evolved for 60 days; the resultant strain was named KO11 E35, which directs the carbon flux through the PP-P and ED-P to lactate and acetate production.
RESULTS:The activities of glucose-6-phosphate dehydrogenase and the ED-P enzymes increased 17-fold and 2-fold, respectively, in KO11 E35 in comparison with KO11. A homoethanologenic derivative was constructed from KO11 E35 by deleting the pta, ack and ldh genes, yielding the KO11 PPAL strain. This strain channels most of the carbon flux from pyruvate to ethanol and increased expression of heterologous pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis allowed us to obtain specific ethanol production rates similar to those found in KO11, but with half the cell mass, i.e. larger ethanol/glucose and ethanol/biomass yields. CONCLUSIONS: These results suggest that it is possible to obtain the same carbon flux using the PP-P and ED-P as when using the Embden-Meyerhof-Parnas pathway for glucose catabolism to ethanol.
Comparison of specific activitiesThe main differences between KO11 E35 and KO11 were that the PFL, PDC and ADH activities were slightly higher in KO11 E35. In contrast, the ZWF and E-D enzyme activities were 17 and 2-fold higher, respectively, in KO11 E35 (Table 2). In accordance with previous reports, 23 as a result of an increase in enzymatic activities, flux redistribution and/or up regulation in existing pathways was observed in response to the adaptive evolution process.J Chem Technol Biotechnol 2017; 92: 990-996
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