Classification and taxonomy of the , with a focus on the genus

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CHAPTER 2 Classification and taxonomy of the Enterobacteriaceae, with a focus on the genus Pantoea

Introduction

The organisms described in the family Enterobacteriaceae are typically facultatively anaerobic, Gram-negative rods and most are motile by means of peritrichous flagella. They grow well at 37 ºC and are oxidase negative and catalase positive, with few exceptions (Brenner and Farmer, 2005). The majority of organisms placed in the Enterobacteriaceae are associated with the digestive tract and human disease, for example, Escherichia, Salmonella and Shigella. However, phytopathogenic bacteria, including Erwinia, Brenneria, Pectobacterium, Dickeya and Pantoea, are also found in this family. In the past 25 years, the number of genera and species within the family Enterobacteriaceae has increased exponentially. When the eighth edition of Bergey’s Manual of Determinative Bacteriology was published in 1974, the Enterobacteriaceae consisted of 12 genera and 36 species (Buchanan and Gibbons, 1974). The latest edition of Bergey’s Manual of Systematic Bacteriology describes 44 genera and 176 species (Brenner and Farmer, 2005). The members of this family can generally be separated into four categories, depending on where they are isolated from: 1) human pathogens, 2) phytopathogens, 3) insect pathogens, symbionts and endosymbionts and environmental, industrial and animals. There are, however, some genera which can overlap several categories (Janda, 2006). One such genus is Pantoea, where the species are primarily known as phytopathogens, but are also regularly isolated from human and clinical samples and from the environment.
The more common enteric species of the family Enterobacteriaceae can be differentiated by phenotypic and biochemical tests, usually with commercialized identification systems. However, infrequently isolated species or environmental strains are more difficult to identify especially if they have an atypical biochemical profile or belong to a rare or novel species. Species belonging to the genus Pantoea are particularly difficult to identify, owing to high phenotypic similarity, a lack of distinguishing characteristics and a somewhat confusing taxonomy. Methods based on genotypic information such as 16S rRNA and protein-encoding gene sequencing, have been employed for differentiation of numerous members of the Enterobacteriaceae. By combining phenotypic and genotypic information in a polyphasic approach, the identification of closely related, or infrequently isolated, enterobacterial species has improved in recent years.
The aim of this review is to examine the current species definition, the techniques used for species delineation and their application in the taxonomic framework of the genus Pantoea.

