Rapid Differentiation, by Polyamine Analysis, of Xanthomonas Strains from Phytopathogenic Pseudomonads and Other Members Of

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INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1991, p. 223-228 Vol. 41, No. 2 0020-7713/91/020223-06$02.00/0 Copyright 0 1991, International Union of Microbiological Societies

Rapid Differentiation, by Polyamine Analysis, of Xanthomonas Strains from Phytopathogenic Pseudomonads and Other Members of the Class Proteobacteria Interacting with Plants GEORG AULING,’” HANS-JURGEN BUSSE,’ FRANK PILZ,’ LESLIE WEBB,2 HELMUT KNEIFEL,2 AND DIETER CLAUS’ Institut fur Mikrobiologie der Universitat, 0-3000 Hannover I ,’Institut fur Biotechnologie, Forschungszentrum Jiilich GmbH, D-5170 Julich,2 and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, 0-3300 Bra~nschweig,~Federal Republic of Germany

A total of 58 strains belonging to phylogenetically assigned or unassigned species of the class Proteobacteria, which are mostly phytopathogenic or might interact with plants, were analyzed for polyamines. The strains of the genus Xanthomonas contained spermidine as the main polyamine. Putrescine was the main polyamine of phytopathogenic strains belonging to the Pseudomonas fluorescens complex, which represents the phylogenetically defined nucleus of the genus Pseudomonas. The genera Azotobacter and Azomonas, which include free-living diazotrophic bacteria that also belong to the P. fluorescens complex, had a polyamine pattern like that of the strains belonging to the P.jluorescens complex. Xylophilus ampelinus (formerly called Xanthomonas ampelina) and unassigned phytopathogenic pseudomonads phylogenetically allocated to the beta subclass of the Proteobacteria contained 2-hydroxyputrescine, the specific polyamine of this subclass. The main polyamine in Rhizobium, Bradyrhizobium, and Phyllobacterium strains and misnamed pseudomonads belonging to the alpha subclass of the Proteobacteria was sym-homospermidine,which was also present in “Azotomonas fluorescens.”

The status of the genus Xanthomonas has been controver- teria now grouped in the class Proteobacteria (26). This sial for decades (7, 20). The rRNA-DNA hybridization data might be the reason why polyamines are recommended as of Palleroni et al. (24) and De Vos and De Ley (8) clearly reliable taxonomic markers within the class Proteobacteria established that Xanthomonas is a genus in its own right. On (21). the other hand, members of the genus Pseudomonas are In this paper we describe the polyamine patterns of distributed over the alpha, beta, and gamma subclasses of Xanthomonas strains, phytopathogenic pseudomonads, and the class Proteobacteria (8,23,24,35). Only the members of certain saprophytic pseudomonads that are not identifiable the Pseudomonas Jluorescens complex, which contains the or are easily misidentified when classical methods are used. type species, Pseudomonas aeruginosa, and belongs to the Previously, we have shown that the well-known problem of gamma subclass of the Proteobacteria, are considered to be phenotypic differentiation between Pseudomonas alcali- members of the authentic genus Pseudomonas (8). genes and Comamonas testosteroni (13), which are members As the plant-pathogenic yellow-pigmented xanthomonads of two different subclasses of the Proteobacteria (33, can be occur in specific habitats, their identification has been re- overcome easily by polyamine analysis (4). Other phyloge- garded as an easy task by many phytobacteriologists. How- netically diverse gram-negative bacteria associated with ever, identification of a gram-negative obligately aerobic plants were included as well. In addition, some effects of bacterium with yellow pigmentation from an uncommon medium composition and osmolarity on polyamine concen- source that is not typical for xanthomonads is much more tration or pattern are described. difficult. For example, a yellow-pigmented ligninolytic bacterium isolated by Kern (17) could be assigned to the genus Xanthomonas only after ubiquinone analysis (18). Likewise, MATERIALS AND METHODS strains belonging to the species formerly called Pseudomo- Strains, growth conditions, and media. The strains were nus maltophilia (now included in the genus Xanthomonas obtained from the Deutsche Sammlung von Mikroorganis- [27]) which occur in soil or clinical specimens may be men und Zellkulturen GmbH, Braunschweig, Federal Re- identified either by rRNA-DNA hybridization or by a public of Germany, and the Laboratorium voor Microbiolo- chemotaxonomic analysis in which quinone or fatty acid gie, Ghent, Belgium. Biomass from Azotobacter vinelandii patterns are used (22). The facultatively autotrophic yellow was obtained from C. Fritzsche. Xanthomonas sp. strain 99 bacteria recently placed in the new genus Hydrogenophaga was isolated by H. Kern (18). Xanthomonas sp. strain I Xan (33) cannot be differentiated from Xanthomonas strains by W was isolated as a white mutant from Xanthomonas their quinone systems, as both taxa have the same ubiqui- campestris NRRL-B 1459 by G. Trilsbach. Pseudomonas none (22, 33). As simple reliable tests are still not available aeruginosa 087 and Pseudomonas Jluorescens 0136 were (16, 32) for allocation of bacterial isolates to and within a isolated by L. Webb. phylogenetically defined genus Xanthomonas, rapid chemo- Generally, biomass was grown in liquid cultures on a taxonomic methods may fill the gap. Recently, it was shown reciprocal shaker at 28°C in Erlenmeyer flasks and harvested by Busse and Auling (4) that polyamine patterns reflect the in the late logarithmic phase. The amount of growth was evolutionary diversity of the majority of gram-negative bac- determined by turbidity measurements. A simple fermentor system consisting of a 1.5-liter glass vessel was used for continuous cultivation. Biomass was harvested when upon * Corresponding author. constant dilution a steady state was maintained for 14 h.

