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Proteobacteria

Emended description of the genus Pantoea, description of four species from human clinical samples, Pantoea septica sp. nov., Pantoea eucrina sp. nov., Pantoea brenneri sp. nov. and Pantoea conspicua sp. nov., and transfer of Pectobacterium cypripedii (Hori 1911) Brenner et al. 1973 emend. Hauben et al. 1998 to the genus as Pantoea cypripedii comb. nov.

Carrie L. Brady, Ilse Cleenwerck, Stephanus N. Venter, Katrien Engelbeen, Paul De Vos, Teresa A. Coutinho

  • 1Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa
  • 2BCCM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium
  • Correspondence
    Teresa A. Coutinho
    teresa.coutinho{at}fabi.up.ac.za

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(10):2430–2440 · https://doi.org/10.1099/ijs.0.017301-0

    View at publisher PubMed

    Abstract

    Bacterial strains belonging to DNA hybridization groups (HG) II, IV and V, in the Erwinia herbicola–Enterobacter agglomerans complex, of Brenner et al. [Int J Syst Bacteriol 34 (1984), 45–55] were suggested previously to belong to the genus Pantoea, but have never been formally described and classified. Additionally, it has been shown in several studies that Pectobacterium cypripedii is more closely related to species of Pantoea than to those of Pectobacterium. In this study, the phylogenetic positions of Brenner's DNA HG II, IV and V and Pectobacterium cypripedii were re-examined by both 16S rRNA gene sequencing and multilocus sequence analyses (MLSA) based on the gyrB, rpoB, atpD and infB genes. The analyses revealed that DNA HG II, IV and V and Pectobacterium cypripedii form five separate branches within the genus Pantoea (strains from HG V were split into two branches). DNA–DNA hybridization data further confirmed that DNA HG II, IV and V constitute four separate species. Pectobacterium cypripedii was shown to be a close phylogenetic relative of Pantoea dispersa and DNA HG IV by both 16S rRNA gene sequence and MLSA analyses. Biochemical analyses performed on strains from DNA HG II, IV and V and Pectobacterium cypripedii confirmed their taxonomic position within the genus Pantoea and revealed phenotypic characteristics that allow the differentiation of these species from each other and from their closest phylogenetic neighbours. It is proposed to emend the description of the genus Pantoea and to describe Pantoea septica sp. nov. for DNA HG II (type strain LMG 5345T =BD 874T =CDC 3123-70T), Pantoea eucrina sp. nov. for DNA HG IV (type strain LMG 2781T =BD 872T =CDC 1741-71T =LMG 5346T), Pantoea brenneri sp. nov. for strains of DNA HG V excluding LMG 24534 (type strain LMG 5343T =BD 873T =CDC 3482-71T) and Pantoea conspicua sp. nov. for the remaining strain of DNA HG V (type strain LMG 24534T =BD 805T =CDC 3527-71T) and to transfer Pectobacterium cypripedii to the genus as Pantoea cypripedii comb. nov. (type strain LMG 2657T =ATCC 29267T =DSM 3873T =LMG 2655T).

    • The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are EU216734–EU216737 (16S rRNA gene), EU145261–EU145273 and FJ187830–FJ187833 (gyrB gene), EU145293–EU145305 and FJ187840–FJ187843 (rpoB gene), EU145245–EU145257 and FJ187825–FJ187828 (atpD gene) and EU145277–EU145289 and FJ187835–FJ187838 (infB gene), as detailed in Supplementary Table S1.

    • Details of sequence accession numbers and a UPGMA dendrogram based on phenotypic characteristics are available as supplementary material with the online version of this paper.