The Genus Pantoea

The genus Pantoea was formed to accommodate two hydridization groups from the Erwinia herbicola–Enterobacter agglomerans complex that did not correlate with either Erwinia or Enterobacter (Gavini et al., 1989). The earliest reports of bacteria that were later included in the complex were isolated from plants, seeds and fruit and assigned the names Bacterium herbicola aureum (Düggeli, 1904 cited by Graham & Hodgkiss, 1967) and Erwinia lathyri (Manns & Daubenhaus, 1913 cited by Graham & Hodgkiss, 1967). The first recorded isolation of these bacteria from humans occurred in 1928 when strains were isolated from stool samples of patients suffering from typhoid fever and named Bacterium typhi flavum (Dresel & Stickl, 1928 cited by Graham & Hodgkiss, 1967). Another species which later joined the Erwinia herbicola–Enterobacter agglomerans complex was Pseudomonas trifolii, which became Xanthomonas trifolii or Xanthomonas herbicola (Hüss, 1907; James, 1955 cited by Graham & Hodgkiss, 1967). In 1964, it was suggested by Dye that X. trifolii and E. lathyri had similar morphological and biochemical characteristics and should be re-classified as Erwinia herbicola (Dye, 1964). Graham and Hodgkiss (1967) noted the similarities between B. typhi flavum and the chromagenic bacteria E. herbicola, E. lathyri, E. ananas, E. cassavae, E. milletiae and E. uredovora. In 1972, Ewing and Fife compared the “herbicola–lathyri bacteria” with isolates implicated in a nosocomial septicaemia outbreak in the U.S.A. in 1971 and proposed that all of those strains should be incorporated into the genus Enterobacter as Enterobacter agglomerans (Ewing & Fife, 1972). The epithet agglomerans (Beijerinck, 1888) having priority over herbicola and trifolii. The names Erwinia herbicola and Enterobacter agglomerans were both included in the Approved Lists of Bacterial Names, resulting in general confusion regarding the correct taxonomy of these bacteria (Skerman et al., 1980). In the next 16 years, several studies were performed on the Erwinia herbicola–Enterobacter agglomerans complex in attempts to resolve the nomenclature of these strains (Gavini et al., 1983, Mergaert et al., 1983, Brenner et al., 1984, Verdonck et al., 1987, Beji et al., 1988). The most successful of these studies was by Brenner et al. (1984), who performed DNA-DNA hybridization on 124 strains belonging to the Erwinia herbicola–Enterobacter agglomerans complex. Ninety strains were divided into 13 hybridization groups (DNA hybridization groups I to XIII) and the remaining 34 strains did not fall into any group. This study paved the way for the description of several new species.
DNA hybridization group XIII contained strains received as Erwinia herbicola ssp. herbicola, Erwinia lathyri, Erwinia milletiae and Xanthomonas trifolii (Brenner et al. 1984). Type strains and reference strains of these species were later hybridized to the type strain of Enterobacter agglomerans (ATCC 27155^T) and demonstrated more than 90 % DNA homology. Based on this DNA hybridization data, as well as protein electropherograms and phenotypic data, the synonymy of Erwinia herbicola, Erwinia milletiae and Enterobacter agglomerans was proposed (Beji et al., 1988). In agreement with Ewing and Fife (1972), the epithet agglomerans had priority, but the placement of the species in a genus was undecided. A year later, a new genus Pantoea was proposed to contain the species agglomerans which included the synonyms Erwinia herbicola and Erwinia milletiae (Gavini et al., 1989). Also described was a new species, Pantoea dispersa containing strains belonging to DNA hybridization group III from Brenner et al. (1984).
In Japan in 1988, bacterial strains that produce 2,5-diketo-D-gluconic acid (DKGA) were isolated from fruit and soils samples. As these strains shared the general characteristics of the genus Erwinia, they were tentatively named “Erwinia citreus”,
“Erwinia punctata” and “Erwinia terreus” (Sonoyama et al., 1988). After further testing, it was concluded that these DKGA-producing strains belonged to the Erwinia herbicola–Enterobacter agglomerans complex, as they were phenotypically related to DNA hybridization groups II, II and IV of Brenner et al. (1984). Following DNA hybridization and further phenotypic tests, the DKGA-producing strains were described and classified in the genus Pantoea as P. citrea, P. punctata and P. terrea (Kageyama et al., 1992).
A year later it was proposed to transfer Erwinia ananas, Erwinia uredovora and Erwinia stewartii to the genus Pantoea following DNA hybridization and protein profiling (Mergaert et al., 1993). E. ananas and E. uredovora were shown to be subjective synonyms and united as a single species which was classified as Pantoea ananas. Several strains from Brenner’s DNA hybridization group VI were found in the same protein profile groups as Pantoea ananas, resolving another group from the Erwinia herbicola–Enterobacter agglomerans complex. The epithet ananas was later corrected to ananatis in accordance with the International Code of Nomenclature of Bacteria (Trüper and De’ Clari, 1997). Two separate subspecies were created within the species Pantoea stewartii (formerly Erwinia stewartii), P. stewartii subsp. stewartii and P. stewartii subsp. indologenes (Mergaert et al., 1993). These two subspecies shared 60 – 83 % DNA homology but were considerably different in biochemical characteristics and fatty acid composition. Out of the 13 DNA hybridization groups of Brenner et al. (1984), three groups (DNA hybridization groups III, VI and XIII) have been conclusively classified as Pantoea species and four groups (DNA hybridization groups I, II, IV and V) provisionally assigned to the genus Pantoea (Grimont and Grimont, 2005). The remaining six DNA hybridization groups have been assigned to other genera within the Enterobacteriaceae.
Species of Pantoea are generally acknowledged as plant-associated bacteria and are widely distributed in the environment. The type species of the genus, P. agglomerans, has been found to cause crown and root gall disease of beet and gypsophila, leaf blight and bulb rot of onions, seed and boll rot of cotton and leaf blight and vascular wilt of maize and sorghum (Cooksey, 1986; Burr et al., 1991; Edens et al., 2006; Medrano and Bell, 2007; Morales-Valenzuela et al., 2007). P. agglomerans is also associated with human and clinical samples, and is regarded as a rare opportunistic pathogen (Bicudo et al., 2007; De Champs et al., 2000; Fulleron et al., 2007; Kratz et al., 2003; Lim et al., 2006). P. dispersa has been isolated from soil, plant surfaces, seed and humans (Gavini et al., 1989; Schmid et al., 2003). P. citrea and P. punctata have both been isolated from mandarin oranges, and P. citrea is the causal agent of pink disease of pineapple whilst P. terrea is found in soil (Kageyama et al., 1992; Cha et al., 1997). ananatis is the most varied species in the genus, causing a variety of diseases on a diverse range of hosts. This bacterium has been identified as the causal agent of brown rot of pineapple fruitlets and soft rot of sugarcane, brown spot of honeydew melon, postharvest disease of cantaloupe fruit, leaf blight, seed stalk rot and bulb decay of onion, necrotic leaf blotch disease of sudangrass, leaf spot of maize, bacterial blight of Eucalyptus, stem necrosis of rice and brown stalk rot of maize (Serrano, 1928; Wells et al., 1987; Bruton et al., 1991; Gitaitis and Gay, 1997; Azad et al., 2000; Paccola-Meirelles et al., 2001; Coutinho et al., 2002; Cother et al., 2004; Goszczynska et al., 2007). The causal agent of Stewart’s vascular wilt of sweet corn is P. stewartii subsp. stewartii (Stewart, 1897) and P. stewartii subsp. indologenes has been linked with leaf spot of millet (Mergaert et al., 1993) and leaf blotch of sudangrass (Azad et al., 2000).