223 224 AULING ET AL. INT. J. SYST. BACTERIOL.

TABLE 1. Effects of different growth media on the polyamine patterns of selected strainsn

~ Concn (pmol/g [dry wt]) of Organism or medium Growth conditionsb HPUT DAP PUT CAD SPD HSPD SPM Pseudomonas alcaligenes DSM 50342 TRY Tr 31.2 1.6 2.0 PEP Tr 87.8 4.3 3.1 PY E“ 0.5 47.2 3.6 13.O 0.9 Pseudomonas fragi DSM 3456T TRY Tr 48.3 Tr 8.2 PEP 0.8 82.8 13.8 0.2 Pseudornonas mendocina DSM 50017 TRY 1.0 94.2 6.5 14.9 0.6 PEP 1.2 108.0 1.2 6.8 t PY E‘ 1.3 61.3 1.6 15.9 0.2 Pseudomonas pseudoalcaligenes DSM 50188 TRY 1.8 121.4 20.8 13.6 PEP 0.4 65 .O 0.8 10.4 Tr PY E“ Tr 25.6 5.8 19.8 Xanthomonas campestris DSM 3586 TRY Tr 0.7 Tr 19.9 PEP Tr 0.3 0.3 15.2 1.1 PYE 0.7 2.1 52.3 3.7 Methylobacterium mesophilicum DSM 1708 TRY 3.4 0.3 0.4 25.1 PEP Tr 11.7 4.8 1.3 18.6 0.5 Methylobacterium radiotolerans DSM 1819 TRY 6.7 0.2 0.4 22.1 Tr PEP Tr 10.2 1.2 1.3 23.1 0.2 Methylobacterium rhodinium DSM 2163 TRY 6.1 Tr 0.3 31.8 Tr PEP Tr 9.9 Tr 2.4 34.0 1.0 “Pseudomonas indigofera” DSM 3303 TRY 7.5 20.2 Tr PEP 9.8 41.6 0.5 Tr 0.7 Tr Deleya marina DSM 50416 MB 7.5 PYE + 2.4% NaCl Tr Tr 12.6 0.3 PYE + 2.0% NaCl Tr Tr 13.9 1.4 PYE + 1.5% NaCl 0.5 0.4 18.7 1.5 PYE + 1.0% NaCl 3.0 0.2 26.8 0.8 TRY mediume Tr 0.2 Tr PEP medium 0.4 1.1 0.4 0.4 Abbreviations: HPUT, 2-Hydroxyputrescine; DAP, 1,3-diaminopropane; PUT, putrescine; CAD, cadaverine; SPD, spermidine; HSPD, sym-homospermidine; SPM, spermine; Tr, trace (less than 0.6 pmol of 2-hydroxyputrescine per g, 0.4 bmol of 1,3-diaminopropane per g, 0.3 pmol of putrescine or cadaverine per g, or 0.2 pmol of spermidine, sym-homospermidine, or spermine per g). DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Federal Republic of Germany; LMG, Culture Collection, Laboratorium voor Microbiologie, Rijksuniversiteit, Ghent, Belgium; NCPPB, National Collection of Plant Pathogenic Bacteria, Hatching Green, England; PDDCC, Plant Diseases Division Culture Collection, Auckland, New Zealand. Growth conditions are described in Materials and Methods. Data from reference 4. T = type strain. ‘ Amounts of polyamines per 10 g (dry weight) are given.