    Since the early 1970s, various attempts have been made to clarify the Erwinia herbicola–Enterobacter agglomerans complex (Ewing & Fife, 1972; Gardner & Kado, 1972; Young et al., 1978; Dye, 1981; Brenner et al., 1984; Verdonck et al., 1987; Beji et al., 1988). The most comprehensive study to date was performed by Brenner et al. (1984), who separated 124 strains belonging to this complex into 13 groups based on their DNA–DNA hybridization relatedness. The majority of strains used in Brenner's study were clinical strains implicated in a nosocomial septicaemia outbreak in 1971 (Maki et al., 1976), although plant-pathogenic strains from the complex were also included. In the years following Brenner's hybridization study, many of the 13 DNA hybridization groups (HG) were further investigated and classified as novel species in existing or novel genera. Gavini et al. (1989) transferred DNA HG XIII and DNA HG III to the novel genus Pantoea as Pantoea agglomerans and Pantoea dispersa, respectively. Mergaert et al. (1993) proposed the name Pantoea ananatis for DNA HG VI, containing strains of Erwinia ananas and its synonym Erwinia uredovora, whilst DNA HG VII–XII were assigned to species within the genera Enterobacter, Rahnella and Leclercia (Grimont & Grimont, 2005). Beji et al. (1988) suggested the inclusion of Brenner's DNA HG V into Enterobacter agglomerans (now Pantoea agglomerans), although only 62 % DNA relatedness was observed between the reference strain (CDC 3482-71 =LMG 5343) and Pantoea agglomerans ATCC 27155T. However, Gavini et al. (1989) opposed this recommendation and proposed to unite Brenner's DNA HG V with protein profile group VII (Beji et al., 1988) in a new genus and species, a proposal that has never been implemented.

    Recently, polyphasic taxonomic studies on strains from Beji's protein profile group VII, Pantoea reference strains as well as possible Pantoea species isolated from eucalyptus and maize resulted in the description of four novel species (Pantoea anthophila, Pantoea vagans, Pantoea eucalypti and Pantoea deleyi) and the transfer of Pantoea citrea, Pantoea punctata and Pantoea terrea to the genus Tatumella (Brady et al., 2009, 2010). Multilocus sequence analysis (MLSA) based on gyrB, rpoB, atpD and infB sequences was used in these studies as a supporting phylogenetic technique, since these genes were previously shown to be useful phylogenetic markers for Pantoea (Brady et al., 2008). In that study, strains from DNA HG II, IV and V (referred to as MLSA groups E, F, H and I) and Pectobacterium cypripedii were suggested to be members of the genus Pantoea. Previous studies have also questioned the taxonomic position of Pectobacterium cypripedii, as it does not cluster with other Pectobacterium species and several different 16S rRNA gene sequences are available for the type strain of this species (Spröer et al., 1999; Samson et al., 2005; Young & Park, 2007; Naum et al., 2008; Yarza et al., 2008). In this study, we propose the taxonomic reclassification of strains from Brenner's DNA HG II, IV and V and Pectobacterium cypripedii within the genus Pantoea.

    Strains

    Strains used in this study are listed in Table 1⇓. Strains belonging to Brenner's DNA HG II, IV and V were kindly provided by Mrs Caroline Mohr from the Centers for Disease Control (CDC), Atlanta, GA, USA. Strains from Brenner's DNA HG I could not be acquired. Details of individual sequence accession numbers are given in Supplementary Table S1, available in IJSEM Online.

    Table 1.

    Strains used in this study

    Collections: LMG, BCCM/LMG Bacteria Collection, Ghent University, Belgium; CDC, Centers for Disease Control, Atlanta, GA, USA; BD, Plant Pathogenic and Plant Protecting Bacteria (PPPPB) Culture Collection, ARC-PPRI, Pretoria, South Africa.

