@article{hinzke_kouris_hughes_strous_kleiner_2019, title={More Is Not Always Better: Evaluation of 1D and 2D-LC-MS/MS Methods for Metaproteomics}, volume={10}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2019.00238}, abstractNote={Metaproteomics, the study of protein expression in microbial communities, is a versatile tool for environmental microbiology. Achieving sufficiently high metaproteome coverage to obtain a comprehensive picture of the activities and interactions in microbial communities is one of the current challenges in metaproteomics. An essential step to maximize the number of identified proteins is peptide separation via liquid chromatography (LC) prior to mass spectrometry (MS). Thorough optimization and comparison of LC methods for metaproteomics are, however, currently lacking. Here, we present an extensive development and test of different 1D and 2D-LC approaches for metaproteomic peptide separations. We used fully characterized mock community samples to evaluate metaproteomic approaches with very long analytical columns (50 and 75 cm) and long gradients (up to 12 hours). We assessed a total of over 20 different 1D and 2D-LC approaches in terms of number of protein groups and unique peptides identified, peptide spectrum matches (PSMs) generated, the ability to detect proteins of low-abundance species, the effect of technical replicate runs on protein identifications and method reproducibility. We show here that, while 1D-LC approaches are faster and easier to set up and lead to more identifications per minute of runtime, 2D-LC approaches allow for a higher overall number of identifications with up to >10,000 protein groups identified. We also compared the 1D- and 2D-LC approaches to a standard GeLC workflow, in which proteins are pre-fractionated via gel electrophoresis. This method yielded results comparable to the 2D-LC approaches, however with the drawback of a much increased sample preparation time. Based on our results, we provide recommendations on how to choose the best LC approach for metaproteomics experiments, depending on the study aims.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Hinzke, Tjorven and Kouris, Angela and Hughes, Rebecca-Ayme and Strous, Marc and Kleiner, Manuel}, year={2019}, month={Feb} }
 @article{hughes_jin_zhang_zhang_tran_williams_lindsey_miller_2018, title={Genome sequence, metabolic properties and cyanobacterial attachment of Porphyrobacter sp. HT-58-2 isolated from a filamentous cyanobacterium–microbial consortium}, volume={164}, ISSN={1350-0872 1465-2080}, url={http://dx.doi.org/10.1099/mic.0.000706}, DOI={10.1099/mic.0.000706}, abstractNote={Tolyporphins are structurally diverse tetrapyrrole macrocycles produced by the cyanobacterial culture HT-58-2. Although tolyporphins were discovered over 25 years ago, little was known about the microbiology of the culture. The studies reported herein expand the description of the community of predominantly alphaproteobacteria associated with the filamentous HT-58-2 cyanobacterium and isolate a dominant bacterium, Porphyrobacter sp. HT-58-2, for which the complete genome is established and growth properties are examined. Fluorescence in situ hybridization (FISH) analysis of the cyanobacterium–microbial community with a probe targeting the 16S rRNA of Porphyrobacter sp. HT-58-2 showed fluorescence emanating from the cyanobacterial sheath. Although genes for the biosynthesis of bacteriochlorophyll a (BChl a) are present in the Porphyrobacter sp. HT-58-2 genome, the pigment was not detected under the conditions examined, implying the absence of phototrophic growth. Comparative analysis of four Porphyrobacter spp. genomes from worldwide collection sites showed significant collinear gene blocks, with two inversions and three deletion regions. Taken together, the results enrich our understanding of the HT-58-2 cyanobacterium–microbial culture.}, number={10}, journal={Microbiology}, publisher={Microbiology Society}, author={Hughes, Rebecca-Ayme and Jin, Xiaohe and Zhang, Yunlong and Zhang, Ran and Tran, Sabrina and Williams, Philip G. and Lindsey, Jonathan S. and Miller, Eric S.}, year={2018}, month={Oct}, pages={1229–1239} }
 @article{hughes_zhang_zhang_williams_lindsey_miller_2017, title={Genome Sequence and Composition of a Tolyporphin-Producing Cyanobacterium-Microbial Community}, volume={83}, ISSN={["1098-5336"]}, DOI={10.1128/aem.01068-17}, abstractNote={ABSTRACT The cyanobacterial culture HT-58-2 was originally described as a strain of Tolypothrix nodosa with the ability to produce tolyporphins, which comprise a family of distinct tetrapyrrole macrocycles with reported efflux pump inhibition properties. Upon reviving the culture from what was thought to be a nonextant collection, studies of culture conditions, strain characterization, phylogeny, and genomics have been undertaken. Here, HT-58-2 was shown by 16S rRNA analysis to closely align with Brasilonema strains and not with Tolypothrix isolates. Light, fluorescence, and scanning electron microscopy revealed cyanobacterium filaments that are