Multilocus sequence analysis, a rapid and accurate identification tool for taxonomic classification, evolutionary relationship and population biology of the genus Shewanella

Abstract

22The genus Shewanella comprises a group of marine-dwelling species with worldwide 23 distribution. Several species are regarded as causative agents of food spoilage and 24 opportunistic pathogens of human diseases. In this study, a standard multilocus 25 sequence analysis (MLSA) based on six protein-coding genes (gyrA, gyrB, infB, recN, 26 rpoA and topA) was established as a rapid and accurate identification tool in fifty-nine 27 The nucleotide sequences of six HKGs are deposited in GenBank nucleotide sequence database under accession numbers of gyrA: MH090144-MH090185; gyrB: MH090186-MH090202; infB: MH090203-MH090244; recN: MH090245-MH090286; rpoA: MH090287-MH090328; and topA: MH090329-MH090370. 2 type Shewanella strains. This method yielded sufficient resolving power in regard to 28 enough informative sites, adequate sequence divergences and distinct interspecies 29 branches. The stability of phylogenetic topology was supported by high bootstrap 30 values and concordance with different methods. The reliability of the MLSA scheme 31 was further validated by identical phylogenies and high correlations of genomes. The 32 MLSA approach provided a robust system to exhibit evolutionary relationships in the 33 Shewanella genus. The split network tree proposed twelve distinct monophyletic 34 clades with identical G+C contents and high genetic similarities. Eighty-six tested 35 strains were investigated to explore the population biology of the Shewanella genus in 36 China. The most prevalent Shewanella species were Shewanella algae, Shewanella 37 xiamenensis, Shewanella chilikensis, Shewanella indica, Shewanella seohaensis and 38 Shewanella carassii. The strains frequently isolated from clinical and food samples 39 highlighted the importance of increasing the surveillance of Shewanella species. 40 Combined with the genetic, genomic and phenotypic analyses, Shewanella upenei 41 should be considered a synonym of S. algae, and Shewanella pacifica should be 42 reclassified as a synonym of Shewanella japonica. 43 IMPORTANCE 44 The MLSA scheme based on six HKGs (gyrA, gyrB, infB, recN, rpoA and topA) is 45 well established as a reliable tool for taxonomic, evolutionary and epidemiological 46 analyses of the genus Shewanella in this study. The standard MLSA method allows 47 48 The robust phylogenetic network of MLSA provides profound insight into the 49 evolutionary structure of the genus Shewanella. The population genetics of 50 Shewanella species determined by the MLSA approach plays a pivotal role in clinical 51 diagnosis and routine monitoring. Further studies on remaining species and genomic 52 analysis will enhance a more comprehensive understanding of the microbial 53 systematics, phylogenetic relationships and ecological status of the genus Shewanella.54

Characterization of putative pathogenic Shewanella algae isolated from ballast water

“…It is known that the rate of molecular evolution of gyrB sequences is faster than 16S rRNA which provides higher phylogenetic resolution. It was reported in several studies that gyrB gene has always been used as a discriminative detection for Shewanella species identification [50][51][52]. The similarities values of greater than 97% observed in both gyrB and 16S rRNA sequences of S. algae isolates with S. haliotis and S. upenei revealed a very close Available at www.veterinaryworld.org/Vol.14/March-2021/18.pdf phylogenetic association between the three species.…”

Establishment and Application of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Detection of Shewanella Genus

“…Since MLSA has been confirmed to be accurate for identifying Shewanella at the species level ( Fang et al, 2019 ), all 125 test strains were analyzed by MLSA, and the results were used to evaluate the effectiveness of MALDI-TOF MS for species identification. Taking the MLSA identification results as the “true species identity” of a test strain, all strains were identified correctly at the genus level, and 116 (92.8%) of the test strains were accurately identified at the species level.…”

“…Genomic DNAs were extracted according to the standardized instructions of the DNA extraction kit (TaKaRa, Dalian, China). Six single-copy housekeeping genes ( gyrA , gyrB , infB , recN , rpoA , and topA ) were selected according to previous studies ( Fang et al, 2019 ). Housekeeping genes of the 36 type strains were obtained from GenBank ( Supplementary Table 1 ).…”

“…The housekeeping gene gyrB was found to have a higher resolution than 16S rRNA for Shewanella species identification ( Bozal et al, 2002 ; Miyazaki et al, 2006 ; Sung et al, 2012 ), but no standardized cutoff value has been established for the identification ( Bozal et al, 2002 ; Miyazaki et al, 2006 ; Sung et al, 2012 ). We previously established that the method of multilocus sequence analysis (MLSA) can accurately identify Shewanella at the species level ( Fang et al, 2019 ). This method requires PCR and sequencing of six housekeeping genes from each isolate, which is time-consuming and costly.…”

Phylogenomic analysis reveals a two‐stage process of the evolutionary transition of Shewanella from the upper ocean to the hadal zone

“…In particular, YLB‐06 has the largest genome among the reported genomes of the Shewanella genus. The chromosomal DNA G + C contents of the three strains were 45.1, 43.6 and 43.5 mol% (Table S1) respectively, which are lower than those of the Benthica clade of Shewanella species (47–49 mol%), as reported previously (Fang et al ., 2019). Moreover, the DNA–DNA hybridization (DDH) and average nucleotide identity (ANI) estimates between strains YLB‐06 and YLB‐08 and their closest type strains were significantly lower than the proposed cutoff level (70% and 95%–96%) for species delineation (Stackebrandt et al ., 2002; Richter and Rosselló‐Móra, 2009), suggesting that strains YLB‐06 and YLB‐08 may represent two novel species in the genus Shewanella (Table S2).…”