    16S rRNA gene sequencing

    Genomic DNA was extracted from strains using an alkali extraction method (Niemann et al., 1997) and stored at −20 °C until further use. Almost-complete 16S rRNA gene sequences were determined for representative strains from each DNA HG, LMG 5345T, LMG 2781T, LMG 5343T, LMG 24534T, and the type strain of Pectobacterium cypripedii, LMG 2657T, using the primers and conditions described by Coenye et al. (1999). The 16S rRNA gene sequence of LMG 2657T was compared to that of Pectobacterium cypripedii DSM 3873T (GenBank accession no. AJ233413), as used in the All Species Living Tree (Yarza et al., 2008), to ascertain whether the sequence was correct, as several different sequences are available for this strain. The sequences were aligned using clustal_x (Thompson et al., 1997) and the overhangs were trimmed. The modeltest 3.7 program (Posada & Crandall, 1998) was then applied to determine the best-fit evolutionary model. Maximum-likelihood and neighbour-joining analyses were performed using Phyml (Guindon & Gascuel, 2003) and paup 4.0b10 (Swofford, 2000), respectively, by applying the models and parameters determined by modeltest (only maximum-likelihood phylogenetic trees are shown). Bootstrap analysis with 1000 replicates was performed to assess the support for these clusters.

    Strains LMG 5343T and LMG 24534T, from Brenner's DNA HG V, demonstrated high 16S RNA gene sequence similarity to each other (>99 %), and to strains of Pantoea agglomerans, Pantoea ananatis, Pantoea anthophila, Pantoea deleyi, Pantoea eucalypti, Pantoea stewartii and Pantoea vagans (>98 %), which are regarded as the ‘core’ Pantoea species. These strains cluster closely with the type strain of Pantoea deleyi (Fig. 1⇓) with a high bootstrap value but on separate branches, suggesting that they belong to different species. Strain LMG 5345T, from Brenner's DNA HG II, shares more than 98 % 16S rRNA gene sequence similarity with LMG 5343T and the type strains of Pantoea agglomerans, Pantoea vagans and Pantoea eucalypti. This strain is situated at the border of the ‘core’ Pantoea group. Strain LMG 2781T, from Brenner's DNA HG IV, demonstrated more than 98 % 16S rRNA gene sequence similarity to the type strains of Pantoea agglomerans and Pantoea dispersa and clusters close to the type strain of the latter species but with low bootstrap support. Pectobacterium cypripedii DSM 3873T shows higher 16S rRNA gene sequence similarity to species of Pantoea (96.8–98.0 %) and Erwinia (94.6–97.7 %) than to the other species of Pectobacterium (95.3–96.4 %). This type strain clusters between the Erwinia and Pantoea clusters, noticeably distant from the other Pectobacterium species. Species of Erwinia, Pectobacterium, Brenneria and Pantoea can be characterized to the genus level by 14–21 signature nucleotides in their 16S rRNA gene sequences according to Hauben & Swings (2005). The 16S rRNA gene sequences of Pectobacterium cypripedii and all recognized Pantoea species were examined closely for the signature nucleotides using the Escherichia coli numbering (Brosius et al., 1978). Pectobacterium cypripedii shares ten common signature nucleotides with Pantoea species and seven with Erwinia species but only four with other Pectobacterium species, whilst three signature nucleotides were shown to be unique to Pectobacterium cypripedii (C at positions 847, 1216 and 1217). Furthermore, Pectobacterium cypripedii was shown to share 20 of the 23 atpD gene signature nucleotides that can be used to characterize species as belonging to Pantoea (Brady et al., 2010).

    Figure image not available in archive
    Fig. 1.

    Maximum-likelihood tree based on almost-complete 16S rRNA gene sequences of members of Pantoea and phylogenetically related species. Bootstrap values after 1000 replicates are expressed as percentages. Escherichia coli ATCC 11775T was included as an outgroup. Bar, 0.1 substitutions per nucleotide position.

    The genus Pantoea is polyphyletic, based on 16S rRNA gene sequence analysis, with Pantoea dispersa, Pectobacterium cypripedii and strain LMG 2781T clustering separately from the remaining ‘core’ species (Brady et al., 2008). MLSA of partial nucleotide sequences of the housekeeping genes gyrB, rpoB, atpD and infB is a more useful technique than 16S rRNA gene sequence analysis for examining the phylogeny of Pantoea species and strains (Brady et al., 2008).

