Dataset Information

High-Resolution Analysis of the Peptidoglycan Composition in Streptomyces coelicolor.

ABSTRACT: The bacterial cell wall maintains cell shape and protects against bursting by turgor. A major constituent of the cell wall is peptidoglycan (PG), which is continuously modified to enable cell growth and differentiation through the concerted activity of biosynthetic and hydrolytic enzymes. Streptomycetes are Gram-positive bacteria with a complex multicellular life style alternating between mycelial growth and the formation of reproductive spores. This involves cell wall remodeling at apical sites of the hyphae during cell elongation and autolytic degradation of the vegetative mycelium during the onset of development and antibiotic production. Here, we show that there are distinct differences in the cross-linking and maturation of the PGs between exponentially growing vegetative hyphae and the aerial hyphae that undergo sporulation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified over 80 different muropeptides, revealing that major PG hydrolysis takes place over the course of mycelial growth. Half of the dimers lacked one of the disaccharide units in transition-phase cells, most likely due to autolytic activity. The deacetylation of MurNAc to MurN was particularly pronounced in spores and strongly reduced in sporulation mutants with a deletion of bldD or whiG, suggesting that MurN is developmentally regulated. Altogether, our work highlights the dynamic and growth phase-dependent changes in the composition of the PG in StreptomycesIMPORTANCE Streptomycetes are bacteria with a complex lifestyle and are model organisms for bacterial multicellularity. From a single spore, a large multigenomic multicellular mycelium is formed, which differentiates to form spores. Programmed cell death is an important event during the onset of morphological differentiation. In this work, we provide new insights into the changes in the peptidoglycan composition and over time, highlighting changes over the course of development and between growing mycelia and spores. This revealed dynamic changes in the peptidoglycan when the mycelia aged, with extensive peptidoglycan hydrolysis and, in particular, an increase in the proportion of 3-3 cross-links. Additionally, we identified a muropeptide that accumulates predominantly in the spores and may provide clues toward spore development.

SUBMITTER: van der Aart LT

PROVIDER: S-EPMC6153666 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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High-Resolution Analysis of the Peptidoglycan Composition in Streptomyces coelicolor.

van der Aart Lizah T LT Spijksma Gerwin K GK Harms Amy A Vollmer Waldemar W Hankemeier Thomas T van Wezel Gilles P GP

Journal of bacteriology 20180924 20

The bacterial cell wall maintains cell shape and protects against bursting by turgor. A major constituent of the cell wall is peptidoglycan (PG), which is continuously modified to enable cell growth and differentiation through the concerted activity of biosynthetic and hydrolytic enzymes. Streptomycetes are Gram-positive bacteria with a complex multicellular life style alternating between mycelial growth and the formation of reproductive spores. This involves cell wall remodeling at apical sites ...[more]

PMID: 30061355

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Project description:Whilst being closely related to the model actinomycete Streptomyces coelicolor A3(2), S. lividans 66 differs from it in several significant and phenotypically observable ways, including antibiotic production. Previous comparative gene hybridization studies investigating such differences have used low-density (one probe per gene) PCR-based spotted arrays. Here we use new experimentally optimised 104,000 × 60-mer probe arrays to characterize in detail the genomic differences between wild-type S. lividans 66, a derivative industrial strain, TK24, and S. coelicolor M145.The high coverage and specificity (detection of three nucleotide differences) of the new microarrays used has highlighted the macroscopic genomic differences between two S. lividans strains and S. coelicolor. In a series of case studies we have validated the microarray and have identified subtle changes in genomic structure which occur in the Asp-activating adenylation domains of CDA non-ribosomal peptide synthetase genes which provides evidence of gene shuffling between these domains. We also identify single nucleotide sequence inter-species differences which exist in the actinorhodin biosynthetic gene cluster. As the glyoxylate bypass is non-functional in both S. lividans strains due to the absence of the gene encoding isocitrate lyase it is likely that the ethylmalonyl-CoA pathway functions as the alternative mechanism for the assimilation of C2 compounds.This study provides evidence for widespread genetic recombination, rather than it being focussed at 'hotspots', suggesting that the previously proposed 'archipelago model' of genomic differences between S. coelicolor and S. lividans is unduly simplistic. The two S. lividans strains investigated differ considerably in genetic complement, with TK24 lacking 175 more genes than its wild-type parent when compared to S. coelicolor. Additionally, we confirm the presence of bldB in S. lividans and deduce that S. lividans 66 and TK24, both deficient in the glyoxylate bypass, possess an alternative metabolic mechanism for the assimilation of C2 compounds. Given that streptomycetes generally display high genetic instability it is envisaged that these high-density arrays will find application for rapid assessment of genome content (particularly amplifications/deletions) in mutational studies of S. coelicolor and related species.

Project description:Nucleoid-associated proteins (NAPs) are small, highly abundant transcriptional regulators with low sequence specificity which are involved in multiple DNA-related processes including gene expression, DNA protection, recombination/repair and nucleoid structuring. Through these functions they are able to regulate important phenotypic properties including virulence, secondary metabolism and stress resistance. However the set of NAPs known within the Actinobacteria is small and incomplete. The missing proteins are likely to be key regulators of virulence in pathogens such as Mycobacterium tuberculosis and also of development and secondary metabolism in industrially-important species such as Streptomyces. Here, we use label-free LC-MS/MS to systematically search for novel NAPs in isolated nucleoids of the model actinomycete Streptomyces coelicolor. Based on the criteria of high abundance (emPAI score) and predicted DNA-binding ability (DNAbinder score) we identified a set of 24 proteins with a high predicted likelihood of being NAPs. The approach was deemed successful as the set included known major NAPs HupA, HupS, sIHF and Lsr2 as well as the global transcriptional regulators BldD and CRP and the pleiotropic response regulator AfsQ1. It also included a number of proteins whose functions are not yet known from recognisable classes of transcription factor (SCO2140, SCO4493, SCO1839, SCO1210, SCO5405, SCO4229, SCO3198) or from uncharacterised protein families (SCO5783, SCO5592, SCO3793, SCO6482) which comprise a valuable set of candidates for further study.In this paper we establish a robust protocol for preparing S. coelicolor nucleoids for mass spectrometric analysis and develop a workflow for identifying novel nucleoid-associated proteins (NAPs) by combining LC-MS/MS with a bioinformatical analysis. The nucleoid-associated proteins of many species are known to be key regulators of virulence, stress tolerance and global patterns of gene expression. Identifying the "missing" nucleoid proteins of S. coelicolor is likely to have important implications for manipulating the production of secondary metabolites such as antibiotics. Candidate NAPs were identified. Several of these are highly conserved in clinically important species such as Mycobacterium and in many commercially important species such as Salinispora and Micromonospora which represent a vital source of novel drugs such as antibiotics, antifungals and anticancer agents.

Dataset Information

High-Resolution Analysis of the Peptidoglycan Composition in Streptomyces coelicolor.

Publications

High-Resolution Analysis of the Peptidoglycan Composition in Streptomyces coelicolor.

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