Amylases catalyze the cleavage of α-d-1,4 and α-d-1,6-glycosidic bonds in starch and related carbohydrates. Amylases are widely distributed in nature and are important in carbohydrate metabolism. This is the release of four single and two double deletions in Salmonella enterica serovar Typhimurium LT2 that are important for glycan degradation during infection

Arabyan, Narine

Huang, Bihua C

Weimer, Bart C

Genome Announcements

English

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UC DavisUC Davis Previously Published WorksTitleAmylases and Their Importance during Glycan Degradation: Genome Sequence Release of Salmonella Amylase Knockout StrainsPermalinkhttps://escholarship.org/uc/item/3z49p515JournalMicrobiology Resource Announcements, 5(20)ISSN2576-098XAuthorsArabyan, NarineHuang, Bihua CWeimer, Bart CPublication Date2017-05-18DOI10.1128/genomea.00355-17 Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaAmylases and Their Importance duringGlycan Degradation: Genome SequenceRelease of Salmonella Amylase KnockoutStrainsNarine Arabyan,a,b Bihua C. Huang,a,b Bart C. Weimera,bDepartment of Population Health and Reproduction, School of Veterinary Medicine, University of California,Davis, Davis, California, USAa; 100K Pathogen Genome Project, UC Davis, Davis, California, USAbABSTRACT Amylases catalyze the cleavage of -D-1,4 and -D-1,6-glycosidic bondsin starch and related carbohydrates. Amylases are widely distributed in nature andare important in carbohydrate metabolism. This is the release of four single and twodouble deletions in Salmonella enterica serovar Typhimurium LT2 that are importantfor glycan degradation during infection.Amylases in Salmonella spp. are poorly characterized glycosyl hydrolases. Theimportance of amylases during Salmonella infection is understudied, especially asnew virulence factors. The genome of Salmonella enterica serovar Typhimurium LT2contains four amylases that are classified under the glycoside hydrolase 13 (GH13)family: malS (periplasmic -amylase), amyA (cytoplasmic -amylase), glgX (glycogen-debranching enzyme), and glgB (1,4--glucan-branching enzyme). Although glgX andglgB are not annotated as amylases, both have a conserved -amylase domain andfunction as amylases. Investigating the genomic diversity of microbial amylases will aidin understanding their importance during pathogenesis. Inhibitors of amylases can benew drug targets.Amylases play an important role by altering the host glycan profiles during infectionto gain access of the host epithelial cells by binding to terminal mannose molecules toinitiate glycan degradation, as was shown by Arabyan et al. (1). That group alsodemonstrated that each of the four mutant amylase strains (ΔmalS, ΔamyA, ΔglgX, andΔglgB mutants) had different invasion phenotypes during the in vitro infection ofdifferentiated colonic epithelial cells (Caco-2) (1); however, only the ΔmalS mutantsignificantly (P  0.05) reduced adhesion and invasion during infection that werecomparable to those seen with a nonpathogenic Salmonella strain (1).The 100K Pathogen Genome Project (http://www.100kgenomes.org) is a large-scalesequencing consortium that offers the use of new next-generation sequencing meth-ods to provide cutting-edge methods for pathogen detection and control in the foodsupply. This project is focused on producing genomes of pathogenic isolates from theenvironment, plants, animals, and humans worldwide, providing new insights into thegenetic diversity of Salmonella and other foodborne pathogens. These amylase mutantstrains (four single-deletion and two double-deletion mutants) were constructed in theWeimer laboratory (UC Davis, Davis, CA) (1), as described by Datsenko and Wanner (2).Cultures were grown on 1.5% Luria-Bertani (LB) agar (Difco, Franklin Lakes, NJ) with10 g/ml chloramphenicol at 37°C and lysed (3); genomic DNA (gDNA) was extracted(4), checked for quality (5), and fragmented (6). Libraries were 350 to 500 bp (7, 8) andwere indexed (96 genomes/lane) and sequenced (Illumina HiSeq 3000; PE150) (9–11) atUC Davis DNA Technologies Core (Davis, CA). Paired-end reads were de novo assembledusing CLC Workbench version 6 (Qiagen), with default parameters. Here, the 100KReceived 23 March 2017 Accepted 27 March2017 Published 18 May 2017Citation Arabyan N, Huang BC, Weimer BC.2017. Amylases and their importance duringglycan degradation: genome sequence releaseof Salmonella amylase knockout strains.Genome Announc 5:e00355-17. https://doi.org/10.1128/genomeA.00355-17.Copyright © 2017 Arabyan et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to Bart C. Weimer,bcweimer@ucdavis.edu.PROKARYOTEScrossmVolume 5 Issue 20 e00355-17 genomea.asm.org 1Pathogen Genome Project has assembled four genomes of single- and two double-amylase-deletion strains of Salmonella enterica serovar Typhimurium LT2.Accession number(s). All sequences are publicly available and can be found at the100K Project BioProject (NCBI PRJNA186441) in the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) and genome assemblies can be found in NCBI GenBank(Table 1).ACKNOWLEDGMENTSWe gratefully acknowledge funding for this project from Mars, Inc., NIH (grants1R01HD065122-01A1 and NIH-U24-DK097154) and an Agilent Technologies ThoughtLeader Award to B.C.W.REFERENCES1. Arabyan N, Park D, Foutouhi S, Weis AM, Huang BC, Williams CC, DesaiP, Shah J, Jeannotte R, Kong N, Lebrilla CB, Weimer BC. 2016. Salmonelladegrades the host glycocalyx leading to altered infection and glycanremodeling. Sci Rep 6:29525. https://doi.org/10.1038/srep29525.2. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomalgenes in Escherichia coli K-12 using PCR products. Proc Natl Acad SciU S A 97:6640 – 6645. https://doi.org/10.1073/pnas.120163297.3. Jeannotte R, Lee E, Kong N, Ng W, Kelly L, Weimer BC. 2014. High-throughput analysis of foodborne bacterial genomic DNA using Agilent2200 TapeStation and genomic DNA ScreenTape system. Applicationnote 5991– 4003EN. Agilent Technologies, Santa Clara, CA. https://www.agilent.com/cs/library/applications/5991-4003EN.pdf .4. Kong N, Ng W, Lee V, Kelly L, Weimer BC. 2013. Production and analysisof high molecular weight genomic DNA for NGS pipelines using AgilentDNA extraction kit (p/n 200600). Application note 5991–3722EN. AgilentTechnologies, Santa Clara, CA https://www.agilent.com/cs/library/applications/5991-3722EN.pdf.5. Kong N, Ng W, Cai L, Leonardo A, Kelly L, Weimer BC. 2014. Integratingthe DNA integrity number (DIN) to assess genomic DNA (gDNA) qualitycontrol using the Agilent 2200 TapeStation system. Application note5991–5442EN. Agilent Technologies, Santa Clara, CA http://www.agilent.com/cs/library/applications/5991-5442EN.pdf.6. Jeannotte R, Lee E, Arabyan N, Kong N, Thao K, Huang BH, Weimer BC.2014. Optimization of Covaris settings for shearing bacterial genomicDNA by focused ultrasonication and analysis using Agilent 2200TapeStation. Application note 5991–5075EN. Agilent Technologies,Santa Clara, CA. http://cn.agilent.com/cs/library/applications/5991-5075EN.pdf .7. Kong N, Ng W, Foutouhi A, Huang BH, Kelly L, Weimer BC. 2014. Qualitycontrol of high-throughput library construction pipeline for KAPA HTPlibrary using an agilent 2200 TapeStation. Application note5991–5141EN. Agilent Technologies, Santa Clara, CA http://www.agilent.com/cs/library/applications/5991-5141EN.pdf.8. Kong N, Thao K, Huang C, Appel M, Lappin S, Knapp L, Kelly L, WeimerBC. 2014. Automated library construction using KAPA library preparationkits on the Agilent NGS workstation yields high-quality libraries forwhole-genome sequencing on the Illumina platform. Application note5991– 4296EN. Agilent Technologies, Santa Clara, CA. http://www.agilent.com/cs/library/applications/5991-4296EN.pdf.9. Lüdeke CH, Kong N, Weimer BC, Fischer M, Jones JL. 2015. Completegenome sequences of a clinical isolate and an environmental isolate ofVibrio parahaemolyticus. Genome Announc 3(2):e00216-15. https://doi.org/10.1128/genomeA.00216-15.10. Weis AM, Clothier KA, Huang BC, Kong N, Weimer BC. 2016. Draftgenome sequences of Campylobacter jejuni strains that cause abortion inlivestock. Genome Announc 4(6):e01324-16. https://doi.org/10.1128/genomeA.01324-16.11. Weis AM, Huang BC, Storey DB, Kong N, Chen P, Arabyan N, Gilpin B,Mason C, Townsend AK, Smith WA, Byrne BA, Taff CC, Weimer BC. 2017.Large-scale release of Campylobacter draft genomes: resources for foodsafety and public health from the 100K pathogen genome project.Genome Announc 5(1):e00925-16. https://doi.org/10.1128/genomeA.00925-16.TABLE 1 Salmonella enterica serovar Typhimurium LT2 amylase deletion mutantsGenBank accessionno.SRA accessionno. Isolate name MutationNo. ofcontigs Coverage ()Total genomesize (bp)No. ofCDSsaMXAY00000000 SRR3622951 BCW_7501 ΔmalS 65 105 4,892,783 4,797MZNJ00000000 SRR5288769 BCW_7502 ΔglgB 64 287 4,896,277 4,809MXAZ00000000 SRR3622952 BCW_7503 ΔglgX 50 130 4,893,261 4,799MZNK00000000 SRR5288768 BCW_7513 ΔinvA::ΔmalS 61 150 4,893,696 4,809MZNL00000000 SRR5288767 BCW_7521 ΔflgJ::ΔmalS 57 139 4,896,778 4,803MZNM00000000 SRR5288770 BCW_8419 ΔamyA 58 150 4,892,055 4,802aCDSs, coding sequences.Arabyan et al.Volume 5 Issue 20 e00355-17 genomea.asm.org 2

Amylases and Their Importance during Glycan Degradation: Genome Sequence Release of Salmonella Amylase Knockout Strains

ABSTRACT
          
            Amylases catalyze the cleavage of α-
            d
            -1,4 and α-
            d
            -1,6-glycosidic bonds in starch and related carbohydrates. Amylases are widely distributed in nature and are important in carbohydrate metabolism. This is the release of four single and two double deletions in
            Salmonella enterica
            serovar Typhimurium LT2 that are important for glycan degradation during infection.
          </jats:p

Narine Arabyan

Bihua C. Huang

Bart C. Weimer

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Amylases and Their Importance during Glycan Degradation: Genome Sequence Release of
            <i>Salmonella</i>
            Amylase Knockout Strains

https://escholarship.org/content/qt3z49p515/qt3z49p515.pdf?t=qakkzn

Amylases and Their Importance during Glycan Degradation: Genome Sequence Release of Salmonella Amylase Knockout Strains.

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