    Analysis of gyrB, rpoB, atpD and infB sequences

    Partial gyrB (742 bp), rpoB (637 bp), atpD (657 bp) and infB (615 bp) gene sequences were determined for all strains (Brady et al., 2008). Sequence analysis and tree construction were performed as described above, and a phylogenetic tree based on concatenated sequences of the four genes is presented in Fig. 2⇓. The phylogenetic tree demonstrates that Brenner's DNA HG II, IV and V and Pectobacterium cypripedii form five separate well-supported clusters within the genus, distinct from the existing Pantoea species with validly published names. Strains from Brenner's DNA HG V cluster close to Pantoea agglomerans, Pantoea eucalypti, Pantoea vagans, Pantoea anthophila and Pantoea deleyi with high bootstrap support (100 %). LMG 24534T clusters distant from the other strains within this group, corroborating the existence of two different species within Brenner's DNA HG V. Brenner et al. (1984) noted that aerogenic and anaerogenic strains were found simultaneously in two DNA hybridization groups only, HG II and V. These DNA groups consisted of anaerogenic strains, except for LMG 24527 (=CDC 1778-80) in Brenner's DNA HG II and LMG 24534T (=CDC 3527-71T) in Brenner's DNA HG V. In Fig. 2⇓, LMG 24527 clusters close to the other strains from Brenner's DNA HG II, whereas LMG 24534T is separated from the rest of Brenner's DNA HG V. Strains from DNA HG II and IV cluster between Pantoea ananatis and Pantoea dispersa, whilst Pectobacterium cypripedii clusters with DNA HG IV and Pantoea dispersa with high bootstrap support. The MLSA data, together with the 16S rRNA gene sequence analysis, suggest that Brenner's DNA HG II, IV and V represent four novel species within the genus Pantoea and indicate that Pectobacterium cypripedii should be transferred to this genus.

    Figure image not available in archive
    Fig. 2.

    Maximum-likelihood tree based on concatenated housekeeping gene sequences of Pantoea strains. Bootstrap values after 1000 replicates are expressed as percentages. Escherichia coli K-12 was included as an outgroup. Gene sequences for Erwinia tasmaniensis Et1/99T, Escherichia coli ATCC 11775T, Shigella dysenteriae Sd197, Klebsiella pneumoniae 342, Citrobacter rodentium ICC168, Citrobacter koseri ATCC BAA-895 and Cronobacter sakazakii ATCC BAA-894 were obtained from genome sequencing databases (, , ). Bar, 0.1 substitutions per nocleotide position.

    DNA–DNA hybridizations

    High-quality DNA for DNA–DNA hybridizations was prepared by the method of Wilson (1987), with minor modifications (Cleenwerck et al., 2002). DNA–DNA hybridizations were performed using the microplate method (Ezaki et al., 1989) with some modifications (Cleenwerck et al., 2002) at a hybridization temperature of 45±1 °C. Reciprocal reactions (A×B and B×A) were performed for every DNA pair from all strains and their variation was within the limits of this method (Goris et al., 1998). The values presented are based on a minimum of four replicates. Strains from each DNA HG were hybridized amongst each group and LMG 24534T (the aerogenic strain from Brenner's DNA HG V) was hybridized to reference strains from these groups (LMG 5345T, LMG 2781T and LMG 5343T). These four reference strains were also hybridized to the type strains of Pantoea agglomerans, Pantoea ananatis, Pantoea anthophila, Pantoea deleyi, Pantoea dispersa, Pantoea eucalypti, Pantoea stewartii and Pantoea vagans. The type strain of Pectobacterium cypripedii was hybridized to the type strain of Pantoea dispersa, the only species with which it shares more than 97 % 16S rRNA gene sequence similarity. A summary of the DNA–DNA hybridization results is presented in Table 2⇓ and reveals that the DNA–DNA relatedness within each DNA HG was greater than 70 %. Strains LMG 5345T, LMG 2781T, LMG 5343T and LMG 24534T exhibited less than 70 % DNA–DNA relatedness with each other and with the type strains of Pantoea species with validly published names. Strain LMG 24534T, the aerogenic strain from Brenner's DNA HG V, exhibited only 55–60 % DNA–DNA relatedness when hybridized with other strains from this DNA HG. This is considerably lower than the 73 % observed by Brenner et al. (1984). The lower values obtained in our study may be due to the different hybridization method used; however, our DNA–DNA hybridization data are congruent with the MLSA data.

