International Journal of Systematic Bacteriology (1 999), 49, 1433-1 438 Printed in Great Britain The phylogenetic position of Serratia, -1 -1 Buttiauxella and some other genera of the family Enterobacteriaceae Cathrin Sprber, Ulrike Mendrock, Jolantha Swiderski, Elke Lang and Erko Stackebrandt Author for correspondence : Erko Stackebrandt. Tel : + 49 53 1 26 16 352. Fax : + 49 53 1 26 16 4 18. e-mail : [email protected] DSMZ - Deutsche Sammlung The phylogenetic relationships of the type strains of 38 species from 15 genera von Mikroorganismen und of the family Enterobacteriaceae were investigated by comparative 16s rDNA Zellkulturen GmbH, Mascheroder Weg 1b, analysis. Several sequences of strains f rom the genera Citrobacter, Erwinia, D-38124Braunschweig, Pantoea, Proteus, Rahnella and Serratia, analysed in this study, have been Germany analysed previously. However, as the sequences of this study differ slightly from the published ones, they were included in the analysis. Of the 23 enterobacterial genera included in an overview dendrogram of relatedness, members of the genera Xenorhabdus, Photorhabdus, Proteus and Plesiomonas were used as a root. The other genera formed two groups which could be separated, although not exclusively, by signature nucleotides at positions 590-649 and 600-638. Group A contains species of Brenneria, Buttiauxella, Citrobacter, Escherichia, Erwinia, Klebsiella, Pantoea, Pectobacterium and Salmonella. All seven type strains of Buttiauxella share 16s rDNA similarities greater than 99 O/O. Group B embraces two phylogenetically separate Serratia clusters, a lineage containing Yersinia species, Rahnella aquatica, Ewingella americana, and also the highly related pair Hafnia alvei and Obesumbacterium pro teus. Keywords: Enterobacteriaceae, Buttiauxella, Serratia, 16s rDNA analysis In contrast to other taxon-rich families, such as the ture of the 16s rDNA, this molecule was not thought Clostridiaceae, the Bacillaceae and the Pseudo- to solve taxonomic problems concerning closely re- monadaceae, members of the Enterobacteriaceae have lated species. Extensive phylogenetic analysis of not been subjected to extensive analysis of 16s rDNA. the genera Yersinia, Salmonella, Photorhabdus and While some genera have been investigated in detail, Erwinia, however, have demonstrated that the variable e.g. Xenorhabdus and Photorhabdus (Szallas et al., and highly variable regions of the 16s rDNA molecule 1997), Yersinia (Ibrahim et al., 1994), Salmonella have sufficient phylogenetic powers of discrimination (Chang et al., 1997; Christensen et al., 1998), Serratia to allow the recognition of the same sets of relatedness (Dauga et al., 1990; Harada et al., 1996), and Erwinia as those unravelled by DNA-DNA reassociation (Kwon et al., 1997; Hauben et al., 1998), other genera studies. Furthermore, the phylogenetic incoherence of have been analysed less extensively, e.g. Enterobacter Erwinia has been demonstrated in the studies of Kwon (Hauben et al., 1998), Proteus (Niebel et al., 1987), et al. (1997) and Hauben et al. (1998). Citrobacter (Maidak et al., 1997), and most of the In this work, 16s rDNA-based analysis of the family monospecific genera. The reason for the dismissal of Enterobacteriaceae is extended by adding sequences of many members of the Enterobacteriaceae may stem 38 type strains to the database. Eleven type strains of from knowledge of the high intrafamily related- species investigated in the course of this study ness (Brenner, 1991), as measured by DNA-DNA Erwinia were published by Hauben et al. (1998), and several of hybridization. Because of the conserved primary struc- these species were subsequently reclassified by these authors as species of Pectobacterium and Brenneria. The EMBL accession numbers for the 165 rDNA sequences analysed in this The organisms investigated in this study, their strain paper are AJ233400-AJ233437. designation, and their 16s rDNA accession numbers 01020 0 1999 IUMS 1433 C. Sproer and others Tabre 1. Strains analysed in this study, and their 165 aligned manually against available sequences for rDNA accession numbers members of the family Enterobacteriaceae. Evolution- ary distances were computed from globally aligned Specieslsubspecies Strain Accession sequences by using the correction of Jukes & Cantor no. (1969), omitting gaps and ambiguous positions. Dendrograms of relatedness were generated by the Budvicia aquatica DSM 5075T A5233407 algorithms of De Soete (1983) and by neighbour- Buttiauxella agrestis DSM 4586T AJ23 3400 joining and maximum-likelihood analyses using the Buttiauxella brennerae DSM 9396T A5233401 programs of the PHYLIP package (Felsenstein, 1993). Buttiauxella ferragu tiae DSM 9390T A5233402 Bootstrap values were determined as described else- But