Transactions of the Illinois State Academy of Science received 11/7/16 (2017) Volume 110, pp. 9-15 accepted 8/28/17 Comparative Analysis of Alternansucrase Genes from Leuconostoc Strains Scott Michael Holt Western Illinois University, Department of Biological Sciences Macomb, IL 61455, [email protected] ABSTRACT Alternansucrase from the bacterial genus Leuconostoc catalyzes the synthesis of alternan, which has many commercial applications. A comparative analysis was performed to build a consensus of molecular features for gene sequences previously annotated as “alternansu- crase.” Alternansucrase genes from L. citreum isolates formed a distinct clade among glucansucrases. The clade had identical predicted gene and protein size. Amino acid sequence features for the signal peptide, key regions within the catalytic domain, and C-terminal repeat structures were also nearly identical for the clade. Alternansucrase gene from L. fallax, however, is more distantly related and possesses features that are clearly distinct from the clade group. Consequently, the gene from L. fallax appears atypical and should be designated so when annotated as an “alternansucrase.” With tremendous influx of new sequence information due to next generation sequencing technology accurate annotation of gene function becomes extremely important particularly for genome mining applications. This study provided a consensus of molecular features for “alternansucrase” that should be considered during annotation of sequence information. Key words: Alternansucrase, Dextransucrase, Leuconostoc, Alternan, α-glucan INTRODUCTION Jeans et al. 1954, Jeans 1979, Seymore et al. features with more recently available asr Glucansucrases made by certain Leucono- 1979). Alternansucrase may serve as a good sequences (putative) reported in online stoc bacterial species catalyze the synthe- model for structure/function studies since databases. Phylogenetic relationships and sis of α-glucans from inexpensive, widely asr genes are probably highly conserved regions of consensus and divergence were available, and renewable agricultural resi- like all GH70 glucansucrases. In addition, identified among these commercially im- dues such as sucrose. Leuconostoc and their the biochemical characteristics of the few portant genes from Leuconostoc isolates corresponding α-glucans have a variety alternan polymers reported in the litera- obtained from across the globe. Informa- of realized (Alsop 1983, Cargill Inc. 2009, ture differ only in their linkage proportion tion generated from this study will provide Dolan et al. 2012, Gronwell and Ingelman (Bounaix et al. 2009, Jeans et al. 1954, Jeans more accurate annotation or prediction of 1948, Monsan and Paul 1995, Paul et al. 1979, Seymore et al. 1979). This difference what constitutes an asr gene. In addition, 1992) and potential commercial applica- of a single characteristic (link proportion) this study identified new potential targets tions (Côté and Holt 2007, Day and Chung among asr gene products may help simpli- for site-directed mutagenesis needed to 2007, Irague et al. 2012, Lappan and Fogler fy future structure/function relationship elucidate structure/function relationships 1995, Vandamme et al. 2003) which include studies of alternansucrases. In order to re- of alternansucrase and possibly other glu- many food, nonfood, and industrial uses. late enzyme structural components (1º and cansucrases. Understanding the diversity and structure/ 2º) with functional characteristics (speci- METHODS ficities), more asr genes must be properly function relationships of glucansucrases Bacterial Strains and Culture Conditions will enhance their biotechnology potential identified and characterized. Until recently, by aiding the development of unique or only one alternansucrase gene sequence Leuconostoc strains B-1498 and B-1501 tailor-made α-glucans for specific applica- (asr1355) has been reported from Leuco- were obtained from the United States De- tions. nostoc strain B-1355 (Arguello-Morales et partment of Agriculture, NRRL culture al. 2000). Since asr1355 has been report- Leuconostoc glucansucrases belong to Gly- collection, Peoria, IL. Leuconostoc strains ed (Arguello-Morales et al. 2000), other B-1498 and B-1501 stock cultures were coside Hydrolase Family 70 (CAZY 2015, sequences described as “alternansucrase” Coutinho and Henrissat 1999) and include stored at -80ºC. A portion of each stock from different Leuconostoc isolates have be- culture was sub-cultured by isolation streak alternansucrase (EC 2.4.1.140) and dex- come available through Sanger or genomic transucrase (EC 2.4.1.5) among others. on MRS agar (Oxoid LTD, Hampshire, En- sequencing (Amari et al. 2014, Bounaix et gland) plates (De Man et al. 1960) and in- Alternansucrase catalyzes the synthesis al. 2009, Kim et al. 2008, Nam et al. 2011). of the α-glucan called alternan [alternat- cubated at 30ºC for 48 h. An isolated colony Thus far, alternan formation, alternansu- from each strain was used to inoculate 25- ing α-(1→6) and α-(1→3) links] which has crase enzyme