Lantibiotic Reductase LtnJ Substrate Selectivity Assessed with a Collection of Nisin Derivatives as Substrates

Lantibiotics are potent antimicrobial peptides characterized by the presence of dehydrated amino acids, dehydroalanine and dehydrobutyrine, and (methyl)lanthionine rings. In addition to these posttranslational modifications, some lantibiotics exhibit additional modifications that usually confer increased biological activity or stability on the peptide. LtnJ is a reductase responsible for the introduction of d-alanine in the lantibiotic lacticin 3147. The conversion of l-serine into d-alanine requires dehydroalanine as the substrate, which is produced in vivo by the dehydration of serine by a lantibiotic dehydratase, i.e., LanB or LanM. In this work, we probe the substrate specificity of LtnJ using a system that combines the nisin modification machinery (dehydratase, cyclase, and transporter) and the stereospecific reductase LtnJ in Lactococcus lactis. We also describe an improvement in the production yield of this system by inserting a putative attenuator from the nisin biosynthesis gene cluster in front of the ltnJ gene. In order to clarify the sequence selectivity of LtnJ, peptides composed of truncated nisin and different mutated C-terminal tails were designed and coexpressed with LtnJ and the nisin biosynthetic machinery. In these tails, serine was flanked by diverse amino acids to determine the influence of the surrounding residues in the reaction. LtnJ successfully hydrogenated peptides when hydrophobic residues (Leu, Ile, Phe, and Ala) were flanking the intermediate dehydroalanine, while those in which dehydroalanine was flanked by one or two polar residues (Ser, Thr, Glu, Lys, and Asn) or Gly were either less prone to be modified by LtnJ or not modified at all. Moreover, our results showed that dehydrobutyrine cannot serve as a substrate for LtnJ.     

Enhancing initiation and proliferation in radiata pine (<Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don) somatic embryogenesis through seed family screening,zygotic embryo staging and media adjustments

In Pinus spp., initiation of somatic embryogenesis (SE) is influenced by the developmental stage of immature embryos, the genotype of the parent trees and the formulation of tissue culture medium. Optimizing all these factors can lead to improved initiation and proliferation response; however, few studies have focused on improving these stages. For this reason, the objectives of this research were to determine the best immature zygotic embryo developmental stage for initiation and to test the effect of different sources of organic nitrogen in the initiation and proliferation steps in Pinus radiata SE. We have determined and verified the optimum zygotic embryo developmental stages 2–4 for embryogenic tissue (ET) initiation and proliferation and identified the most responsive seed families in two consecutive years. Besides EDM (Walter et al. 1998), medium with high gellan gum content during ET proliferation maintained the embryogenic tissue in a better micro-morphological arrangement for a longer time.     

Zirex: a Novel Zinc-Regulated Expression System for Lactococcus lactis

Here, we report a new zinc-inducible expression system for Lactococcus lactis, called Zirex, consisting of the pneumococcal repressor SczA and P_czcD. P_czcD tightly regulates the expression of green fluorescent protein in L. lactis. We show the applicability of Zirex together with the nisin-controlled expression system, enabling simultaneous but independent regulation of different genes.     

Characterization of bacterial communities associated with Brassica napus L. growing on a Zn-contaminated soil and their effects on root growth

The interaction between plant growth-promoting bacteria (PGPB) and plants can enhance biomass production and metal tolerance of the host plants. This work aimed at isolating and characterizing the cultivable bacterial community associated with Brassica napus growing on a Zn-contaminated site, for selecting cultivable PGPB that might enhance biomass production and metal tolerance of energy crops. The effects of some of these bacterial strains on root growth of B. napus exposed to increasing Zn and Cd concentrations were assessed. A total of 426 morphologically different bacterial strains were isolated from the soil, the rhizosphere, and the roots and stems of B. napus. The diversity of the isolated bacterial populations was similar in rhizosphere and roots, but lower in soil and stem compartments. Burkoholderia, Alcaligenes, Agrococcus, Polaromonas, Stenotrophomonas, Serratia, Microbacterium, and Caulobacter were found as root endophytes exclusively. The inoculation of seeds with Pseudomonas sp. strains 228 and 256, and Serratia sp. strain 246 facilitated the root development of B. napus at 1,000 µM Zn. Arthrobacter sp. strain 222, Serratia sp. strain 246, and Pseudomonas sp. 228 and 262 increased the root length at 300 µM Cd.     

Serum interleukin-6 in renal osteodystrophy: relationship with serum PTH and bone remodeling markers.

Interleukin-6, synthesized by osteoblasts in response to PTH, stimulates osteoclastogenesis and bone resorption in vitro, and it has been implicated in the pathogenesis of bone loss in several clinical situations. The aim of this study was to evaluate whether serum levels of interleukin-6 were increased in patients with renal osteodystrophy, and to investigate the possible relationships between serum interleukin-6 and PTH levels on one hand, and serum interleukin-6 and bone remodeling markers on the other. Serum interleukin-6 (IL-6), intact PTH, osteocalcin, bone alkaline phosphatase (BAP) and carboxyterminal telopeptide of Type 1 collagen (ICTP) were measured in 86 uremic patients. IL-6 (median [range] 16.5 [1.0-430] pg/ml), PTH (279.8 [11-2004] pg/ml), osteocalcin (143.8 [8-921] ng/ml), BAP (20.9 [6-169] U/I) and ICTP (38.8 [1.5-181.5] microg/l) were higher than normal. IL-6 levels correlated with PTH (r= 0.22, p = 0.04) and with ICTP (r = 0.31, p = 0.004). A stronger correlation was found between PTH and circulating bone remodeling markers (r = 0.66 for osteocalcin, r = 0.56 for BAP, and r = 0.39 for ICTP). The correlation between PTH and IL-6 was stronger in those patients (n = 15) with severe secondary hyperparathyroidism (r= 0,71, p = 0.003). On the other hand, in the group of patients (n = 41) with PTH lower than 250 pg/ml, there was no correlation between IL-6 and PTH, while IL-6 correlated with ICTP (r = 0.44, p = 0.006). Serum IL-6 correlates with ICTP which suggests that it may mediate bone resorption in renal osteodystrophy.     

Somatic embryogenesis in Pinus halepensis Mill.: an important ecological species from the Mediterranean forest

Pinus halepensis Mill. is a common forest species in the Mediterranean area and it is important for environmental conservation. This study established a method of regenerating Pinus halepensis Mill. through somatic embryogenesis. The effect of culture medium (mineral salts, nitrogen source and plant growth regulators), collection date and seed family on embryogenic tissue initiation and proliferation in Pinus halepensis was analysed during the first steps of embryogenesis process. This study showed a marked effect of the culture medium tested as well as some significant differences among collection dates. Furthermore, the embryogenic tissue initiation was affected by the amino acid mixture in the culture medium and the proliferation stage was significantly affected by the combination of plant growth regulators. At the end of the maturation phase the presence of activated charcoal was also evaluated. Finally, maturation of embryogenic tissue was affected by the nitrogen source in the culture medium and these results were different for high and low mature embryo producing cell lines. To the best of our knowledge, this is the first report on Aleppo pine somatic embryogenesis describing a simple and efficient procedure for large-scale somatic embryo production.