Genetic manipulation revealing an unusual N-terminal region in a stand-alone non-ribosomal peptide synthetase involved in the biosynthesis of ramoplanins

Ramoplanins produced by Actinoplanes are new structural class of lipopeptide and are currently in phase III clinical trials for the prevention of vancomycin-resistant enterococcal infections. The depsipeptide structures of ramoplanins are synthesized by non-ribosomal peptide synthetases (NRPS). Romo-orf17, a stand-alone NRPS, is responsible for the recruitment of Thr into the linear NRPS pathways for which the corresponding adenylation domain is absent. Here, systematical gene inactivation and complementation have been carried out in a Actinoplanes sp. using homologous recombination and site-specific integration methods. A hybrid gene coding for the N-terminal region of the stand-alone NRPS and the A-PCP domains of a heterologous NRPS restored production of ramoplanins. The results elucidate the unusual N-terminal region which is essential for the biosynthesis of ramoplanins.     

Functional identification of the gene encoding the enzyme involved in mannosylation in ramoplanin biosynthesis in Actinoplanes sp.

Ramoplanin is a lipopeptide antibiotic active against multi-drug-resistant, Gram-positive pathogens. Structurally, it contains a di-mannose moiety attached to the peptide core at Hpg¹¹. The biosynthetic gene cluster of ramoplanin has already been reported and the assembly of the depsipeptide has been elucidated but the mechanism of transferring sugar moiety to the peptide core remains unclear. Sequence analysis of the biosynthetic gene cluster indicated ramo-orf29 was a mannosyltransferase candidate. To investigate the involvement of ramo-orf29 in ramoplanin biosynthesis, gene inactivation and complementation have been conducted in Actinoplanes sp. ATCC 33076 by homologous recombination. Metabolite analysis revealed that the ramo-orf29 inactivated mutant produced no ramoplanin but the ramoplanin aglycone. Thus, ramo-orf29 codes for the mannosyltransferase in the ramoplanin biosynthesis pathway. This lays the foundation for further exploitation of the ramoplanin mannosyltransferase and aglycone in combinatorial biosynthesis.     

Distinct intrinsic and synaptic properties of pre‐sympathetic and pre‐parasympathetic output neurons in Barrington's nucleus

Barrington's nucleus (BN), commonly known as the pontine micturition center, controls micturition and other visceral functions through projections to the spinal cord. In this study, we developed a rat brain slice preparation to determine the intrinsic and synaptic mechanisms regulating pre‐sympathetic output (PSO) and pre‐parasympathetic output (PPO) neurons in the BN using patch‐clamp recordings. The PSO and PPO neurons were retrogradely labeled by injecting fluorescent tracers into the intermediolateral region of the spinal cord at T13‐L1 and S1‐S2 levels, respectively. There were significantly more PPO than PSO neurons within the BN. The basal activity and membrane potential were significantly lower in PPO than in PSO neurons, and A‐type K⁺ currents were significantly larger in PPO than in PSO neurons. Blocking A‐type K⁺ channels increased the excitability more in PPO than in PSO neurons. Stimulting μ‐opioid receptors inhibited firing in both PPO and PSO neurons. The glutamatergic EPSC frequency was much lower, whereas the glycinergic IPSC frequency was much higher, in PPO than in PSO neurons. Although blocking GABA_A receptors increased the excitability of both PSO and PPO neurons, blocking glycine receptors increased the firing activity of PPO neurons only. Furthermore, blocking ionotropic glutamate receptors decreased the excitability of PSO neurons but paradoxically increased the firing activity of PPO neurons by reducing glycinergic input. Our findings indicate that the membrane and synaptic properties of PSO and PPO neurons in the BN are distinctly different. This information improves our understanding of the neural circuitry and central mechanisms regulating the bladder and other visceral organs.     

Exosomes from Retinal Astrocytes Contain Antiangiogenic Components That Inhibit Laser-induced Choroidal Neovascularization

Exosomes released from different types of host cells have different biological effects. We report that exosomes released from retinal astroglial cells (RACs) suppress retinal vessel leakage and inhibit choroidal neovascularization (CNV) in a laser-induced CNV model, whereas exosomes released from retinal pigmental epithelium do not. RAC exosomes inhibit the migration of macrophages and the tubule forming of mouse retinal microvascular endothelial cells. Further, we analyzed antiangiogenic components in RAC exosomes using an angiogenesis array kit and detected several endogenous inhibitors of angiogenesis exclusively present in RAC exosomes, such as endostatin. Moreover, blockade of matrix metalloproteinases in the cleavage of collagen XVIII to form endostatin using FN-439 reverses RAC exosome-mediated retinal vessel leakage. This study demonstrates that exosomes released from retinal tissue cells have different angiogenic effects, with exosomes from RACs containing antiangiogenic components that might protect the eye from angiogenesis and maintain its functional integrity. In addition, by identifying additional components and their functions of RAC exosomes, we might improve the antiangiogenic therapy for CNV in age-related macular degeneration and diabetic retinopathy.     