Azotobacter chroococcum and Xylophilus ampelinus were below were obtained by using cells from batch cultures. grown on AZO agar or GYCA agar (see below) for 1 week at When the polyamines of a few selected species obtained 25°C. after growth in batch and continuous cultures were ana- The media used had the following compositions: PYE, lyzed, similar concentrations of single polyamines were 0.3% meat peptone and 0.3% yeast extract; PEP, 0.1% meat found (data not shown). These observations indicate that we peptone; TRY, 1.0% Trypton; PYG, 1%peptone, 0.5% yeast used a suitable time of harvesting for cells from batch extract, and 2% glucose; GYCA agar, 0.5% yeast extract, cultures. However, biomass harvested from solid medium 1% glucose, 3% CaCO,, and 2% agar; AZO, 1% glucose, for practical reasons, where cells in different growth states 0.025% CaCO,, and a salt solution (10 ml/liter) containing were mixed, generally contained lower concentrations of 1% CaCl, . 2H,O, 1% MgS0,. 7H20, 0.05% Na,MoO, . polyamines, as we observed for Xylophilus ampelinus and 2H,O, 9% K,HPO,, 1% KH2P0,, and 0.1% FeSO, - 7H,O Azotobacter chroococcurn. (pH 7.3); DP, as described previously (3,but supplemented A cellular component which is proposed for use as a with 0.5% glucose instead of mannitol; MB, 37.4 g of Marine chemotaxonomic marker should be a stable characteristic Broth 2216 (Difco) per liter. that is not affected too much by growth on different media. Quantitative analysis of polyamines. Extraction, analysis, To investigate the effects of different media on the polyamine and quantification of polyamines were carried out as de- patterns, several representative species belonging to the scribed previously by Scherer and Kneifel (25) and Busse authentic genus Pseudomonas as defined by De Vos and De and Auling (4). Ley (8) and the genus Xanthomonas and some misnamed pseudomonads belonging to the beta and alpha subclasses of RESULTS AND DISCUSSION the Proteobacteria were grown on different media (Table 1). No important changes in the polyamine patterns specifically Effect of growth conditions and medium composition on induced by growth on a particular medium were observed. polyamines. Generally, proliferating cells have higher poly- Only spermine seemed not to be present in several species amine concentrations than cells harvested from the station- when the cells were grown in TRY medium, in contrast to ary phase of growth (4, 29). The polyamine data presented the negligible amounts observed in cells which had been VOL.41, 1991 DIFFERENTIATION BY POLYAMINE ANALYSIS 225

TABLE 2. Polyamine patterns of members of the gamma subclass of the Proteobacteria“

