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      Microbial isolation and characterization from two flex lines from the urine processor assembly onboard the international space station

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          Abstract

          Urine, humidity condensate, and other sources of non-potable water are processed onboard the International Space Station (ISS) by the Water Recovery System (WRS) yielding potable water. While some means of microbial control are in place, including a phosphoric acid/hexavalent chromium urine pretreatment solution, many areas within the WRS are not available for routine microbial monitoring. Due to refurbishment needs, two flex lines from the Urine Processor Assembly (UPA) within the WRS were removed and returned to Earth. The water from within these lines, as well as flush water, was microbially evaluated. Culture and culture-independent analysis revealed the presence of Burkholderia, Paraburkholderia, and Leifsonia. Fungal culture also identified Fusarium and Lecythophora. Hybrid de novo genome analysis of the five distinct Burkholderia isolates identified them as B. contaminans, while the two Paraburkholderia isolates were identified as P. fungorum. Chromate-resistance gene clusters were identified through pangenomic analysis that differentiated these genomes from previously studied isolates recovered from the point-of-use potable water dispenser and/or current NCBI references, indicating that unique populations exist within distinct niches in the WRS. Beyond genomic analysis, fixed samples directly from the lines were imaged by environmental scanning electron microscopy, which detailed networks of fungal-bacterial biofilms. This is the first evidence of biofilm formation within flex lines from the UPA onboard the ISS. For all bacteria isolated, biofilm potential was further characterized, with the B. contaminans isolates demonstrating the most considerable biofilm formation. Moreover, the genomes of the B. contaminans revealed secondary metabolite gene clusters associated with quorum sensing, biofilm formation, antifungal compounds, and hemolysins. The potential production of these gene cluster metabolites was phenotypically evaluated through biofilm, bacterial-fungal interaction, and hemolytic assays. Collectively, these data identify the UPA flex lines as a unique ecological niche and novel area of biofilm growth within the WRS. Further investigation of these organisms and their resistance profiles will enable engineering controls directed toward biofilm prevention in future space station water systems.

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          The Sequence Alignment/Map format and SAMtools

          Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: http://samtools.sourceforge.net Contact: rd@sanger.ac.uk
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            CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes

            Large-scale recovery of genomes from isolates, single cells, and metagenomic data has been made possible by advances in computational methods and substantial reductions in sequencing costs. Although this increasing breadth of draft genomes is providing key information regarding the evolutionary and functional diversity of microbial life, it has become impractical to finish all available reference genomes. Making robust biological inferences from draft genomes requires accurate estimates of their completeness and contamination. Current methods for assessing genome quality are ad hoc and generally make use of a limited number of “marker” genes conserved across all bacterial or archaeal genomes. Here we introduce CheckM, an automated method for assessing the quality of a genome using a broader set of marker genes specific to the position of a genome within a reference genome tree and information about the collocation of these genes. We demonstrate the effectiveness of CheckM using synthetic data and a wide range of isolate-, single-cell-, and metagenome-derived genomes. CheckM is shown to provide accurate estimates of genome completeness and contamination and to outperform existing approaches. Using CheckM, we identify a diverse range of errors currently impacting publicly available isolate genomes and demonstrate that genomes obtained from single cells and metagenomic data vary substantially in quality. In order to facilitate the use of draft genomes, we propose an objective measure of genome quality that can be used to select genomes suitable for specific gene- and genome-centric analyses of microbial communities.
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              Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads

              The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce complete genome assemblies, but the sequencing is more expensive and error-prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate “hybrid” assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler uses a novel semi-global aligner to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long-read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.
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                Author and article information

                Contributors
                Journal
                Biofilm
                Biofilm
                Biofilm
                Elsevier
                2590-2075
                02 March 2023
                December 2023
                02 March 2023
                : 5
                : 100108
                Affiliations
                [a ]JES Tech, Houston, TX, USA
                [b ]KBR, Houston, TX, USA
                [c ]Space Systems Department, NASA Marshall Space Flight Center, Huntsville, AL, USA
                [d ]Exploration Research and Technology, NASA Kennedy Space Center, Merritt Island, FL, USA
                [e ]KBR, Moffett Field, CA, USA
                [f ]Jacobs, Huntsville, AL, USA
                [g ]Biomedical Research and Environmental Sciences Division, NASA Johnson Space Center, Houston, TX, USA
                [h ]Department of Bioengineering, Department of Physics, Northeastern University, Boston, MA, USA
                Author notes
                []Corresponding author. sarah.wallace@ 123456nasa.gov
                Article
                S2590-2075(23)00005-9 100108
                10.1016/j.bioflm.2023.100108
                10020673
                36938359
                61465510-b83c-4cf2-bba6-bbab5f6d11de
                © 2023 Published by Elsevier B.V.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 28 October 2022
                : 13 January 2023
                : 16 February 2023
                Categories
                Article

                spaceflight,biofilms,environmental microbiology,international space station,pangenome

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