Influence of Shifting Positions of Ser, Thr, and Cys Residues in Prenisin on the Efficiency of Modification Reactions and on the Antimicrobial Activities of the Modified Prepeptides

Since the recent discovery that the nisin modification and transport machinery can be used to produce and modify peptides unrelated to nisin, specific questions arose concerning the specificity of the modification enzymes involved and the limits of their promiscuity with respect to the dehydration and cyclization processes. The nisin leader peptide has been postulated to fulfill a recognition and binding function required for these modifications. Here, we investigated whether the relative positions of the modifiable residues in the nisin prepeptide, with respect to the leader peptide, could influence the efficiency of their modification. We conducted a systematic study on the insertion of one to four alanines in front of either ring A or ring D to change the “reading frame” of modifiable residues, resulting in altered distance and topology of the modifiable residues relative to the leader. The insertion of N-terminal and hinge-located Ala residues had only a modest influence on the modification efficiency, demonstrating that the “phasing” of these residues relative to the leader peptide is not a critical factor in determining modification. However, in all cases, but especially with the N-terminal insertions, the antimicrobial activities of the fully modified nisin species were decreased.     

Mutagenesis of nisin’s leader peptide proline strongly modulates export of precursor nisin

The lantibiotic nisin is produced by Lactococcus lactis as a precursor peptide comprising a 23 amino acid leader peptide and a 34 amino acid post-translationally modifiable core peptide. We previously demonstrated that the conserved FNLD part of the leader is essential for intracellular enzyme-catalyzed introduction of lanthionines in the core peptide and also for transporter-mediated export, whereas other positions are subject to large mutational freedom. We here demonstrate that, in the absence of the extracellular leader peptidase, NisP, export of precursor nisin via the modification and transporter enzymes, NisBTC, is strongly affected by multiple substitutions of the leader residue at position -2, but not by substitution of positions in the vicinity of this site. Export levels of precursor nisin increased by more than 70% for position -2 mutants Asp, Thr, Ser, Trp, Lys, Val and decreased more than 70% for Cys, His, Met. In a strain with leader peptidase, the Pro-2Lys and Pro-2Asp precursor nisins were less efficiently cleaved by NisP than wild type precursor nisin. Taken together, the wild type precursor nisin with a proline at position -2 allows balanced export and cleavage efficiencies by precursor nisin’s transporter and leader peptidase.     

Major gene‐regulatory mechanisms operating in ribosomally synthesized and post‐translationally modified peptide (RiPP) biosynthesis

Post‐translationally modified peptides commonly display antimicrobial activity, but can also aid the development of bacterial colonies, giving a competitive advantage in the ecological niche. The production of post‐translationally modified peptides by bacteria is a complex and energetically costly process that is strictly orchestrated in the cell. The onset of peptide production is linked to the different enzymes that take part during maturation, the transporters and the immunity determinants (if required). Thus, the population can make optimal use of available resources and obtain the benefits of production at an advantageous moment during growth, avoiding toxicity to itself. The timing and level of expression of the different operons is controlled by diverse (complex) regulatory pathways in response to environmental changes, stress or master regulators during specific growth transition phases. In this review, we highlight the basic principles and mechanisms of regulation of expression of post‐translationally modified peptides and the relationship with the overall culture developmental processes and/or cellular differentiation. We also discuss the biotechnological consequences derived from the understanding of regulatory networks involved in the biosynthesis of these natural products.     

Expression of prophage-encoded endolysins contributes to autolysis of Lactococcus lactis

Analysis of autolysis of derivatives of Lactococcus lactis subsp. cremoris MG1363 and subsp. lactis IL1403, both lacking the major autolysin AcmA, showed that L. lactis IL1403 still lysed during growth while L. lactis MG1363 did not. Zymographic analysis revealed that a peptidoglycan hydrolase activity of around 30 kDa is present in cell extracts of L. lactis IL1403 that could not be detected in strain MG1363. A comparison of all genes encoding putative peptidoglycan hydrolases of IL1403 and MG1363 led to the assumption that one or more of the 99 % homologous 27.9-kDa endolysins encoded by the prophages bIL285, bIL286 and bIL309 could account for the autolysis phenotype of IL1403. Induced expression of the endolysins from bIL285, bIL286 or bIL309 in L. lactis MG1363 resulted in detectable lysis or lytic activity. Prophage deletion and insertion derivatives of L. lactis IL1403 had a reduced cell lysis phenotype. RT-qPCR and zymogram analysis showed that each of these strains still expressed one or more of the three phage lysins. A homologous gene and an endolysin activity were also identified in the natural starter culture L. lactis subsp. cremoris strains E8, Wg2 and HP, and the lytic activity could be detected under growth conditions that were identical as those used for IL1403. The results presented here show that these endolysins of L. lactis are expressed during normal growth and contribute to autolysis without production of (lytic) phages. Screening for natural strains expressing homologous endolysins could help in the selection of strains with enhanced autolysis and, thus, cheese ripening properties.