~~ Growth Concn (prnol/g [dry wt]) of: Organism’ conditions DAP PUT CAD SPD SPM Azomonas macrocytogenes (“Azotobacter macrocytogenes”) DSM 721T AZO 31.5 1.3 5.5 Azotobacter beijerinckii DSM 37gT AZO Tr 28.4 1.2 2.2 Azotobacter chroococcum DSM 2286T AZO Tr 17.8 0.5 4.5 Azotobacter vinelandii DSM 2289T DP 7.0 42.8 9.2 Deleya marina (“Pseudomonas marina”) DSM 50416 MB 7.5 Deleya aquamarina (“Alcaligenes aquamarinus”) DSM 30101T MB Tr Tr 18.2 Pseudomonas agarici NCPPB 2289 PYE 1.0 66.0 8.2 13.6 2.2 Pseudomonas aeruginosa Webb 087 PEP 1.8 58.0 16.0 2.7 Pseudomonas cichorii NCPPB 943T PYE 2.0 83.3 4.2 19.4 3.5 Pseudomonas citronellolis DSM 50332T PEP 0.3 38.1 4.4 3.2 Pseudomonas Jluorescens Webb 0136 PEP 0.2 22.3 9.3 2.9 Pseudomonas fragi DSM 3456T PEP 0.8 82.8 13.8 0.2 Pseudomonas marginalis PDDCC 3553T PYE 0.8 82.0 0.8 15.2 3.3 Pseudomonas oleovorans DSM 1045T TRY 0.3 36.5 45 .O 6.0 Pseudomonas syringae (‘ ‘Pseudomonas morsprunorum”) DSM 50277 PEP 5.7 2.8 Pseudomonas syringae (“Pseudomonas phaseolicola”) DSM 50282 PEP 11.4 3.1 Tr Pseudomonas syringae DSM 50307 PEP 12.9 2.6 0.3 Pseudomonas tolaasii NCPPB 2192= PYE 0.2 54.5 0.3 17.9 2.9 Pseudomonas sp. strain LMG 2357 (“Pseudomonas viridijava” NCPPB 2024) PY E 1.6 71.7 0.5 13.9 4.5 Xanthomonas albilineans LMG 494T PYG 42.8 0.9 Xanthomonas axonopodis LMG 568(tl)T PY E 1.2 Tr 7.0 3.8 Xanthomonas fragariae LMG 70gT PYE Tr Tr 10.1 4.0 Xanthomonas campestris NCPPB 52gT PY E 0.7 2.1 52.3 3.7 Xanthomonas campestris (“Pseudomonas gardneri”) LMG 962 PYE 3 .O 5.9 55.1 6.1 Xanthomonas campestris (“Xanthornonas pelargonii”) DSM 1350 PEP Tr 0.5 29.1 26.8 1.2 Xanthomonas campestris (“Xanthomonas pelargonii”) DSM 50857 PEP 0.2 0.7 0.2 25.2 1.o Xanthomonas campestris DSM 1049 PEP 0.2 1.2 1.2 37.4 1.2 Xanthomonas campestris DSM 50852 PEP 0.7 0.2 20.6 0.6 Xanthomonas campestris DSM 1050 PEP 0.7 0.7 26.7 1.4 Xanthomonas campestris NRRL-B1459 PEP 0.3 0.2 34.0 1.3 Xanthomonas campestris pv. graminis LMG 726 PY E Tr 41.8 2.3 Xanthomonas maltophilia DSM 50170T PEP 0.2 0.6 37.9 33 .O 1.4 Xanthomonas maltophilia DSM 50173 PEP 54.0 23.6 0.7 Xanthomonas maltophilia (“Pseudomonus betle”) LMG 978 PYE 0.4 7.4 61.4 4.2 Xanthomonas oryzae pv. oryzae LMG 5047T PYE Tr 67.8 6.5 Xanthomonas oryzae pv. oryzicola LMG 797 PYE Tr 57.6 4.1 Xanthomonas sp. strain 99 (Kern) PEP Tr 0.2 38.5 30.0 1.3 Xanthomonas sp. strain I Xan W PEP 0.3 18.0

a For abbreviations see Table 1, footnote a. ’The polyamine patterns of the type strains of Xanthomonas albilineans and Xanthomonas fragariae, not the phylogenetically investigated strains, were analyzed.

grown in PEP or PYE medium. Interestingly, another effect coli (19) and two Vibrio species (36), we investigated the of TRY medium was the large increases in the concentration effect of increasing the sodium chloride concentration on of cadaverine in Pseudomonas pseudoalcaligenes and Pseu- polyamine content in Deleya marina DSM 50416. In this domonas mendocina cells. In the other media tested the species the amount of the main component (spermidine) cadaverine concentration was lower than the concentration increased twofold when the sodium chloride supplement in of each of the residual polyamines detected. Pseudomonas PYE medium was decreased from 2.4 to 1%.However, no alcaligenes, which is closely related to Pseudomonas important changes occurred in the concentrations of the pseudoalcaligenes and Pseudomonas mendocina (35), did other poly amines. not exhibit an enhanced cadaverine concentration when it Differentiation between the genera Xanthomonas and Pseu- was grown in TRY medium. As cadaverine is a product of domonas by polyamine pattern. The polyamine data for the decarboxylation of lysine, it would be interesting to assay phylogenetically determined strains (8, 9, 11, 28, 35) shown these species for differences in either uptake of lysine and in Table 2 clearly demonstrate that the genera Xanthomonas cadaverine or activity of the lysine decarboxylase. Never- and Pseudomonas (phylogenetically defined) could be differ- theless, the intracellular cadaverine concentrations detected entiated merely by their polyamine patterns. Within the in Pseudomonas pseudodalcaligenes and Pseudomonas genus Xanthomonas the common feature was the presence mendocina exceeded by far the amount of cadaverine which of spermidine as a major polyamine in the species Xanth- could be taken up from TRY medium. In the classical omonas albilineans, Xanthomonas axonopodis, Xanthomo- taxonomic literature (15) the three species discussed above nas campestris, Xanthomonas fragariae, Xanthomonas have been reported to be negative for lysine decarboxylase maltophilia, and Xanthomonas oryzae (recently created as a or negative for utilization of lysine (23). new species of this genus) (28). This compound was also a As there have been reports of an inverse relationship major polyamine in the ligninolytic isolate Xanthomonas sp. between polyamine content and osmolarity in Escherichia strain 99, which was previously assigned to the genus 226 AULING ET AL. INT. J. SYST. BACTERIOL.

TABLE 3. Polyamine patterns of species belonging to the alpha and beta subclasses of the Proteobacteriaa

Growth Concn (prnol/g [dry wtl) of Organism conditions HPUT DAP PUT CAD SPD HSPD SPM Alpha subclass “Azotomonas fluorescens” LMG 3027 PYE 6.2 22.9 12.2 Tr Bradyrhizobium japonicum Webb 007 PEP 1.2 0.2 29.3 Bradyrhizobium japonicum Webb 008 PEP 2.0 0.4 26.6 Methylobacterium mesophilicum (“Pseudomonas mesophilica”) DSM 170gT PEP Tr 11.7 4.8 1.3 18.6 0.5 Methylobacterium radiotolerans (“Pseudomonas radiora”) DSM 1819T PEP Tr 10.2 1.2 1.3 23.1 0.2 Methylobacterium rhodinium (“Pseudomonas rhodos”) DSM 2163T PEP Tr 9.9 Tr 2.4 34.0 1.0 Phyllobacterium myrcinacearum LMG 2(tl)T PYE 1.4 24.6 6.9 27.5 0.9 Rhizobium leguminosarurn Webb 080 PEP 1.4 1.3 Tr 15.3 Rhizobium meliloti 2011 WT PYE 7.8 3.4 25.7 24.1 3.6 Rhizobium trifolii Webb 079 PEP 2.0 0.4 25.7 Beta subclass “Pseudomonas andropogonis” NCPPB 934T PYE 16.5 0.2 60.2 12.4 11.2 7.1 “Pseudomonas avenue” (“Pseudomonas alboprecipitans”) NCPPB lollT PYE 13.1 37.0 Tr 1.0 0.5 “Pseudomonas gladioli” (b ‘Pseudomonas marginata”) ATCC 1024gT PYE 33.2 Tr 74.7 1.6 0.7 “Pseudomonas glumae” NCPPB 2981T PYE 34.6 72.7 1.1 1.4 1.7 “Pseudomonas indigofera” DSM 3303T PEP 9.8 41.6 0.5 Tr 0.7 Tr “Pseudomonas pickettii” LMG 5942T PYE 28.9 Tr 67.2 “Pseudomonas pseudoalcaligenes subsp. citrulli” PDDCC 6521 PYE 22.0 Tr 53.6 2.1 3.1 1.5 “Pseudomonas rubrilineans” NCPPB 920T PYE 27.4 59.4 2.1 1.1 “Pseudomonas rubrisubalbicans” NCPPB 1027* PYE 29.6 89.4 0.7 3.8 1.3 “Pseudomonas woodsii” NCPPB 968‘r PYE 15.8 Tr 50.9 13.8 11.8 5.7 Xylophilus ampelinus (Xanthomonas ampelina) NCPPB 2217T GYCA 2.9 26.2 1.0 0.8 For abbreviations see Table 1, footnote a.

