Analysis of two distinct mycelial populations in liquid-grown Streptomyces cultures using a flow cytometry-based proteomics approach

Veluw, G. Jerre van; Petrus, Marloes L. C.; Gubbens, Jacob; Graaf, Richard de; Jong, Inez P. de; Wezel, Gilles P. van; Wösten, Han A. B.; Claessen, Dennis

doi:10.1007/s00253-012-4490-5

Cited by 39 publications

(38 citation statements)

References 51 publications

(59 reference statements)

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“…CslA belongs to the glycosyltransferase family 2, which includes cellulose and chitin synthases, among others (32). Deletion of cslA abolishes pellet formation in liquid-grown cultures and also blocks the formation of reproductive aerial hyphae on solid medium (28,29). GlxA is a newly characterized member of the AA5 family of mono-copper oxidase possessing a Tyr-Cys cross-linked redox cofactor (30).…”

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confidence: 99%

Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases

Chaplin

Wilson

Hough

et al. 2016

Journal of Biological Chemistry

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Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.Lytic polysaccharide monooxygenases (LPMOs) 3 are enzymes that utilize copper as their functional active site metal and act to enhance the depolymerization of recalcitrant polysaccharides in nature such as cellulose and chitin (1, 2). Identification and characterization of members of this cuproenzyme family from fungal and bacterial species have gathered pace in recent years due in part to their promising uses in biorefinery applications of biomass to derive second generation biofuels (2, 3). LPMOs are classed into auxiliary activity (AA) families in the CAZy (carbohydrate active enzyme) database with four AA families, AA9, AA10, AA11, and AA13 identified so far (4). AA9 (formerly GH61) and AA10 (formerly CBM33) families have been the most extensively studied, with AA9 members having activity for cellulose, hemicellulose, and cellodextrin substrates (5-8) and AA10 members having activity with either cellulose or chitin substrates (9, 10). The AA11 and AA13 families are the most recently discovered, and so far have been determined to have activity with chitin and starch substrates, respectively (11, 12). All LPMOs coordinate a single copper ion by three nitrogen ligands to from the active site. Two of these are provided from the amino group (Nt) and side chain of the N-terminal His with the third from a second His side chain to giv...

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confidence: 99%

Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases

Chaplin

Wilson

Hough

et al. 2016

Journal of Biological Chemistry

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“…These include the DosS-like (SCO0203), DosR-like (SCO0204), and DosT-like (SCO0211) genes which represent orthologs of M. tuberculosis, where DosS and DosT act as sensors of redox stress (induced by respiration-impairing gases such as NO) and hypoxia for the DosR transcriptional factor (37). Interestingly, proteome comparisons in S. coelicolor populations of small and large pellets revealed that proteins encoded by genes flanking the dosR-like gene (SCO0204) were overrepresented in large pellets, where a higher level of oxygen stress is assumed to occur (38). In Mycobacterium spp., DosR controls the expression of genes necessary for dormancy survival (32,37).…”

Section: Discussionmentioning

confidence: 99%

AterDDomain-Encoding Gene (SCO2368) Is Involved in Calcium Homeostasis and Participates in Calcium Regulation of a DosR-Like Regulon in Streptomyces coelicolor

et al. 2015

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bAlthough Streptomyces coelicolor is not resistant to tellurite, it possesses several TerD domain-encoding (tdd) genes of unknown function. To elucidate the function of tdd8, the transcriptomes of S. coelicolor strain M145 and of a tdd8 deletion mutant derivative (the ⌬tdd8 strain) were compared. Several orthologs of Mycobacterium tuberculosis genes involved in dormancy survival were upregulated in the deletion mutant at the visual onset of prodiginine production. These genes are organized in a putative redox stress response cluster comprising two large loci. A binding motif similar to the dormancy survival regulator (DosR) binding site of M. tuberculosis has been identified in the upstream sequences of most genes in these loci. A predicted role for these genes in the redox stress response is supported by the low NAD ؉ /NADH ratio in the ⌬tdd8 strain. This S. coelicolor gene cluster was shown to be induced by hypoxia and NO stress. While the tdd8 deletion mutant (the ⌬tdd8 strain) was unable to maintain calcium homeostasis in a calcium-depleted medium, the addition of Ca 2؉ in ⌬tdd8 culture medium reduced the expression of several genes of the redox stress response cluster. The results shown in this work are consistent with Tdd8 playing a significant role in calcium homeostasis and redox stress adaptation.

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“…Actinobacteria, especially streptomycetes, form mycelial pellets or clumps when grown in shaken liquid medium; these clumps consist of interconnected hyphae of different sizes in streptomycetes (van Veluw et al, 2012). Clumps of mycobacteria are formed by aggregates of individual cells (Scherr & Nguyen, 2009).…”

Section: Discussionmentioning

confidence: 99%

Morphological and proteomic analysis of early stage air–liquid interface biofilm formation in Mycobacterium smegmatis

et al. 2014

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We studied the early stages of pellicle formation by Mycobacterium smegmatis on the surface of a liquid medium [air-liquid interface (A-L)]. Using optical and scanning electron microscopy, we showed the formation of a compact biofilm pellicle from micro-colonies over a period of 8-30 h. The cells in the pellicle changed size and cell division pattern during this period. Based on our findings, we created a model of M. smegmatis A-L early pellicle formation showing the coordinate growth of cells in the micro-colonies and in the homogeneous film between them, where the accessibility to oxygen and nutrients is different. A proteomic approach utilizing high-resolution two-dimensional gel electrophoresis, in combination with mass spectrometry-based protein identification, was used to analyse the protein expression profiles of the different morphological stages of the pellicle. The proteins identified formed four expression groups; the most interesting of these groups contained the proteins with highest expression in the biofilm development phase, when the floating micro-colonies containing long and more robust cells associate into flocs and start to form a compact pellicle. The majority of these proteins, including GroEL1, are involved in cell wall synthesis or modification, mostly through the involvement of mycolic acid biosynthesis, and their expression maxima correlated with the changes in cell size and the rigidity of the bacterial cell wall observed by scanning electron microscopy.

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Analysis of two distinct mycelial populations in liquid-grown Streptomyces cultures using a flow cytometry-based proteomics approach

Cited by 39 publications

References 51 publications

Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases

Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases

AterDDomain-Encoding Gene (SCO2368) Is Involved in Calcium Homeostasis and Participates in Calcium Regulation of a DosR-Like Regulon in Streptomyces coelicolor

Morphological and proteomic analysis of early stage air–liquid interface biofilm formation in Mycobacterium smegmatis

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