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Proposal of Lysobacter Pythonis Sp. Nov. Isolated from Royal Pythons ଝ Systematic and Applied Microbiology 42 (2019) 326–333 Contents lists available at ScienceDirect Systematic and Applied Microbiology jou rnal homepage: http://www.elsevier.com/locate/syapm Proposal of Lysobacter pythonis sp. nov. isolated from royal pythons ଝ (Python regius) a,∗ b a a Hans-Jürgen Busse , Christopher Huptas , Sandra Baumgardt , Igor Loncaric , a b b c Joachim Spergser , Siegfried Scherer , Mareike Wenning , Peter Kämpfer a Institut für Mikrobiologie, Veterinärmedizinische Universität Wien, A-1210 Wien, Austria b Lehrstuhl für Mikrobielle Ökologie, ZIEL – Institute for Food & Health, Technische Universität München, Weihenstephaner Berg 3, D-85354 Freising, Germany c Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany a r t i c l e i n f o a b s t r a c t Article history: The bacterial strains 4284/11T and 812/17 isolated from the respiratory tract of two royal pythons in 2011 Received 7 December 2018 and 2017, respectively were subjected to taxonomic characterization. The 16S rRNA gene sequences of the Received in revised form 31 January 2019 T two strains were identical and showed highest sequence similarities to Lysobacter tolerans UM1 (97.2%) Accepted 8 February 2019 T and Luteimonas aestuarii DSM 19680 (96.7 %). The two strains were identical in the sequences of the 16S-23S rRNA internal transcribed spacer (ITS) and partial groEL gene sequences and almost identical in Keywords: T genomic fingerprints. In the ITS sequence Ly. tolerans DSM 28473 and in the groEL nucleotide sequence Lysobacter pythonis T Luteimonas mephitis DSM 12574 showed the highest similarity. In silico DDH analyses using genome Phylogeny T Chemotaxonomy sequence based ANIb and gANI similarity coefficients demonstrated that strain 4284/11 represents a T Physiology novel species and revealed Ly. tolerans UM1 as the next relative (ANIb = 76.2 %, gANI = 78.0 %). Based T on the topology of a core gene phylogeny strain 4284/11 could be assigned to the genus Lysobacter. Chemotaxonomic characteristics including polyamine pattern, quinone system, polar lipid profile and fatty acid profile were in accordance with the characteristics of the genera Lysobacter and Luteimonas. T Strains 4284/11 and 812/17 could be differentiated from the type strains of the most closely related species by several physiological tests. In conclusion we are here proposing the novel species Lysobacter T T T T pythonis sp. nov. The type strain is 4284/11 (= CCM 8829 = CCUG 72164 = LMG 30630 ) and strain 812/17 (CCM 8830) is a second strain of this species. © 2019 Elsevier GmbH. All rights reserved. The genera Lysobacter and Luteimonas were placed in the fam- chemotaxonomic characteristics such as fatty acids, polar lipids, ily “Xanthomonadaceae” by Saddler and Bradbury [39]. The name quinones and polyamines are not useful because so far no charac- of the family Xanthomonadaceae is illegitimate because it con- teristics were found distinguishing these genera. tains the genus Lysobacter which is the type genus of the family During routine microbiological diagnosis of human and veteri- Lysobacteraceae [47]. Phylogenetic studies indicate that both gen- nary specimens often only the supposed causative agent of the era Lysobacter and Luteimonas are not monophyletic. For instance, diagnosed disease is identified whereas commensals detected after Lysobacter panaciterrae is found next to several Luteimonas species primary cultivation are usually ignored. However, for treatment of [27,49,52] and might be reclassified in the genus Luteimonas. Unfor- the disease identification of the commensals is of no relevance if the tunately, for establishment of a stable taxonomy within this group causative agent can be detected in the specimen. On the other hand, commensal isolates appear to be an interesting source of unrec- ognized bacterial species and hence they are most interesting for bacterial taxonomists. ଝ The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene, the groEL, In this study we are reporting on the taxonomic characterization the 16S-23S internal transcribed spacer sequences (ITS) and the genome assem- of two bacterial strains isolated from royal python (Python regius). bly are: 16S rRNA gene LM994044 and MG760578; groEL LM994049, LM994050, T Strain 4284/11 was isolated in 2011 from the trachea of a royal LM994051, LM994052, MH105811 and MH105812; ITS LM994045, LM994046, LM994047, LM993048, MG760579 and MG760580; the Genbank accession of the python suffering from respiratory tract infection after antibiotic genome of strain 4284/11T is GCA 003697345.1. treatment and strain 812/17 was isolated in 2017 from the tra- ∗ Corresponding author. E-mail address: [email protected] (H.