Species Definitions and Concepts

The past 20 years have seen many taxonomical rearrangements within the family Enterobacteriaceae, as well as an exponential increase in the number of genera and species described. This can be largely attributed to the advances in molecular microbiology, including PCR and sequencing. Species which were previously indiscernible in their phenotype have been shown to be phylogenetically unrelated. As the techniques used for species differentiation and description have improved over time, so the species concept for prokaryotes has developed. The original species definitions based on morphological characteristics have been proven to be inadequate, but improved definitions have been developed based on new information units such as chemotaxonomic markers, DNA properties and rRNA sequences. A prokaryotic species is presently defined as “a category that circumscribes a (preferably) genomically coherent group of individual isolates/strains sharing a high degree of similarity in (many) independent features, comparatively tested under highly standardized conditions” (Rosselló-Mora and Amann, 2001). Practically, a species can currently be defined as “a group of strains, including the type strain, sharing greater than 70 % DNA-DNA relatedness and with 5 °C or less DTm” (Wayne et al., 1987).

Summary 
Acknowledgements 
Contents 
List of abbreviations 
Prepared manuscripts 
1 Introduction
2 Literature Review: Classification and taxonomy of the , with a focus on the genus
2.1 Introduction
2.2 The Genus
2.3 Species Definitions and Concepts
2.4 Phenotypic Information
2.5 Genomic Information
2.6 Conclusions
2.7 References
Chapter 3 Phylogeny and identification of species associated with the environment, humans and plants based on multilocus sequence analysis
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Tables
Figures
Summary
Chapter 4 Pantoea vagens sp. nov., Pantoea eucalypti sp. nov., Pantoea deleyii sp. nov. and Pantoea anthophila sp. nov., four novel species belonging to the genus Pantoea
Introduction
Methods and Discussion
Description of sp. nov.
References
Tables
Figures
Isolation of from showing symptoms of bacterial  blight and dieback in Uruguay
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Table
Figures
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