    Table 2.

    DNA–DNA hybridization amongst strains belonging to the novel species and reference strains of Pantoea species with validly published names

    DNA G+C content

    The DNA G+C content of the type strains proposed for the four novel species were determined by HPLC (Mesbah et al., 1989) and are as follows: LMG 5345T, 59.3 mol%; LMG 2781T, 56.5 mol%; LMG 5343T, 55.4 mol%; and LMG 24534T, 55.7 mol%. These values are consistent with the DNA G+C contents of other members of the genus Pantoea (Brady et al., 2009; Grimont & Grimont, 2005).

    Phenotypic assays

    Physiological and biochemical tests were performed on the type strains proposed for the four novel species (LMG 5345T, LMG 2781T, LMG 5343T and LMG 24534T) using API 50CHB/E and Biotype-100 (bioMérieux) according to the manufacturer's instructions. Strains LMG 5345T, LMG 2781T and LMG 5343T were used as controls to compare results generated in this study to those reported in Bergey's Manual of Systematic Bacteriology (Grimont & Grimont, 2005) to ensure that the data agreed with those available in literature. Strain LMG 24534T was included to provide phenotypic discrimination between the two species in Brenner's DNA HG V. Generally, a good correlation was observed between the two datasets, although some characteristics reported as variable by Grimont & Grimont (2005) were positive or negative for the type strains in our study. A certain level of phenotypic variability within species of the genus Pantoea has been observed previously (Delétoile et al., 2009), and may account for these minor differences. API 50 CHB/E (bioMérieux) and GN2 MicroPlate (Biolog) tests were performed on all Pectobacterium cypripedii strains used in this study, as very little phenotypic information was available for Pectobacterium cypripedii. Relevant phenotypic results are given in the species descriptions. A selection of 50 phenotypic characteristics was used to construct a UPGMA dendrogram (Supplementary Fig. S1, available in IJSEM Online) in BioNumerics 5.0 (Applied Maths) to demonstrate the phenotypic relationship between the strains. The four novel species and Pectobacterium cypripedii clustered in the midst of the ‘core’ Pantoea species, showing them to be phenotypically related but clearly distinct taxa of the genus. The four novel species can be differentiated from each other by their reactions to dulcitol, erythritol, raffinose, l-tartrate and meso-tartrate. Pectobacterium cypripedii shares multiple phenotypic similarities with Pantoea species, including production of glucose dehydrogenase and gluconate dehydrogenase, acid production from l-arabinose, fructose, maltose and trehalose and the lack of H2S and pectinase production. The most distinctive phenotypic characteristics that can differentiate between the four novel species, Pectobacterium cypripedii and their closest phylogenetic relatives are listed in Table 3⇓. The biochemical profile of Pantoea conspicua sp. nov. is currently based on a single strain; as more strains of this species are isolated and tested, the profile may change slightly.

    Table 3.

    Phenotypic characteristics that distinguish the novel members of Pantoea from each other and from their closest phylogenetic neighbours

    Species/subspecies: 1, Pantoea agglomerans; 2, Pantoea ananatis; 3, Pantoea dispersa; 4, Pantoea stewartii subsp. stewartii; 5, Pantoea stewartii subsp. indologenes; 6, Pantoea vagans; 7, Pantoea eucalypti; 8, Pantoea deleyi; 9, Pantoea anthophila; 10, Pantoea cypripedii comb. nov.; 11, Pantoea septica sp. nov.; 12, Pantoea eucrina sp. nov.; 13, Pantoea brenneri sp. nov.; 14, Pantoea conspicua sp. nov. +, 90–100 % strains positive in 1–2 days; (+), 90–100 % strains positive in 1–4 days; d, 11–89 % strains positive in 1–4 days; (d), 11–89 % strains positive in 3–4 days; −, negative; nd, not determined. Data were taken from the following sources: columns 1–5, Grimont & Grimont (2005) and Brady et al. (2009); 6–9, Brady et al. (2009); 10, 14, this study; 11–13, Grimont & Grimont (2005) and this study.