HIV Populations Are Large and Accumulate High Genetic Diversity in a Nonlinear Fashion

HIV infection is characterized by rapid and error-prone viral replication resulting in genetically diverse virus populations. The rate of accumulation of diversity and the mechanisms involved are under intense study to provide useful information to understand immune evasion and the development of drug resistance. To characterize the development of viral diversity after infection, we carried out an in-depth analysis of single genome sequences of HIV pro-pol to assess diversity and divergence and to estimate replicating population sizes in a group of treatment-naive HIV-infected individuals sampled at single (n = 22) or multiple, longitudinal (n = 11) time points. Analysis of single genome sequences revealed nonlinear accumulation of sequence diversity during the course of infection. Diversity accumulated in recently infected individuals at rates 30-fold higher than in patients with chronic infection. Accumulation of synonymous changes accounted for most of the diversity during chronic infection. Accumulation of diversity resulted in population shifts, but the rates of change were low relative to estimated replication cycle times, consistent with relatively large population sizes. Analysis of changes in allele frequencies revealed effective population sizes that are substantially higher than previous estimates of approximately 1,000 infectious particles/infected individual. Taken together, these observations indicate that HIV populations are large, diverse, and slow to change in chronic infection and that the emergence of new mutations, including drug resistance mutations, is governed by both selection forces and drift.     

Rapid development of molecular markers by next-generation sequencing linked to a gene conferring phomopsis stem blight disease resistance for marker-assisted selection in lupin (Lupinus angustifolius L.) breeding

Selection for phomopsis stem blight disease (PSB) resistance is one of the key objectives in lupin (Lupinus angustifolius L.) breeding programs. A cross was made between cultivar Tanjil (resistant to PSB) and Unicrop (susceptible). The progeny was advanced into F₈ recombinant inbred lines (RILs). The RIL population was phenotyped for PSB disease resistance. Twenty plants from the RIL population representing disease resistance and susceptibility was subjected to next-generation sequencing (NGS)-based restriction site-associated DNA sequencing on the NGS platform Solexa HiSeq2000, which generated 7,241 single nucleotide polymorphisms (SNPs). Thirty-three SNP markers showed the correlation between the marker genotypes and the PSB disease phenotype on the 20 representative plants, which were considered as candidate markers linked to a putative R gene for PSB resistance. Seven candidate markers were converted into sequence-specific PCR markers, which were designated as PhtjM1, PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7. Linkage analysis of the disease phenotyping data and marker genotyping data on a F₈ population containing 187 RILs confirmed that all the seven converted markers were associated with the putative R gene within the genetic distance of 2.1 CentiMorgan (cM). One of the PCR markers, PhtjM3, co-segregated with the R gene. The seven established PCR markers were tested in the 26 historical and current commercial cultivars released in Australia. The numbers of “false positives” (showing the resistance marker allele band but lack of the putative R gene) for each of the seven PCR markers ranged from nil to eight. Markers PhtjM4 and PhtjM7 are recommended in marker-assisted selection for PSB resistance in the Australian national lupin breeding program due to its wide applicability on breeding germplasm and close linkage to the putative R gene. The results demonstrated that application of NGS technology is a rapid and cost-effective approach in development of markers for molecular plant breeding.     

ku70 and ku80 null mutants improve the gene targeting frequency in Monascus ruber M7

Normally, gene targeting by homologous recombination occurs rarely during a transformation process since non-homologous recombination is predominant in filamentous fungi. In our previous researches, the average gene replacement frequency (GRF) in Monascus ruber M7 was as low as 15 %. To develop a highly efficient gene targeting system for M. ruber M7, two M. ruber M7 null mutants of ku70 (MrΔku70) and ku80 (MrΔku80) were constructed which had no apparent defects in the development including vegetative growth, colony phenotype, microscopic morphology and spore yield compared with M. ruber M7. In addition, the production of some significant secondary metabolites such as pigments and citrinin had no differences between the two disruptants and the wild-type strain. Further results revealed that the GRFs of triA (encoding a putative acetyltransferase) were 42.2 % and 61.5 % in the MrΔku70 and MrΔku80 strains, respectively, while it was only about 20 % in M. ruber M7. Furthermore, GRFs of these two disruptants at other loci (the pigE, fmdS genes in MrΔku70 and the ku70 gene in MrΔku80) were investigated, and the results indicated that GRFs in the MrΔku70 strain and the MrΔku80 strain were doubled and tripled compared with that in M. ruber M7, respectively. Therefore, the ku70 and ku80 null mutants of M. ruber M7, especially the ku80-deleted strain, will be excellent hosts for efficient gene targeting.