activity, or an alternansu- novel chemical properties (Côté and Robyt ml MRS broth which was incubated at 30ºC crase gene sequence has only be reported overnight with shaking at 200 rpm. 1982). Alternan polymers from different from Leuconostoc (Alsop 1983, Côté and Leuconostoc isolates are structurally very Robyt 1982, Jeans et al. 1954, Kim et al. Source of Alternansucrase (asr) Genes similar with the exception that the alter- 2008, Nam et al. 2011). nating α-(1→6) and α-(1→3) links are in Alternansucrase (asr) genes are rare. At the different proportions (Bounaix et al. 2009, The goal of this study was to perform a time of this study, only six asr genes (in- comparative analysis of asr1355 sequence Comparative Analysis of Alternansucrase Genes from Leuconostoc Strains 10 Scott Michael Holt asr1501 were assembled using Codon- Code and DNAbaser. Comparative anal- Table 1. Putative alternansucrase genes used in this study, source information, and char- yses of asr1355 with putative asr1498 and acteristics. asr1501 genes (this study) and three other Source Strain Accession Number Gene Size AA Mol. Wt glucan linkage % sequences described as “alternansucrase” Organism Origin Genbank (bp) (kDa) α(1→3) ; α(1→6) (asrKM20, L. citreum KM20; asrC11, L. cit- 1 2 1,3,4 L. citreum FL, USA AJ250173 6174 2057 228,971 43 ; 57 reum LBAE C11; asr3537, L. fallax KCTC NRRL B-1355 L. citreum LA, USA5 KF360257 6174 2057 228,798 38 ; 62 1,3,4 3537) available from online databases were NRRL B-1498 Holt, this study analyzed by BLAST, ORF Finder, PrediSi, L. citreum LA, USA5 KF360258 6174 2057 229,113 33 ; 67 1,3,4 ClustalW2, CD-Domain, and C-DART. At NRRL B-1501 Holt, this study the time of this study, only six alternansu- L. citreum Korea6 NC_0104717 6174 2057 228,864 Not determined KM20 crase gene sequences have been reported L. citreum France⁸ NZ_CAGF010000089 6174 2057 229,094 40 ; 60 9 in the literature or in online databases. For LBAE C11 phylogenetic analyses shown in figures 2, 3, L. fallaxT Germany10 NZ_AEIZ0100000211 6525 1874 208,192 Not determined S1 and S2, full-length amino acid sequenc- KCTC 3537 es of putative alternansucrase genes and 1Jeans et al. 1954, 2Arguello-Morales et al. 2000, 3Jeans 1979, 4Seymore et al. 1979, 5McCleskey et al. 1947, 6Choi et al. 2003, 7Kim et al. 2008, 8Gabriel et al. 1999, 9Bounaix et al. 2009, 10Martinez-Murcia and Collins 1991, 11Nam other select glucoside hydrolase family 70 et al. 2011. enzymes (available from online databases) were first aligned by MUSCLE and files were converted to MEGA (version6) file cluding asr1355) were reported in the lit- regions of previously characterized asr1355 format. MEGA6 software was then used erature or online databases and are listed in cloned from Leuconostoc strain B-1355 to prepare unrooted phylogenetic trees Table 1. All asr genes were considered puta- (Arguello-Morales et al. 2000) (Forward from the aligned full-length amino acid tive except asr1355 which has been cloned, 5‘-TAT ATG GGT GAT AGA TGC AC-3’ sequences by Neighbor-Joining and Max- sequenced, and its protein product bio- and Reverse 5‘-ATA ATG TTA CCC TCC imum Likelihood methods. The Neigh- chemically characterized (Arguello-Mo- TTT GT-3’). The predicted PCR product bor-Joining algorithm parameters “com- rales et al. 2000). asr gene sequences from size based on the asr1355 gene is 6270 bp, plete deletion,” “amino acid p-distance,” strains Leuconostoc B-1498 and 1501 were which included non-coding regions. PCR and “bootstrap” (1000 replications) were sequenced for this study. asr gene sequenc- reaction included ddH2O, AccuTaq LA 10x used in the analyses. The Maximum Like- es from Leuconostoc strains KM20, C11, buffer, DNA (100 ng), MgSO4 (1.0 mM), lihood algorithm parameters of “complete and KCTC 3537 were determined through primers (12.0 pm), dNTP mix (200 µM), deletion,” “amino acid substitution mod- genome sequencing from previous studies and JumpStart RedAccutaq LA DNA Poly- el” (Jones-Taylor-Thornton (JTT) model), (Amari et al. 2014, Kim et al. 2008, Nam et merase (Sigma Aldrich, St Louis, MO, 0.5 “Nearest-Neighbor-Interchange (NNI) tree al. 2011). The linkage distribution from al- unit). PCR conditions were performed as inference option”, and “bootstrap” (1000 ternan products for strains B-1355, B-1498, previously reported (Kossman et al. 2000). replications) were used in the analyses. No and B-1501 (Jeans et al. 1954, Jeans 1979, Initial denaturation at 95ºC for 2 min pre- bootstrap “cutoff” value was used during Seymour et al. 1979), and C11 (Bounaix et ceded cycle conditions. Cycle conditions the preparation of phylogenetic trees so al. 2009) were reported from previous stud- (35 cycles) included denaturation at 95ºC that all data can be shown. ies. The alternan products reported for the for 45 s, primer annealing at 50ºC for 45 previous studies were prepared from par- s, primer extension at 68ºC using ramped RESULTS AND DISCUSSION tially purified enzymatic culture extracts time starting at 7 m ending at 8 min 42 s. Five gene sequences described as “alternan- (Bounaix et al.