    Conclusions

    DNA HG II, IV and V from the hybridization study performed on the Erwinia herbicola–Enterobacter agglomerans complex by Brenner et al. (1984) are among the last of the original 13 DNA groups to be classified. This is in part due to the difficulties previously experienced in differentiating Pantoea species, both phenotypically and phylogenetically. These difficulties can be overcome by applying MLSA to support data from phenotypic tests and 16S rRNA gene sequencing. The results presented in this study indicate that Pectobacterium cypripedii should be transferred to the genus Pantoea, of which the description should be emended, as Pantoea cypripedii comb. nov. and that Brenner's DNA HG II, IV and V represent four novel species within the genus, Pantoea septica sp. nov. (for HG II), Pantoea eucrina sp. nov. (for HG IV), Pantoea brenneri sp. nov. (for most strains of HG V) and Pantoea conspicua sp. nov. (for strain LMG 24534T from HG V).

    Emended description of the genus Pantoea Gavini et al. 1989 emend. Mergaert et al. 1993

    Pantoea [Pan.to′e.a. Gr. adj. pantoios of all sorts and sources; N.L. fem. n. Pantoea (bacteria) from diverse (geographical and ecological) sources].

    The description is based on the data of Gavini et al. (1989), Mergaert et al. (1993), Grimont & Grimont (2005) and Brady et al. (2009, 2010) and data obtained in this study. Straight rods, 0.5–1.3×1.0–3.0 μm. Non-encapsulated. Non-spore-forming. Some strains form symplasmata. Most strains are motile by means of peritrichous flagella. Gram-negative. Colonies on nutrient agar are smooth, translucent and convex with entire margins or heterogeneous in consistency and adhering to the agar. Colonies are yellow, beige or non-pigmented. Facultatively anaerobic. Optimum growth temperature between 28 and 30 °C. Oxidase-negative. Glucose dehydrogenase and gluconate dehydrogenase are produced and are active without an added cofactor. Lysine and ornithine are not decarboxylated. Urease-negative. Pectate is not degraded. H2S is not produced from thiosulphate. Most strains are Voges–Proskauer-positive and indole-negative. Acid is produced from the fermentation of l-arabinose, d-ribose, d-xylose, d-galactose, d-fructose, l-rhamnose, d-mannitol, N-acetylglucosamine, maltose and trehalose. Carbon sources utilized at 28 °C (Biotype-100) are d-glucose, d-fructose, d-galactose, trehalose, d-mannose, cellobiose, 1-O-methyl β-d-glucopyranoside, l-arabinose, glycerol, inositol, d-saccharate, cis-aconitate, d-glucuronate, d-galacturonate, N-acetylglucosamine, d-gluconate, dl-lactate, l-histidine, l-aspartate, l-glutamate, l-alanine and l-serine. Carbon sources not utilized at 28 °C (Biotype-100) are l-sorbose, palatinose, melezitose, maltitol, turanose, tricarballylate, 4-hydroxybenzoate, gentisate, 3-hydroxybenzoate, benzoate, 3-phenylproprionate, m-coumarate, histamine, caprate, caprylate, glutarate, 5-aminovalerate, ethanolamine, tryptamine, itaconate, 3-hydroxybutyrate, propionate and l-tyrosine. Strains have been isolated from plants, seeds, fruits, soils and water and from humans (urine, blood, wounds, internal organs) and other animals. Strains of several species are phytopathogenic on a wide range of plant and agricultural hosts. The G+C content of the DNA ranges from 52.7 to 60.6 mol%. The type species is Pantoea agglomerans (Ewing and Fife 1972) Gavini et al. 1989.

    Description of Pantoea septica sp. nov.