decorated with attached bacteria and associated with free bacteria. Metagenomic surveys of HT-58-2 cultures revealed a diversity of bacteria dominated by Erythrobacteraceae , 97% of which are Porphyrobacter species. A dimethyl sulfoxide washing procedure was found to yield enriched cyanobacterial DNA (presumably by removing community bacteria) and sequence data sufficient for genome assembly. The finished, closed HT-58-2Cyano genome consists of 7.85 Mbp (42.6% G+C) and contains 6,581 genes. All genes for biosynthesis of tetrapyrroles (e.g., heme, chlorophyll a , and phycocyanobilin) and almost all for cobalamin were identified dispersed throughout the chromosome. Among the 6,177 protein-encoding genes, coding sequences (CDSs) for all but two of the eight enzymes for conversion of glutamic acid to protoporphyrinogen IX also were found within one major gene cluster. The cluster also includes 10 putative genes (and one hypothetical gene) encoding proteins with domains for a glycosyltransferase, two cytochrome P450 enzymes, and a flavin adenine dinucleotide (FAD)-binding protein. The composition of the gene cluster suggests a possible role in tolyporphin biosynthesis. IMPORTANCE A worldwide search more than 25 years ago for cyanobacterial natural products with anticancer activity identified a culture (HT-58-2) from Micronesia that produces tolyporphins. Tolyporphins are tetrapyrroles, like chlorophylls, but have several profound structural differences that reside outside the bounds of known biosynthetic pathways. To begin probing the biosynthetic origin and biological function of tolyporphins, our research has focused on studying the cyanobacterial strain, about which almost nothing has been previously reported. We find that the HT-58-2 culture is composed of the cyanobacterium and a community of associated bacteria, complicating the question of which organisms make tolyporphins. Elucidation of the cyanobacterial genome revealed an intriguing gene cluster that contains tetrapyrrole biosynthesis genes and a collection of unknown genes, suggesting that the cluster may be responsible for tolyporphin production. Knowledge of the genome and the gene cluster sharply focuses research to identify related cyanobacterial producers of tolyporphins and delineate the tolyporphin biosynthetic pathway.}, number={19}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Hughes, Rebecca-Ayme and Zhang, Yunlong and Zhang, Ran and Williams, Philip G. and Lindsey, Jonathan S. and Miller, Eric S.}, year={2017}, month={Oct} }
 @article{zhang_zhang_hughes_dai_gurr_williams_miller_lindsey_2017, title={Quantitation of Tolyporphins, Diverse Tetrapyrrole Secondary Metabolites with Chlorophyll-Like Absorption, from a Filamentous Cyanobacterium-Microbial Community}, volume={29}, ISSN={0958-0344}, url={http://dx.doi.org/10.1002/pca.2735}, DOI={10.1002/pca.2735}, abstractNote={Abstract Introduction Tolyporphins are unusual tetrapyrrole macrocycles produced by a non‐axenic filamentous cyanobacterium (HT‐58‐2). Tolyporphins A–J, L, and M share a common dioxobacteriochlorin core, differ in peripheral substituents, and exhibit absorption spectra that overlap that of the dominant cyanobacterial pigment, chlorophyll a . Identification and accurate quantitation of the various tolyporphins in these chlorophyll‐rich samples presents challenges. Objective To develop methods for the quantitative determination of tolyporphins produced under various growth conditions relative to that of chlorophyll a . Methodology Chromatographic fractionation of large‐scale (440 L) cultures afforded isolated individual tolyporphins. Lipophilic extraction of small‐scale (25 mL) cultures, HPLC separation with an internal standard, and absorption detection enabled quantitation of tolyporphin A and chlorophyll a , and by inference the amounts of tolyporphins A–M. Absorption spectroscopy with multicomponent analysis of lipophilic extracts (2 mL cultures) afforded the ratio of all tolyporphins to chlorophyll a . The reported absorption spectral data for the various tolyporphins required re‐evaluation for quantitative purposes. Results and Discussion The amount of tolyporphin A after 50 days of illumination ranged from 0.13 nmol/mg dry cells (media containing nitrate) to 1.12 nmol/mg (without nitrate), with maximum 0.23 times that of chlorophyll a . Under soluble‐nitrogen deprivation after 35–50 days, tolyporphin A represents 1/3–1/2 of the total tolyporphins, and the total amount of tolyporphins is up to 1.8‐fold that of chlorophyll a . Conclusions The quantitative methods developed herein should facilitate investigation of the biosynthesis of tolyporphins (and other tetrapyrroles) as well as examination of other strains for production of tolyporphins. Copyright © 2017 John Wiley & Sons, Ltd.}, number={2}, journal={Phytochemical Analysis}, publisher={Wiley}, author={Zhang, Yunlong and Zhang, Ran and Hughes, Rebecca-Ayme and Dai, Jingqiu and Gurr, Joshua R. and Williams, Philip G. and Miller, Eric S. and Lindsey, Jonathan S.}, year={2017}, month={Nov}, pages={205–216} }