-J. Busse). https://doi.org/10.1016/j.syapm.2019.02.002 0723-2020/© 2019 Elsevier GmbH. All rights reserved. H.-J. Busse, C. Huptas, S. Baumgardt, et al. / Systematic and Applied Microbiology 42 (2019) 326–333 327 Xanthomonas campestris ATCC 33913T (AE008922) Luteimonas terr ae THG-MD21T (KJ769177) Luteimona s aestuarii B9T (EF660758) Lysobacter toleran s UM1T (FTLW01000003) T 62 Lysobacter pythonis 4284/11 (LM994044) 100 Lysob acter pythonis 812/17 (MG760578) Lysobacter ginseng isoli Gsoil 357T (AB245363) T 99 Lysobacter enzymogenes ATCC29487 (jgi.1095734) T 83 Lysobacter antibioticus ATCC 29479 (CP013141) Lysobacter cap sici YC5194T (JAAN01000045) Lysobacter gummosus KCTC 12132T (AB161361) 90 Luteimona s cucumeris CGMCC 1.1082T (jgi.1041429) Luteimonas vado sa KMM9005T (AB704915) Luteimona s terricola BZ92rT (FJ948107) 100 Luteimona s padinae CDR SL15T (LN879436) Lysobacter pana citerrrae Gsoil 068T (AB245359) Luteimonas aquatica RIB1-20T (EF626688) Luteimona s mephitis DSM 12574T (AULN01000012) Luteimonas marina FR1330T (EU295459) 62 Luteimona s huabeiensis HB2T (JAAN01000045) Fig. 1. Maximum-likelihood tree based on 16S rRNA gene-sequences showing the relatedness of the strains 8284/11 and 812/17 with reference species of the genera T Lysobacter and Luteimonas sharing at least 95.0% 16S rRNA gene sequence similarity. S. maltophilia IAM 12423 was used as an outgroup. Bootstrap values (%) >60 based on 100 replicates are given at nodes. Bar indicates 0.1 substitutions per nucleotide position. T chea of another royal python displaying symptoms of respiratory similarities of 4284/11 and 812/17 to the type species of Lysobacter distress. suggest that they could be assigned to this genus. For phyloge- T For preliminary classification, the 16S rRNA genes were ampli- netic analyses the 16S rRNA gene sequences of strains 4284/11 and fied using universal primers 27f and 1494r [23]. The PCR reaction 812/17 were aligned with sequences of type strains of all Lysobacter ␮ mix consisted of 30 l REDTaq ReadyMix PCR Reaction Mix with and Luteimonas species, which shared more than 95.0% similarity, T MgCl2 (Sigma–Aldrich), 37.5 pmol of each primer (Invitrogen), and Lu. mephitis DSM 12574 , Stenotrophomonas maltophilia IAM T T ␮ ␮ 2.50 l DNA and 26.20 l sterile water. Amplification started with 12423 and Xanthomonas campestris ATCC 33913 using clustal x ◦ an initial denaturation step at 95 C for 5 min, followed by 30 cycles [44]. The sequences were manually edited for ambiguous bases and ◦ ◦ of denaturation at 94 C for 1.5 min, annealing at 53 C for 1.5 min gaps using Bioedit [14]. Phylogenetic trees were calculated apply- ◦ ◦ and elongation at 72 C for 5 min, and a final elongation at 72 C for ing the algorithms maximum likelihood, maximum parsimony and 10 min. neighbor joining and standard adjustments implemented in the ® The PCR products were purified using Wizard SV Gel und PHYLIP package [10] were applied. In the maximum likelihood tree T PCR Clean-Up System (Promega) according to the manufacturer’s (Fig. 1) the two strains formed, together with Ly. tolerans UM1 , instructions and sequenced by LGC Genomics (Berlin) resulting in a lineage deeply branching from the neighboring lineage with Ly. T T 16S rRNA gene sequences of strains 4284/11 and 812/17 with enzymogenes ATCC 29487 , and clearly separate from Lu. mephi- T length of 1447 and 1442 nucleotides. The two 16S rRNA gene tis DSM 12574 . Also, the phylogenetic trees calculated applying sequences were 100% identical. Sequence comparisons were car- the maximum parsimony and neighbor joining algorithms showed ried out using the EzTaxon-e server [50] and identified Lysobacter these branching patterns (results not shown). On the other hand, T T tolerans UM1 [27,34] and Luteimonas aestuarii B9 [37] as the phylogenetic analyses did not suggest a very close relatedness to next related species sharing 97.2 and 96.7% similarity, respec- the type species of the genera Luteimonas or Lysobacter. It is worth tively. Sequence similarities with the type species of the genera to mention that only a minority of branchings were supported from T Lysobacter and Luteimonas, Lysobacter enzymogenes DSM 2043 and bootstrap values (>70%) in all trees calculated. T Luteimonas mephitis DSM 12574 , were 95.7% and 93.3%, respec- In order to gain more genetic information concerning genus T tively. Surprisingly, among the species sharing in the 16S rRNA gene affiliation of strain 4284/11 the partial groEL sequence of the T sequence more than 95.0% similarity nine Luteimonas species and isolate and the reference species Ly. tolerans DSM 28473 , Lu. aes- T T six Lysobacter species were found. However, the higher sequence tuarii DSM 19680 , Lu. mephitis CIP 107229 and Ly. enzymogenes 328 H.-J. Busse, C. Huptas, S. Baumgardt, et al. / Systematic
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