    Pantoea septica (sep′ti.ca. N.L. fem. adj. septica from Gr. adj. septikos putrefying, decaying or septic, referring to the septicaemia outbreak associated with these strains).

    Displays the following properties in addition to those in the emended genus description. Cells are motile, 0.9×1.5–3.0 μm, occurring singly or in pairs. Colonies are beige, round, convex and smooth with entire margins. Indole is not produced. Phenylalanine deaminase reaction is weakly positive. Acid is produced from the substrates listed in the genus description; some strains can also produce acid from glycerol, inositol, cellobiose and lactose. Carbon sources utilized at 28 °C are as listed in the genus description and d-xylose, l-rhamnose, d-mannitol and d-malate. Some strains can utilize melibiose, sucrose, raffinose, maltotriose, maltose, lactose, lactulose, gentiobiose, l-fucose, d-arabitol, xylitol, dulcitol (weakly), d-sorbitol (weakly), l-tartrate, meso-tartrate and citrate. Carbon sources not utilized at 28 °C are as listed in the genus description and l-arabitol, d-tagatose, adonitol, erythritol, 3-O-methyl d-glucopyranose, d-tartrate, quinate and betaine. The DNA G+C content of the type strain is 59.3 mol%. Strains belonging to this species were implicated in a nationwide septicaemia outbreak in the USA in 1971.

    The type strain is LMG 5345T (=BD 874T =CDC 3123-70T), isolated from a human stool sample in New Jersey, USA.

    Description of Pantoea eucrina sp. nov.

    Pantoea eucrina (eu.cri′na. N.L. fem. adj. eucrina from Gr. adj. eukrines well-separated, referring to the clear separation of the strains from other species within the genus).

    Displays the following properties in addition to those in the emended genus description. Cells are motile, 0.9×1.5–3.0 μm, occurring singly or in pairs. Colonies are beige, round, convex and smooth with entire margins. Indole is not produced. Phenylalanine deaminase reaction is weakly positive. Acid is produced from the substrates listed in the genus description; some strains can also produce acid from inositol, cellobiose and lactose. Carbon sources utilized at 28 °C are as listed in the genus description and sucrose, maltotriose, maltose, d-arabitol, l-arabitol, xylitol, d-mannitol, adonitol and citrate. Some strains can utilize gentiobiose, l-rhamnose and erythritol. Carbon sources not utilized at 28 °C are as listed in the genus description and melibiose, raffinose, lactose, lactulose, d-xylose, l-fucose, dulcitol, d-tagatose, d-sorbitol, 3-O-methyl d-glucopyranose, l-, d- and meso-tartrate, d-malate, quinate and betaine. The DNA G+C content of the type strain is 56.5 mol%. Strains belonging to this species were implicated in a nationwide septicaemia outbreak in the USA in 1971.

    The type strain is LMG 2781T (=BD 872T =CDC 1741-71T =LMG 5346T), isolated from a human trachea in Connecticut, USA.

    Description of Pantoea brenneri sp. nov.

    Pantoea brenneri (bren′ne.ri. N.L. gen. n. brenneri of Brenner, named after Don J. Brenner, in recognition of his contribution in resolving the Erwinia herbicola–Enterobacter agglomerans complex).

    Displays the following properties in addition to those in the emended genus description. Cells are motile, 0.9×1.5–3.0 μm, occurring singly or in pairs. Colonies are beige, round, convex and smooth with entire margins. Indole is not produced. Phenylalanine deaminase reaction is weakly positive. Acid is produced from the substrates listed in the genus description; some strains can also produce acid from glycerol, dulcitol, inositol, d-sorbitol, cellobiose, lactose and raffinose. Carbon sources utilized at 28 °C are as listed in the genus description and sucrose, maltotriose, maltose, d-xylose, l-rhamnose, d-mannitol, meso-tartrate, d-malate and citrate. Some strains can utilize raffinose, lactose (weakly), lactulose, l-fucose, d-arabitol and l-tartrate. Carbon sources not utilized at 28 °C are as listed in the genus description as well as melibiose, gentiobiose, l-arabitol, xylitol, dulcitol, d-tagatose, d-sorbitol, adonitol, erythritol, 3-O-methyl d-glucopyranose, d-tartrate, quinate and betaine. The DNA G+C content of the type strain is 55.4 mol%. Strains belonging to this species were implicated in a nationwide septicaemia outbreak in the USA in 1971.