tiauxella guviniae DSM 9393’ A5233403 where (Felsenstein, 1993). The following reference But tiauxella izardii DSM 9397T A 5233404 sequences were taken from the EMBL database : Buttiauxella noackiae DSM 9401T A5233405 Escherichia coli (501695, Brosius et al., 1978); Pecto- Bu ttiauxella warmboldiae DSM 9404T A5233406 bacterium cacticidum LMG 17936T (AJ223409) ; Brenneria alni DSM 11811T A 5233409 Erwinia persicinus ATCC 3599gT (U80205) ; Erwinia Brenneria quercina DSM 4561T A5233416 psidii LMG 7034 (296085) ; Erwinia tracheiphila LMG Brenneria rubrifaciens DSM 4483T A5233418 2906T (Y 13250) ; Ewingella americana NCPPB 3905 Brenneria salicis DSM 3016tjT A5233419 (X88848, entry cited unpublished) ; Hafnia alvei ATCC Citrobacter freundii DSM 30039T A5233408 13337T (M59 155, entry cited unpublished) ; Plesio- Erwinia amylovora DSM 30165T A52334 10 monas shigelloides ATCC 14029T(M59 159, entry cited Erwinia mallotivora DSM 4565T A52334 14 unpublished) ; Salmonella typhimurium ATCC 19430T Erwinia nigrzjhens DSM 30175T A5233415 (247544) ; Yersinia pestis D-28 (X75274), Xenorhabdus Er w inia rhapon t ici DSM 4484T A52334 17 nematophilus DSM 3370T (X8225 l), Photorhabdus Klebsiella pneumoniae subsp. DSM 30104T A5233420 luminescens DSM 3368T (X82248) and Plesiomonas pneumoniae shigelloides ATCC 14029T (X74688). Leminorella grimon t ii DSM 5078T A5233421 Almost complete 16s rDNA sequences were analysed Obesumbacteriumproteus DSM 2777T A5233422 for the type strains of 38 species from the genera Pan toea agglomerans DSM 3493T A5233423 Brenneria, Budvicia, Buttiauxella, Citrobacter, Pectobacterium carotovorum DSM 30168T A523341 1 Erwinia, Klebsiella, Leminorella, Obesumbacterium, subsp. carotovorum Pantoea, Pectobacterium, Pragia, Rahnella, Serratia Pectobacterium chrysanthemi DSM 4610T A5233412 and Tatumella. The lengths of the sequences ranged Pectobacterium cypripedii DSM 3873T A5233413 between 1493 and 1516 nucleotides, corresponding to Pragia fontium DSM 5563T A5233424 95.5 and 98.4Y0, respectively, of the Escherichia coli Proteus vulgaris DSM 301 18T A5233425 sequence. Some of the sequences of type strains of Rahnella aquatica DSM 4594T A5233426 Erwinia, Serratia, Citrobacter, Pan toea and Rahnella Serratia en tomophila DSM 12358T A5233427 species analysed in this study have been published Serratia jicariu DSM 4569T A5233428 recently (Dauga et al., 1990; Harada et al., 1996; Serra tia fonticola DSM 4576T A5233429 Kwon et al., 1997; Maidak et al., 1997), but, as there Serratia grimesii DSM 30063T A5233430 are some nucleotide differences in sequences orig- Serratia marcescens DSM 30121T A523343 1 inating from the same strain (98.8-99-9 Yo sequence Serratia odorifera DSM 4582T A5233432 similarity), sequences determined in this study were Serratia plymuthica DSM 4540T A5233433 deposited in the EMBL database. Sequence similarities Serratia proteamaculans DSM 4543T A5233434 obtained for the sequences of the 11 type strains of subsp. proteamaculans Erwinia, Brenneria and Pectobacterium, published, Serratia proteamaculans DSM 4597T A5233435 during this study, by Hauben et al. (1998), ranged subsp. quinovora between 98.5 and 100 YO.A significant deviation from Serratia rubidaea DSM 4480T A5233436 the data of Kwon et at. (1997) refers to Brenneria Tatumella ptyseos DSM 5000T A5233437 salicis. While the 16s rDNA sequences of strains DSM 30166T (this study) and LMG 269gT (Hauben et al., 1998) are identical, they share only 94-5Yo sequence similarity with strain ATCC 157 12T. The branching are listed in Table 1. Extraction of genomic DNA and the amplification of 16s rDNA were performed as point of Pectobacterium cypripedii DSM 3873Twithin the radiation of authentic Erwinia species differs from described previously (Rainey et al., 1996). The PCR the position of the type strain LMG 5657T of the same products were purified by using the Prep-A-Gene kit (Bio-Rad), as described by the manufacturer. The species within the radiation of cluster 111, which, DyeDeoxy Terminator Cycle Sequencing kit (Applied consequently, has been reclassified as Pectobacterium cypripedii (Hauben et al., 1998). Certainly, the auth- Biosystems) was used for direct sequencing of the PCR enticity of the type strains of these two species requires products, as described by the manufacturer. The investigation. sequence reactions were electrophoresed on an Applied Biosystems 373A DNA Sequencer. Sequences were Comparison of the sequences generated in this study 1434 International Journal of Systematic Bacteriology 49 Phylogeny of enterobacterial genera Escherichia coli Salmonella typhimurium ATCC 19430 T Pantoea agglomerans DSM 3493T Eminia cluster I Etwinia tracheiphila LMG 2906T Pectobacterium cypn'pedii DSM 3873T Ewinia mellotivora DSM 4565 T Elwinie psi& LMG 7034T L-