     

Differential effects of pharmacological liver X receptor activation on hepatic and peripheral insulin sensitivity in lean and ob/ob mice

Liver X receptor (LXR) agonists have been proposed to act as anti-diabetic drugs. However, pharmacological LXR activation leads to severe hepatic steatosis, a condition usually associated with insulin resistance and type 2 diabetes mellitus. To address this apparent contradiction, lean and ob/ob mice were treated with the LXR agonist GW-3965 for 10 days. Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp studies. Hepatic glucose production (HGP) and metabolic clearance rate (MCR) of glucose were determined with stable isotope techniques. Blood glucose and hepatic and whole body insulin sensitivity remained unaffected upon treatment in lean mice, despite increased hepatic triglyceride contents (61.7 +/- 7.2 vs. 12.1 +/- 2.0 nmol/mg liver, P < 0.05). In ob/ob mice, LXR activation resulted in lower blood glucose levels and significantly improved whole body insulin sensitivity. GW-3965 treatment did not affect HGP under normo- and hyperinsulinemic conditions, despite increased hepatic triglyceride contents (221 +/- 13 vs. 176 +/- 19 nmol/mg liver, P < 0.05). Clamped MCR increased upon GW-3965 treatment (18.2 +/- 1.0 vs. 14.3 +/- 1.4 ml x kg(-1) x min(-1), P = 0.05). LXR activation increased white adipose tissue mRNA levels of Glut4, Acc1 and Fasin ob/ob mice only. In conclusion, LXR-induced blood glucose lowering in ob/ob mice was attributable to increased peripheral glucose uptake and metabolism, physiologically reflected in a slightly improved insulin sensitivity. Remarkably, steatosis associated with LXR activation did not affect hepatic insulin sensitivity.     

Phosphatases modulate the bistable sporulation gene expression pattern in Bacillus subtilis

Summary Spore formation in the Gram-positive bacterium Bacillus subtilis is a last resort adaptive response to starvation. To initiate sporulation, the key regulator in this process, Spo0A, needs to be activated by the so-called phosphorelay. Within a sporulating culture of B. subtilis, some cells initiate this developmental program, while other cells do not. Therefore, initiation of sporulation appears to be a regulatory process with a bistable outcome. Using a single cell analytical approach, we show that the autostimulatory loop of spo0A is responsible for generating a bistable response resulting in phenotypic variation within the sporulating culture. It is demonstrated that the main function of RapA, a phosphorelay phosphatase, is to maintain the bistable sporulation gene expression. As rapA expression is quorum regulated, it follows that quorum sensing influences sporulation bistability. Deletion of spo0E, a phosphatase directly acting on Spo0A approximately P, resulted in abolishment of the bistable expression pattern. Artificial induction of a heterologous Rap phosphatase restored heterogeneity in a rapA or spo0E mutant. These results demonstrate that with external phosphatases, B. subtilis can use the phosphorelay as a tuner to modulate the bistable outcome of the sporulating culture. This shows that B. subtilis employs multiple pathways to maintain the bistable nature of a sporulating culture, stressing the physiological importance of this phenomenon.     

The farnesoid X receptor modulates hepatic carbohydrate metabolism during the fasting-refeeding transition

The liver plays a central role in the control of blood glucose homeostasis by maintaining a balance between glucose production and utilization. The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor. Hepatic FXR expression is regulated by glucose and insulin. Here we identify a role for FXR in the control of hepatic carbohydrate metabolism. When submitted to a controlled fasting-refeeding schedule, FXR(-/-) mice displayed an accelerated response to high carbohydrate refeeding with an accelerated induction of glycolytic and lipogenic genes and a more pronounced repression of gluconeogenic genes. Plasma insulin and glucose levels were lower in FXR(-/-) mice upon refeeding the high-carbohydrate diet. These alterations were paralleled by decreased hepatic glycogen content. Hepatic insulin sensitivity was unchanged in FXR(-/-) mice. Treatment of isolated primary hepatocytes with a synthetic FXR agonist attenuated glucose-induced mRNA expression as well as promoter activity of L-type pyruvate kinase, acetyl-CoA carboxylase 1, and Spot14. Moreover, activated FXR interfered negatively with the carbohydrate response elements regions. These results identify a novel role for FXR as a modulator of hepatic carbohydrate metabolism.