    The type strain is LMG 5343T (=BD 873T =CDC 3482-71T), isolated from a human urethra in Montana, USA.

    Description of Pantoea conspicua sp. nov.

    Pantoea conspicua (con.spi′cu.a. L. fem. adj. conspicua conspicuous, referring to the conspicuous separation from other strains within DNA HG V).

    Displays the following properties in addition to those in the emended genus description. Cells are motile, 0.9×1.5–3.0 μm, occurring singly or in pairs. Colonies are beige, round, convex and smooth with entire margins. Aerogenic. Indole is not produced. Phenylalanine deaminase reaction is weakly positive. Acid is produced from the substrates listed in the genus description and from dulcitol, inositol, cellobiose and lactose. Carbon sources utilized at 28 °C are as listed in the genus description as well as maltotriose, maltose, lactose, gentiobiose, d-ribose, d-xylose, l-rhamnose, l-fucose, d-arabitol, dulcitol, d-mannitol, l-tartrate, d-malate and citrate. Carbon sources not utilized at 28 °C are as listed in the genus description as well as melibiose, sucrose, raffinose, lactulose, l-arabitol, xylitol, d-tagatose, d-sorbitol, adonitol, erythritol, 3-O-methyl d-glucopyranose, d-tartrate, meso-tartrate, quinate and betaine. The DNA G+C content of the type strain is 55.7 mol%.

    The type strain is LMG 24534T (=BD 805T =CDC 3527-71T), isolated from a human blood sample in Paris, France.

    Description of Pantoea cypripedii (Hori 1911) comb. nov.

    Pantoea cypripedii (cyp.ri.pe′di.i. N.L. n. Cypripedium botanical name for a genus of orchid; N.L. gen. n. cypripedii of cypripedium orchids).

    Basonym: Pectobacterium cypripedii (Hori 1911) Brenner et al. 1973.

    Other synonyms: ‘Bacillus cypripedii’ Hori 1911, ‘Erwinia carotovora var. cypripedii’ (Hori 1911) Dye 1969, Erwinia cypripedii (Hori 1911) Bergey et al. 1923.

    Displays the following properties in addition to those in the emended genus description. Cells are motile, 0.5–1.0×1.0–3.0 μm, occurring singly or in pairs. Colonies are beige, round, convex and smooth with entire margins. Indole is not produced. Phenylalanine deaminase reaction is weakly positive. Acid is produced from the substrates listed in the genus description and from glycerol (weakly), inositol (weakly) and cellobiose. Carbon sources utilized at 28 °C are as listed in the genus description as well as melibiose, sucrose, maltose, lactose, gentiobiose, d-ribose, d-xylose, l-rhamnose, d-arabitol, d-mannitol, d-sorbitol (weakly), d-tartrate, d-malate, citrate and quinate. Carbon sources not utilized at 28 °C are as listed in the genus description as well as raffinose, lactulose, l-fucose, l-arabitol, xylitol, dulcitol, d-tagatose, adonitol, erythritol and betaine. The DNA G+C content is 54.1–54.6 mol%.

    The type strain, LMG 2657T (=ATCC 29267T =DSM 3873T =LMG 2655T), was isolated from a cypripedium orchid in California, USA.

    Acknowledgments

    This study was partially supported by the South African-Flemish Bilateral Agreement, the National Research Foundation (NRF), the Tree Protection Co-operative Programme (TPCP) and the THRIP support programme of the Department of Trade and Industry, South Africa. The BCCM/LMG Bacteria collection is supported by the Federal Public Planning Service-Science Policy, Belgium. The authors wish to acknowledge Katrien Vandemeulebroecke for technical assistance and Mrs Caroline Mohr for providing us with the CDC strains.

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