Mycelium Differentiation and Development of <em>Streptomyces</em> in Liquid Nonsporulating Cultures: Programmed Cell Death, Differentiation, and Lysis Condition Secondary Metabolite Production

Streptomycetes are mycelium-forming sporulating bacteria that produce two thirds of clinically relevant secondary metabolites. Secondary metabolite production is activated at specific developmental stages of Streptomyces life cycle. Despite this, Streptomyces differentiation in liquid nonsporulating cultures (flasks and industrial bioreactors) tends to be underestimated and the most important parameters managed are only indirectly related to differentiation: modifications to the culture media, optimization of productive strains by random or directed mutagenesis, analysis of biophysical parameters, etc. In this chapter, we review the relationship between differentiation and antibiotic production in liquid cultures. Morphological differentiation in liquid cultures is comparable to that occurring during pre-sporulation stages in solid cultures: an initial compartmentalized mycelium suffers a programmed cell death, and remaining viable segments then differentiate to a second multinucleated antibiotic-producing mycelium. Differentiation is one of the keys to interpreting biophysical fermentation parameters and to rationalizing the optimization of secondary metabolite production in liquid cultures

The DNA cytosine methylome revealed two methylation motifs in the upstream regions of genes related to morphological and physiological differentiation in Streptomyces coelicolor A(3)2 M145

DNA methylation is an epigenetic modification detected in both prokaryotic and eukaryotic genomic DNAs. In bacteria, the importance of 5-methylcytosine (m5C) in gene expression has been less investigated than in eukaryotic systems. Through dot-blot analysis employing m5C antibodies against chromosomal DNA, we have previously demonstrated that m5C influences the differentiation of Streptomyces coelicolor A(3)2 M145 in solid sporulating and liquid non-sporulating complex media. Here, we mapped the methylated cytosines of the M145 strain growing in the defined Maltose Glutamate (MG) liquid medium. Sequencing of the M145 genome after bisulfite treatment (BS-sequencing) evidenced 3360 methylated cytosines and the two methylation motifs, GGCmCGG and GCCmCG, in the upstream regions of 321 genes. Besides, the role of cytosine methylation was investigated using the hypo-methylating agent 5'-aza-2'-deoxycytidine (5-aza-dC) in S. coelicolor cultures, demonstrating that m5C affects both growth and antibiotic biosynthesis. Finally, quantitative reverse-transcription polymerase-chain-reaction (RT-qPCR) analysis of genes containing the methylation motifs in the upstream regions showed that 5-aza-dC treatment influenced their transcriptional levels and those of the regulatory genes for two antibiotics. To the best of our knowledge, this is the first study that reports the cytosine methylome of S. coelicolor M145, supporting the crucial role ascribed to cytosine methylation in controlling bacterial gene expression

A fatal case of Nocardia otitidiscaviarum pulmonary infection and brain abscess: taxonomic characterization by molecular techniques

We report on a rare case of pulmonary Nocardiosis and brain abscess caused by Nocardia otitidiscaviarum in an elderly woman with chronic obstructive pulmonary disease. Taxonomic identification involved phenotypic testing, restriction fragment length polymorphism (RFLP), and complete 16S rRNA gene sequencing

Genetic testing to predict weight loss and diabetes remission and long-term sustainability after bariatric surgery : a pilot study

Introduction: The aim of this pilot study was to assess genetic predisposition risk scores (GPS) in type 2 diabetic and non-diabetic patients in order to predict the better response to bariatric surgery (BS) in terms of either weight loss or diabetes remission. Research Design and Methods: A case-control study in which 96 females (47 with type 2 diabetes) underwent Roux-en-Y gastric by-pass were included. The DNA was extracted from saliva samples and SNPs were examined and grouped into 3 GPS. ROC curves were used to calculate sensitivity and specificity. Results: A highly sensitive and specific predictive model of response to BS was obtained by combining the GPS in non-diabetic subjects. This combination was different in diabetic subjects and highly predictive of diabetes remission. Additionally, the model was able to predict the weight regain and type 2 diabetes relapse after 5 years' follow-up. Conclusions: Genetic testing is a simple, reliable and useful tool for implementing personalized medicine in type 2 diabetic patients equiring BS

The SCO1731 methyltransferase modulates actinorhodin production and morphological differentiation of Streptomyces coelicolor A3(2)

Streptomyces coelicolor is a Gram-positive microorganism often used as a model of physiological and morphological differentiation in streptomycetes, prolific producers of secondary metabolites with important biological activities. In the present study, we analysed Streptomyces coelicolor growth and differentiation in the presence of the hypo-methylating agent 5′-aza-2′-deoxycytidine (5-aza-dC) in order to investigate whether cytosine methylation has a role in differentiation. We found that cytosine demethylation caused a delay in spore germination, aerial mycelium development, sporulation, as well as a massive impairment of actinorhodin production. Thus, we searched for putative DNA methyltransferase genes in the genome and constructed a mutant of the SCO1731 gene. The analysis of the SCO1731::Tn5062 mutant strain demonstrated that inactivation of SCO1731 leads to a strong decrease of cytosine methylation and almost to the same phenotype obtained after 5-aza-dC treatment. Altogether, our data demonstrate that cytosine methylation influences morphological differentiation and actinorhodin production in S. coelicolor and expand our knowledge on this model bacterial system

Recombinant Cyclophilins Lack Nuclease Activity

Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins

Streptomyces Development in Colonies and Soils▿

Streptomyces development was analyzed under conditions resembling those in soil. The mycelial growth rate was much lower than that in standard laboratory cultures, and the life span of the previously named first compartmentalized mycelium was remarkably increased

Mycelium development in <it>Streptomyces antibioticus </it>ATCC11891 occurs in an orderly pattern which determines multiphase growth curves

Abstract Background The current model for the developmental cycle of Streptomyces confluent cultures on agar surface is based on the assumption that the only differentiation takes place along the transverse axis (bottom-up): a vegetative (substrate) mycelium grows completely live and viable on the surface and inside the agar until it undergoes a death process and differentiates to a reproductive (aerial) mycelium which grows into the air. Hence, this vertical description assumes that the development in the pre-sporulating phases is more or less homogeneous in all zones of the plate surface. Results The work presents a detailed analysis of the differentiation cycle in Streptomyces antibioticus ATCC11891 considering a different spatial dimension: the longitudinal axes, represented by the plate surface. A previously unsuspected complexity during the substrate mycelial phase was detected. We have demonstrated that the young substrate hyphae suffer an early death round that has not been previously described. Subsequently, the remaining mycelium grows in successive waves which vary according to the density of the spore inoculum. In the presence of dense inocula (1.5 × 106 spores per plate), the hyphae develop in regular circles, approximately 0.5 cm in diameter. By contrast, with highly diluted inocula (6 × 103 spores per plate), aerial mycelium develops initially in the form of islands measuring 0.9 mm in diameter. Further mycelial development occurs between the circles or islands until the plate surface is totally covered. This pattern persists throughout the entire developmental cycle including the sporulation phases. Conclusion An early death round during the substrate mycelial phase of Streptomyces antibioticus ATCC11891 takes place prior to successive growth periods in surface cultures. These developmental periods in turn, determine the shape of the complex multiphase growth curves observed. As shown here, these results also apply to other Streptomyces strains and species. Understanding these peculiarities of the Streptomyces developmental cycle is essential in order to properly interpret the morphological/biochemical data obtained from solid cultures and will expand the number of potential phenotypes subject to study.</p