Xanthomonas on the basis of nonphylogenetic criteria (18), daceae (12) and belong to the gamma subclass of the and the white mutant derived from Xanthomonas campestris Proteobacteria (11). Both of these species could be clearly NRRL-B 1459. Minor polyamines in these Xanthomonas distinguished from members of the authentic genus Pseudo- species were spermine , putrescine , and 1,3-diaminopropane monas, but not from the genus Xanthomonas, because they (trace amount). The polyamine contents of Xanthomonas had spermidine as the main polyamine and low levels of fragariae and Xanthomonas axonopodis were relatively low. putrescine (Table 2). These two species also cluster together on the basis of Exclusion of misnamed pseudomonads from the authentic phenotypic features (31). genus Pseudomonas by polyamine analysis. The misnamed We showed previously that the members of the authentic Pseudomonas species and the Xylophilus ampelinus (for- genus Pseudomonas have a polyamine pattern with pu- merly Xanthomonas ampelina) strain listed in Table 3 which trescine as the main component and relatively large amounts belong to the beta subclass of the Proteobacteria (9, 10, 34) of spermidine (3, 4). Table 2 shows that other members of had 2-hydroxyputrescine and putrescine as their main com- this genus have the same pattern. The three strains of ponents. Variable amounts of cadaverine, spermidine, and Pseudomonas syringae had significantly lower concentra- spermine and traces of l73-diarninopropane were also de- tions of the single polyamines. Nevertheless, they had the tected. These results are in accordance with the results of typical polyamine pattern of the authentic genus Pseudomo- Busse and Auling (4), who showed that 2-hydroxyputrescine nas. Additional studies will show whether this property can is highly characteristic for species belonging to the beta be exploited for rapid identification of strains belonging to subclass. On the basis of the polyamine pattern alone we this species. Polyamine patterns also indicated the close propose that “Pseudomonas indigofera” DSM 3303 , which relationship between Pseudomonas pseudoalcaligenes and has not been phylogenetically classified yet, is also a mem- Pseudomonas oleovorans (13), as both of these species had ber of the beta subclass. high concentrations of cadaverine when cells were grown in The presence of 2-hydroxyputrescine in Xylophilus am- TRY medium (Tables 1 and 2). Pseudomonas citronellolis pelinus (formerly Xanthomonas ampelina) confirms that this DSM 50332 has not been investigated phylogenetically yet; organism is not a member of the genus Xanthomonas as on the basis of its polyamine pattern (Table 2), we place this shown previously by rRNA-DNA hybridization data (34), strain in the authentic genus Pseudomonas. and justifies its placement in a new genus of the beta The genera Azomonas and Azotobacter cannot be clearly subclass. Therefore, a close relationship between this spe- distinguished from the authentic genus Pseudomonas solely cies and Xanthornonas fragariae and Xanthomonas axonop- on the basis of melting temperature values obtained from odis, as indicated by protein gel electropherograms (31), is rRNA-DNA-hybridizations (8). Representative Azomonas not likely. and Azotobacter strains had putrescine as their main poly- The polyamine approach can also help solve the problem amine compound (Table 2), analogous to the situation in the within the species Pseudomonas pseudoalcaligenes and its genus Pseudomonas, and thus the close phylogenetic rela- two subspecies, Pseudomonas pseudoalcaligenes subsp. tionship is also displayed by the polyamine patterns of these pseudoalcaligenes and Pseudomonas pseudoalcaligenes organisms. subsp. citrulli. While Pseudomonas pseudoalcaligenes Deleya marina (formerly Pseudomonas marina [l])and subsp. pseudoalcaligenes, which contains the type strain of Deleya aquamarina are members of the family Halomona- the species, belongs to the authentic genus Pseudomonas VOL. 41. 1991 DIFFERENTIATION BY POLYAMINE ANALYSIS 227

(i.e., to the gamma subclass of the Proteobacteria [S]), different approaches for identification of xenobiotic-degrading Pseudomonas pseudoalcaligenes subsp. citrulli is a member pseudomonads. Appl. Environ. Microbiol. 551578-1583. of the beta subclass (9) and is closely related to the genus 4. Busse, J., and G. Auling. 1988. Polyamine pattern as a chemo- Comamonas (10). The evolutionary distance between the taxonomic marker within the Proteubucteria. Syst. Appl. Mi- two subspecies is also reflected in the polyamine patterns, crobiol. l l:1-8. 5. Dalton, H., and J. R. Postgate. 1969. Growth and physiology of which are characteristic of the correct phylogenetic taxa. Azotobacter chroococcurn in continuous culture. J. Gen. Micro- The symbiotic N,-fixing bacteria belonging to the genera biol. 56:307-319. Rhizobium and Bradyrhizobium are members of the alpha 6. De Ley, J., W. Mannheim, P. Segers, A. Lievens, M. 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Bacteriol. 33:487-509. strains of the genera mentioned above listed in Table 3 had 9. De Vos, P., M. Goor, M. Gillis, and J. De Ley. 1985. Ribosomal sym-homospermidine as the main polyamine compound and ribonucleic acid cistron similarities of phytopathogenic Pseudo- low, but varying concentrations of the other polyamines. rnonas species. Int. J. Syst. Bacteriol. 35169-184. Only Rhizobium meliloti 2011 WT had spermidine concen- 10. De Vos, P., K. Kersters, E. Falsen, B. Pot, M. Gillis, P. Segers, trations as high as sym-homospermidine concentrations. In and J. De Ley. 1985. Cornamonas Davis and Park 1962 gen. nov. “Azotomonas jiuorescens” the main component was sper- rev. emend., and Cornamonas terrigena Hugh 1962 sp. nom. rev. Int. J. Syst. Bacteriol. 35443453. midine, but significant amounts of sym-homospermidine 11. De Vos, P., A. Van Landschoot, P. Segers, R. Tytgat, M. Gillis, were also present. All of these polyamine patterns are M. Bauwens, R. Rossau, M. Goor, B. Pot, K. Kersters, P. characteristic of many members of the alpha subclass of the Lizzaraga, and J. De Ley. 1989. Genotypic relationships and Proteobacteria (4, 14). taxonomic localization of unclassified Pseudornonas and Pseu- Our data demonstrate the potential of polyamines for use dumonas-like strains by deoxyribonucleic acid-ribosomal ribo- as chemotaxonomic markers for rapid differentiation of the nucleic acid hybridizations. Int. J. Syst. Bacteriol. 39:35-49. genera Pseudomonas and Xanthomonas in the absence of 12. Franzmann, P. D., U. Wehmeyer, and E. Stackebrandt. 1988. reliable phenotypic parameters. The members of the authen- Halomonadaceae fam. nov., a new family of the class Proteo- tic genus Pseudomonas have putrescine as their main com- bacteria to accommodate the genera Halornonas and Deleya. Syst. Appl. Microbiol. 11:16-19. ponent, while the members of the genus Xanthomonas are 13. Gavini, F., B. Holmes, D. Izard, A. Beji, A. 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