INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1988, p. 303-315 Vol. 38, No. 3 0020-7713/88/030303-13$02.00/0 Copyright 0 1988, International Union of Microbiological Societies Pedomicrobium americanum sp. nov. and Pedomicrobium australicum sp. nov. from Aquatic Habitats, Pedomicrobium gen. emend. and Pedomicrobium ferrugineum sp. emend. -t RAINER GEBERS” AND MARITA BEESE Institut fur Allgemeine Mikrobiologie, Universitat Kiel, Biologiezentrum, 0-2300 Kiel, Federal Republic of Germany Five new strains of budding bacteria of the genus Pedornicrobiurn were isolated from freshwater habitats. These strains formed two groups, both of which fitted the description of the genus Pedomicrobium, but their characteristics did not match the description of any existing species. Pedornicrobiurn americanum is proposed for strains IFAM G-1381 (= ATCC 43612), IFAM BA-868 (= ATCC 43613), and IFAM BA-869 (= ATCC 43615), with strain IFAM G-1381 as the type strain. Strains IFAM ST-1306 (= ATCC 43611) and IFAM WD-1355 (= 43614) are named Pedomicrobium australicum, with strain IFAM ST-1306 as the type strain. The descriptions of the genus Pedomicrobiurn and of the type species Pedomicrobium ferrugineum are emended. Since the first pedomicrobia were discovered in podzolic with 2 g of starch per liter or 4 g of gelatin per liter. Nitrate soils by Aristovskaya (1, 2), these hyphal, budding bacteria reduction was demonstrated in PSM to which 10 mM KNO, have been observed in other types of soil (18, 19) and in and 1.7 g of Bacto-Agar per liter were added. The presence aquatic habitats (16,17, 21). In 1974, iron-depositing strains of nitrite was detected semiquantitatively with Merckoquant were isolated from podzolic soils in northern Germany, and nitrite test sticks (E. Merck AG, Darmstadt, Federal Repub- a manganese-depositing strain was isolated from a quartzite lic of Germany). Hemolysis was tested on PSM agar supple- rock pool in France (8). Subsequently, these strains were mented with 100 ml of sheep blood per liter. Amino acid identified as Pedomicrobium ferrugineum and Pedomicro- utilization was determined by comparisons of quantitative bium manganicum, and an emended description was given analyses of fresh PSM or PYVM medium and of the same by Gebers (7). Further investigations demonstrated the spent medium after 6 days of growth. Bacterial cells were genetic relatedness between these Pedomicrobium strains removed by centrifugation, and the supernatant was dehy- and their relationship to other genera of hyphal, budding drated in vacuo. The pellets were dissolved in 4 N HCl and bacteria (9-12, 20). In these studies, five isolates from hydrolyzed at 100°C for 16 h. The hydrolysates were dried in aquatic habitats in North America and Australia (12) proved vacuo and were then dissolved in 0.2 N citrate buffer. These to be two new Pedomicrobium species. Their descriptions preparations were then run through an amino acid analyzer and taxonomy are presented here. The two new species also (Multichrom B; Beckman Instruments, Inc., Fullerton, were compared with “Pedomicrobium podsolicum,” which Calif.). All cultures were incubated aerobically in the dark at was described by Aristovskaya in 1963 (2) but was omitted 30°C. from the 1980 Approved Lists of Bacterial Names (23). Stimulation or inhibition of growth by carbon sources and growth dependence on vitamins, sodium chloride, tempera- MATERIALS AND METHODS ture, pH, or buffer were determined in liquid cultures. Strains and cultivation. All of the bacterial strains were Cultures were pregrown in PSM or PYVM medium. In the supplied by the Culture Collection of the Institut fur Allge- logarithmic growth phase, 1 volume of culture was trans- meine Mikrobiologie (IFAM), Kiel, Federal Republic of ferred to 100 volumes of the test medium and incubated. Germany (Tables 1 and 2). The origins, isolation, and Again from cultures in the logarithmic growth phase, cell cultivation of these strains have been described previously suspensions were inoculated 1: 100 (vol/vol) into fresh test (5, 7, 12). PYVM medium for strains IFAM BA-869 and medium. At the end of the log phase of growth, turbidity, IFAM ST-1306* (T = type strain) contained (per liter) 0.25 g flocculation, growth on glass walls, and cell morphology of peptone (Difco Laboratories, Detroit, Mich.), 0.25 g of were noted. The cultures were checked for purity by streak- yeast extract (Difco), 10 ml of a vitamin solution (24), 20 ml ing onto PSM and nutrient agar (Difco). For protein deter- of Hutner basal salts (4),and 10 mM DL-malate; the final pH minations 4.5-ml portions of cell suspensions were hydro- was 7.5. lyzed in 0.1 N NaOH at 60°C for 90 min. Protein Growth characteristics and physiology. Humic gel agar (7) concentrations were determined in triplicate with the Bio- contained (per liter) 5 g (wet weight) of humic gel (i.e., fulvic Rad protein assay (Bio-Rad Laboratories, Munich, Federal acid iron sesquioxide complexes) and 20 g of Bacto-Agar Republic of Germany). The reference protein used was (Difco); the final pH was 7.2 to 7.5. Fe(II1) was detected by albumin. The growth of P. ferrugineum IFAM S-1290T in the Prussian blue reaction. Mn(1V) was identified with the modified PSM (containing 0.1% yeast extract) supplemented benzidine reagent (6). Catalase, cytochrome oxidase, gelatin with various concentrations of acetate, growth in modified liquefaction, and fermentation of glucose were detected as PSM (pH 8.0) supplemented with various concentrations of described by Skerman (22). The growth medium used for HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfon~c these tests was Pedomicrobium standard medium (PSM) (7) acid) buffer (Sigma Chemie, Taufkirchen, Federal Republic solidified with 18 g of Bacto-Agar per liter and supplemented of Germany), and growth in PSM supplemented with 10 mM phenol, 10 mM benzoic acid, or 10 mM toluene were * Corresponding author. followed optically by using a Klett-Summerson colorimeter. ? Dedicated to Peter Hirsch on the occasion of his 60th birthday. The generation times in PSM or PYVM medium were 303 304 GEBERS AND BEESE INT. J. SYST.BACTERIOL. TABLE 1. Levels of DN A-DNA homology among Pedornicrobiurn and selected Hyphornicrobium strains Source of unlabeled DNA" % Homology with labeled DNA from strain:' IFAM ATCC IFAM IFAM IFAM IFAM IFAM Taxon strain no. strain no. S-12907' G-1381T BA-869 ST-13 06T E-1129T P.ferrugineurn S-1290T 33119T 100 16 14 11 14 Q-1197 33117 99' 17 13 5 12 T-1130 33120 97' 18 11 10 9 F-1225 33122 104' 28 14 13 31 P. arnericanurn BA-869 43615 22 82 100 20 6 G-1381T 43612T 100 100 28 8 BA-868 43613 18 91 94 8 P. australicurn ST-1306T 43611* 18 25 26 100 8 WD-1355 43614 17 25 29 87 8 P. rnanganicurn E-1129= 33121T 13' 10 7 3 100 Hyphomicrobium MC-750 27500 7"(4)J 2 (4) (5) 2 (5) T-854 5 2 1 IFAM, Institut fur Allgemeine Mikrobiologie, Kiel, Federal Republic of Germany; ATCC, American Type Culture Collection, Rockville, Md. ' Mean of at least two reactions corrected for the background values obtained with the self-reassociation controls. The self-reassociation values obtained by hybridization with unrelated Escherichia coli K-12 DNA were as follows: strain IFAM S-1290T, 6.3%; strain IFAM G-1381T, 1.7%; strain IFAM BA-869, 1.3%; strain IFAM ST-1306T, 4.7%; strain IFAM E-1129T, 1.7%. Data from reference 11. The homology values in parentheses are the values for reciprocal reactions and are included for comparison (10). determined by following the optical densities of 150-ml If not stated otherwise, chemicals and vitamins were cultures colorimetrically at 620 to 640 nm (Lange, Berlin, obtained from E. Merck AG; calcium panthothenate came Federal Republic of Germany) for 161 h. Generation times from Sigma Chemie. were calculated from semilogarithmic graphs of optical den- Morphology. Cell morphology and fine structure were sity versus time. investigated by transmission electron microscopy, using a Antibiotic susceptibility was tested in liquid PSM or Philips model EM-300 microscope. Pictures were taken with PYVM medium supplemented with 1, 10, or 100 pg of type 4489 electron microscope film (Eastman Kodak Co., antibiotic per ml. Ampicillin, penicillin G, cycloserine, poly- Rochester, N.Y .). Negative staining, platinum-carbon shad- myxin B, neomycin, chloramphenicol, and streptomycin owing, and thin-section preparation were performed as de- were obtained from Serva, Heidelberg, Federal Republic of scribed previously (7, 8). Germany, cephalothin was obtained from Sigma Chemie, DNA-DNA hybridization. The methods used for cell wall and sulfanilamide was obtained from E. Merck AG. Inhibi- disintegration and deoxyribonucleic acid (DNA) extraction tion of growth was calculated from the protein concentra- and purification have been described previously (12). Shear- tions of triplicate 10-ml test cultures in relation to the protein ing of DNA and radioactive labeling were carried out as concentrations of cultures in the same medium without described by Gebers et al. (10, 11). DNA reassociation inhibitors. procedures and S1 nuclease treatment were performed as TABLE 2. Levels of DNA-DNA homology of P. ferrugineurn IFAM S-1290T with other hyphal, budding bacteria Source of unlabeled DNA" % Homology with labeled DNA from Taxon IFAM ATCC strain no. strain no. strain IFAM S-1290Tb ~~ P. ferrugineurn S-1290T 33119T 100 Hyphomicrobium spp. NQ-521gr 27483 6 CO-582 27492 5 1-551 27489 4 EA-617 4 WH-563 3 MEV-5 33gr 27488 3 KB-677 27498 3 CO-558 27491 3 zv-580 2 SW-808 0 "Genus F" SCH-1315 6 "Genus T" 1300 4 ST-1307 3 Hyphornonas polyrnorpha PS-72gT 33881T 3 Hyphomonas oceanitis SCH-1325T 33879T 3 Hyphornonas sp. SW-814 2 Hyphomonas neptunium LE-670T 15444T 0 Rhodornicrobiurn vannielii DSM 162T 117gT 17100T 0 DSM, Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany.