Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1988, p. 303-315 0020-7713/88/030303-13$02.00/0 Copyright 0 1988, International Union of Microbiological Societies Vol. 38, No. 3 Pedomicrobium americanum sp. nov. and Pedomicrobium australicum sp. nov. from Aquatic Habitats, Pedomicrobium gen. emend. and Pedomicrobium ferrugineum sp. emend. -t RAINER GEBERS” A N D 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 soils by Aristovskaya (1, 2), these hyphal, budding bacteria have been observed in other types of soil (18, 19) and in aquatic habitats (16,17, 21). In 1974, iron-depositing strains were isolated from podzolic soils in northern Germany, and a manganese-depositing strain was isolated from a quartzite rock pool in France (8). Subsequently, these strains were identified as Pedomicrobium ferrugineum and Pedomicrobium manganicum, and an emended description was given by Gebers (7). Further investigations demonstrated the genetic relatedness between these Pedomicrobium strains and their relationship to other genera of hyphal, budding bacteria (9-12, 20). In these studies, five isolates from aquatic habitats in North America and Australia (12) proved to be two new Pedomicrobium species. Their descriptions and taxonomy are presented here. The two new species also were compared with “Pedomicrobium podsolicum,” which was described by Aristovskaya in 1963 (2) but was omitted from the 1980 Approved Lists of Bacterial Names (23). MATERIALS AND METHODS Strains and cultivation. All of the bacterial strains were supplied by the Culture Collection of the Institut fur Allgemeine Mikrobiologie (IFAM), Kiel, Federal Republic of Germany (Tables 1 and 2). The origins, isolation, and cultivation of these strains have been described previously (5, 7, 12). PYVM medium for strains IFAM BA-869 and IFAM ST-1306* (T = type strain) contained (per liter) 0.25 g of peptone (Difco Laboratories, Detroit, Mich.), 0.25 g of yeast extract (Difco), 10 ml of a vitamin solution (24), 20 ml of Hutner basal salts (4),and 10 mM DL-malate; the final pH was 7.5. Growth characteristics and physiology. Humic gel agar (7) contained (per liter) 5 g (wet weight) of humic gel (i.e., fulvic acid iron sesquioxide complexes) and 20 g of Bacto-Agar (Difco); the final pH was 7.2 to 7.5. Fe(II1) was detected by the Prussian blue reaction. Mn(1V) was identified with the benzidine reagent (6). Catalase, cytochrome oxidase, gelatin liquefaction, and fermentation of glucose were detected as described by Skerman (22). The growth medium used for these tests was Pedomicrobium standard medium (PSM) (7) solidified with 18 g of Bacto-Agar per liter and supplemented * Corresponding author. ? Dedicated to Peter Hirsch on the occasion of his 60th birthday. with 2 g of starch per liter or 4 g of gelatin per liter. Nitrate reduction was demonstrated in PSM to which 10 mM KNO, and 1.7 g of Bacto-Agar per liter were added. The presence of nitrite was detected semiquantitatively with Merckoquant nitrite test sticks (E. Merck AG, Darmstadt, Federal Republic of Germany). Hemolysis was tested on PSM agar supplemented with 100 ml of sheep blood per liter. Amino acid utilization was determined by comparisons of quantitative analyses of fresh PSM or PYVM medium and of the same spent medium after 6 days of growth. Bacterial cells were removed by centrifugation, and the supernatant was dehydrated in vacuo. The pellets were dissolved in 4 N HCl and hydrolyzed at 100°C for 16 h. The hydrolysates were dried in vacuo and were then dissolved in 0.2 N citrate buffer. These preparations were then run through an amino acid analyzer (Multichrom B; Beckman Instruments, Inc., Fullerton, Calif.). All cultures were incubated aerobically in the dark at 30°C. Stimulation or inhibition of growth by carbon sources and growth dependence on vitamins, sodium chloride, temperature, pH, or buffer were determined in liquid cultures. Cultures were pregrown in PSM or PYVM medium. In the logarithmic growth phase, 1 volume of culture was transferred to 100 volumes of the test medium and incubated. Again from cultures in the logarithmic growth phase, cell suspensions were inoculated 1:100 (vol/vol) into fresh test medium. At the end of the log phase of growth, turbidity, flocculation, growth on glass walls, and cell morphology were noted. The cultures were checked for purity by streaking onto PSM and nutrient agar (Difco). For protein determinations 4.5-ml portions of cell suspensions were hydrolyzed in 0.1 N NaOH at 60°C for 90 min. Protein concentrations were determined in triplicate with the BioRad protein assay (Bio-Rad Laboratories, Munich, Federal Republic of Germany). The reference protein used was albumin. The growth of P. ferrugineum IFAM S-1290T in modified PSM (containing 0.1% yeast extract) supplemented with various concentrations of acetate, growth in modified PSM (pH 8.0) supplemented with various concentrations of HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfon~c acid) buffer (Sigma Chemie, Taufkirchen, Federal Republic of Germany), and growth in PSM supplemented with 10 mM phenol, 10 mM benzoic acid, or 10 mM toluene were followed optically by using a Klett-Summerson colorimeter. The generation times in PSM or PYVM medium were 303 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 304 INT. J. SYST.BACTERIOL. GEBERS AND BEESE TABLE 1. Levels of DN A-DNA homology among Pedornicrobiurn and selected Hyphornicrobium strains % Homology with labeled DNA from strain:' Source of unlabeled DNA" Taxon P . ferrugineurn P. arnericanurn P . australicurn P. rnanganicurn Hyphomicrobium IFAM strain no. ATCC strain no. IFAM S-12907' IFAM G-1381T IFAM BA-869 S-1290T Q-1197 T-1130 F-1225 BA-869 G-1381T BA-868 ST-1306T WD-1355 E-1129= MC-750 T-854 33119T 33117 33120 33122 43615 43612T 43613 43611* 43614 33121T 27500 100 99' 97' 104' 22 16 17 18 28 82 100 91 25 25 10 14 13 11 14 100 100 94 26 29 7 2 (4) 2 18 18 17 13' 7"(4)J 5 IFAM ST-1306T IFAM E-1129T 14 12 9 31 6 8 8 8 8 100 2 (5) 1 11 5 10 13 20 28 100 87 3 (5) 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 cultures colorimetrically at 620 to 640 nm (Lange, Berlin, Federal Republic of Germany) for 161 h. Generation times were calculated from semilogarithmic graphs of optical density versus time. Antibiotic susceptibility was tested in liquid PSM or PYVM medium supplemented with 1, 10, or 100 pg of antibiotic per ml. Ampicillin, penicillin G, cycloserine, polymyxin B, neomycin, chloramphenicol, and streptomycin were obtained from Serva, Heidelberg, Federal Republic of Germany, cephalothin was obtained from Sigma Chemie, and sulfanilamide was obtained from E. Merck AG. Inhibition of growth was calculated from the protein concentrations of triplicate 10-ml test cultures in relation to the protein concentrations of cultures in the same medium without inhibitors. If not stated otherwise, chemicals and vitamins were obtained from E. Merck AG; calcium panthothenate came from Sigma Chemie. Morphology. Cell morphology and fine structure were investigated by transmission electron microscopy, using a Philips model EM-300 microscope. Pictures were taken with type 4489 electron microscope film (Eastman Kodak Co., Rochester, N.Y .). Negative staining, platinum-carbon shadowing, and thin-section preparation were performed as described previously (7, 8). DNA-DNA hybridization. The methods used for cell wall disintegration and deoxyribonucleic acid (DNA) extraction and purification have been described previously (12). Shearing of DNA and radioactive labeling were carried out as described by Gebers et al. (10, 11). DNA reassociation procedures and S1 nuclease treatment were performed as TABLE 2. Levels of DNA-DNA homology of P. ferrugineurn IFAM S-1290Twith other hyphal, budding bacteria Source of unlabeled DNA" Taxon IFAM strain no. ATCC strain no. S-1290T NQ-521gr CO-582 1-551 EA-617 WH-563 MEV-533gr KB-677 CO-558 zv-580 SW-808 SCH-1315 1300 ST-1307 PS-72gT SCH-1325T SW-814 LE-670T 117gT 33119T 27483 27492 27489 % Homology with labeled DNA from strain IFAM S-1290Tb ~~ P . ferrugineurn Hyphomicrobium spp. "Genus F" "Genus T" Hyphornonas polyrnorpha Hyphomonas oceanitis Hyphornonas sp. Hyphomonas neptunium Rhodornicrobiurn vannielii DSM 162T 27488 27498 27491 100 6 5 4 4 3 3 3 3 2 0 33881T 33879T 15444T 17100T 6 4 3 3 3 2 0 0 DSM, Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany. Mean of at least two reactions corrected for the background values obtained with the self-reassociation controls. The self-reassociation value obtained by hybridization with unrelated E. coli K-12 DNA was 6.3%. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 VOL.38, 1988 PEDOMICROBIUM SPP. 305 TABLE 3. Conditions used for DNA-DNA hybridization reactions Labeled DNA from strain: IFAM IFAM IFAM IFAM IFAM S-1290T G-1381T BA-869 ST-1306T E-1129T T,, temp in o.17 NaCl (oc) Radioactive 95.5 95.0 95.1 95.4 95.2 3H 'I '*'I *''I '1 Sp act after labeling (cp;~~,) of Sp act for Amt of labeled hybridization DNA per (cpm/pg of hybridization DNA) reaction (pg) Ratio of labeled DNA to unlabeled DNA 19,348 2.40 X lo6 7.44 x lo6 2.99 x lo6 2.73 x lo6 19,348 0.12 or 0.19 2.40 X lo6 0.10 0.77 x lo6 0.10 2.99 x lo6 0.10 0.79 x lo6 0.10 1:1,250 or 1:790 1:1,500 1:1,500 1:1,500 1:1,500 HL:g- Iwa&a;gn temp (u) rC) 65 70 70 10 70 ~~~1~~~ 16 20 20 20 20 1,250' 100d 50d 100" SO@ Incubation Filter temp K) type 51 56 56 56 56 GFIC' BA83' BA83' BA83' BA83' Amersham Buchler, Braunschweig, Federal Republic of Germany. Miles Laboratories, Inc., Elkhart, Ind. Whatman, Maidstone, Kent, United Kingdom. Bethesda Research Laboratories, Karlsruhe, Federal Republic of Germany Schleicher & Schiill, Dassel, Federal Republic of Germany. Sigma Chernie, Taufkirchen, Federal Republic of Germany. described previously (10). The specific conditions used for hybridization reactions are shown in Table 3. same time. Mature buds either separate from the hyphae as uniflagellated swarmers or remain attached. Buds may arise in an intercalary fashion by localized hyphal swelling. Occasionally, direct budding of mother cells or division of single mother cells is observed (1, 3). (ii) Cellular structure and composition. Cells stain gram negative, although older cells may stain gram variable. Extracellular polymers stain with ruthenium red. Oxidized iron compounds or manganese compounds or both are deposited primarily on mother cells and also on hyphae. Intracytoplasmic membranes are observed in cells of manganese-depositing species. Up to three granules of poly-phydroxybutyric acid are stored per cell. Small, dense granules stainable with Loefffer methylene blue suggest the presence of polyphosphates. Liquid cultures grown in the dark and in synthetic light may be yellowish white and reddish orange, respectively. (iii) Colonial morphology. Two colony types may develop on solid media. Type 1 colonies are round and convex and have entire or rhizoid edges; they are yellowish red to dark brown, sometimes with concentric rings. These colonies have a soft consistency. Type 2 colonies are round and flat; they pit the agar so that their surfaces remain near the agar level. Their edges are entire or rhizoid. Type 2 colonies are yellowish red to dark brown, and the cells in the center of the colonies are often lysed, giving a granular appearance to the colonies. These colonies have a cartilaginous consistency and may be removed from the agar intact. After restreaking on solid media, type 1 or 2 colonies give rise to both types of colonies. With periodic transfers to fresh medium type 1 colonies tend to predominate. RESULTS AND DISCUSSION The description of the genus Pedomicrobium (7) was based on six strains of P . ferrugineum and one strain of P . manganicum. Five new strains from aquatic habitats exhibit the major characteristics of the genus; i.e., the cells form threadlike outgrowths (hyphae), multiply by budding, and produce extracellular polymers that serve as matrices for deposition of iron oxides or manganese oxides or both. However, these strains are unlike the previously described species P . ferrugineum and P , manganicum, as well as " P . podsolicum" (2, 3 ) and "P.manganicum subsp. sacchalinicum" (18, 19). Our data (see below) allow emendation of the genus description, an emended description of the type species, P . ferrugineum, and descriptions of two new species. Pedomicrobium Aristovskaya 1961 (Gebers 1981) genus emend. (i) Morphology. Cells are spherical, oval, tetrahedral, or rod, pear, bean, or spindle shaped. Up to five or more prosthecae (i.e., cellular outgrowths [hyphae] of constant diameter) are formed per cell; these are 0.15 to 0.3 pm in diameter and vary in length with cultural conditions. At least one hypha originates laterally; other hyphae may appear polarly or subpolarly. True branching of the hyphae occurs. Multiplication is primarily by budding at the hyphal tips, where local swellings form young buds which elongate perpendicularly to the hyphal axis and enlarge up to the size of a mother cell. Hyphal insertion of the buds is lateral in all cases. Up to three buds per mother cell may occur at the TABLE 4. Growth in PYVM medium or in PSM with or without buffers added" Buffer added to broth shift PH None 10 mM HEPES + NaOH 35 mM Tris + HCl' None 6.618.7 7.017.5 7.017.7 7.518.5 P . austrcllicum strains P . nmerictinum strains P . ferrugineum IFAM s-1290T IFAM G-1381T Relative growth Relative growth (%) (%I 100 71 52 67 100 44 57 100 6.618.8 1.017.5 7.018.0 7.518.9 IFAM BA-868 gt: 6.118.5 1.017.2 6.817.0 7.518.5 Relative growth (%I 100 18 3 52 IFAM BA-869h gt: Relative growth IFAM ST-130bTh PH shift 97 100 49 90 IFAM WD-1355 i:t Relative gr,ooth 6.718.3 7.017.1 6.817.0 7.5J8.5 50 8 2 100 (%I (%I 6.918.8 6.818.6 6.917.4 7.619.0 Relative growth 6.918.9 6.818.3 6.917.1 7.619.0 loo 53 23 97 Growth was calculated individually for each strain relative to the culture yielding maximum protein (= 100%). pH shift, pH of medium shortly after inoculation/pH after 6 to 14 days. Strains IFAM BA-869 and IFAM ST-13MT were grown in PYVM medium. Tris, Tris( hydrox ymet hy1)aminomethane. (I Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 306 INT. GEBERS AND BEESE J. SYST. BACTERIOL. 1 1501 h .-cnc c 3 -Y b c Q Y L. . I 2 =8 I 8 *- b % 260 5' 0 m 40 I 0 300 1 600 1 50 I 0 time(h) 0 I 5' 0 I I 100 200 I time (h) FIG. 1. Growth of P . ferrugineurn IFAM S-1290T in modified PSM (pH 8) supplemented with HEPES buffer. Symbols: A, no HEPES added, pH 8.7 at the end of growth; A,50 mM HEPES, pH 8.5; B, 100 mM HEPES, pH 7.9. 0, 200 mM HEPES, pH 7.9. (iv) Physiological properties. Cells are microaerophilic to aerobic and heterotrophic. Slow, poor growth occurs on 0.1 to 1%fulvic acid iron sesquioxide complexes as sole carbon and nitrogen sources; good growth occurs with 10 mM acetate, 10 mM malate, 10 mM succinate, or 10 mM gluconate as the carbon source and a nitrogen source such as FIG. 2. Growth of P . ferrugineum IFAM S-1290T in PSM supplemented with aromatic compounds. Symbols: x , no supplement, pH 8.7 at the end of growth; 0 , l O mM phenol added; pH 8.6; 0 , l O mM benzoic acid, pH 8.9; B, 10 mM toluene, pH 8.8. yeast extract, peptone, Casamino Acids (Difco), or Soytone (Difco) at a concentration of 0.05%. Vitamin mixtures stimulate growth; lack of vitamins produces pleomorphic cells. Good growth occurs in the presence of 0.1% NaC1; no growth, but survival, occurs in the presence of 1or 2.5% NaCl; 5% NaCl is bactericidal for the species that have been described (7). Gelatin and starch are not hydrolyzed. Neither acid nor gas is produced anaerobically from glucose. Anaerobic growth is not observed in stab cultures. Catalase is pro- TABLE 5. Growth on carbon sources" Growth index for: Carbon source (10 mM)' P . umericunum strains P. ferrugineurn IFAM S-1290T IFAM G-1381' IFAM BA-868 21 100 24 100 17 100 15 Oi 16 30 33 13 43 7i 21 102 36 17 12i 29 41 8i 28 P . uustrulicum strains IFAM BA-869 IFAM ST-1306T IFAM WD-1355 49 100 1% 42i 44i 51 109 56 63 20i 74 156 89 34 NT 1Oi 32i 52 NT 6 100 24 Oi 18 15 25 4 Oi 7 23 30 4 12 Oi 11 56 2i 18 ~ ControlC Acetate Caproate Citrate Ethanol Formate Gluconate D-Glucose Glycerol D-Lactate Lactose DL-Malate D-Mannitol Methanol Phenol Propanol Pyruvate D-Ribose Succinate a 44 3i 1Oi 14i 18 1li Oi 8i 15i 22 24 19 Oi 8i 70 lli 37 5id Oi 5i 30 35 6i 30 20 7i 23 21 21 1Oi 8i 60 5i 66 61 100 14i 92 44i 75 84 4% 69 23i 41i 121 52i 37i NT' 58 39i 48i 108 A growth index was calculated from protein measurements individually for each strain as a percentage of the growth in medium to which acetate was added. ' Carbon sources were added to PYVM medium (for strains IFAM BA-869 and IFAM ST-13MT) or to PSM without acetate (for strains IFAM S-12!NT, IFAM G-1381T, IFAM WD-1355, and IFAM BA-868). No additional carbon source was added to the basic media. i, Inhibition of growth compared with control. ' NT, Not tested. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 VOL. 38, 1988 PEDOMICROBIUM SPP. 307 TABLE 6. Utilization of amino acids" % Remaining after 6 days of growth Amino acid DL-Alanine Ammonia L-Arginine L-Aspartic acid L-Glutamic acid Glycine L-Histidine L-Isoleucine L-Leucine L-Lysine L-Pheny lalanine L-Proline L-Serine L-Threonine L-Tyrosine D-Valine P . americanum strains P . austrulicum strains P. ferrugineum IFAM S-1290T IFAM G-1381T IFAM BA-868 IFAM BA-869 IFAM ST-1306T IFAM WD-1355 18 62 50' 36 24 46 44 22 18 54 27 56 35 39 55 13 14 57 26' 34 21 47 40 19 17 50 14 99 32 38 41 10 21 67 37' 36 25 52 71 22 20 51 22 42 36 41 78' 13 64 68 62 82 32 49 22 42 82 75 86 36 72 57 57 ND 56 23 67 84' 42 45 47 75 106 88 102 92 65 73 71 56 91 55 69 56 49 19 28 29 37 55 30 76 59 53 ND' 25 Avg 35 62 58 39 36 39 50 47 43 66 37 68 49 50 58 35 " Quantitative amino acid analysis of fresh PSM or PYVM medium (strains IFAM BA-869 and IFAM ST-1306T) compared with the same spent medium. The amino acid concentrations in the fresh media were taken t o be 100%. Approximate values. ND, Not determined. TABLE 7. Inhibition of growth by antibiotics ~~ % Inhibition of growth of" Antibiotic Ampicillin Penicillin G Cephalothin Cycloserine Polymyxin B Neom y cin Chloramphenicol Streptomycin Sulfanilamide Concn (&ml) 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100 P. ferrugineum IFAM S-1290T P . uustrulicum strains P . americunum strains IFAM G-1381T IFAM BA-868 IFAM BA-869 IFAM ST-1306' IFAM WD-1355 19 91 95 0 0 93 0 0 77 16 89 95 80 17 74 91 10 59 90 19 45 26 43 91 23 98 97 0 41 59 59 92 33 0 17 94 18 + + + 3 78 93 73 73 97 4 60 68 63 90 96 75 61 91 91 69 78 95 76 75 84 71 77 95 55 89 82 81 84 74 83 79 85 76 86 85 84 43 90 92 84 88 84 85 82 74 94 98 73 69 77 90 84 5 47 88 78 83 80 85 83 76 78 98 84 77 96 95 95 81 85 82 85 82 5 3 16 0 0 3 16 20 22 48 49 47 13 13 8 15 19 33 22 65 82 3 82 87 18 81 +b 12 73 82 5 82 + + + + + + + + + + + + + + + " Inhibition was evaluated by comparing the protein concentrations in cultures grown in the presence of antibiotics with the concentrations in untreated cultures. Higher concentrations of cephalothin, polymyxin B, and streptomycin caused a yellow color reaction during protein hydrolysis with 0.1 N NaOH. Inhibition was estimated by visual comparison of culture turbidity. +, Strong inhibition (approximately 60 to 100%). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 308 INT. J. SYST.BACTERLOL. GEBERS AND BEESE FIG. 3. Electron micrograph of p . americanum IFAM G-1381* negatively stained with 0.5% phosphotungstic acid: oval-shaped imother cell with lateral hypha and bud. The arrows indicate extracellular polymers incrusted with iron oxides or manganese oxides or both. Bar = 1 bm. FIG. 5 . Electron micrograph of P. arnericanurn IFAM G-1381T negatively stained with 0.5% phosphotungstic acid: mother cell with polar hypha and bud. Note beginning outgrowth of second, subpolar hypha (arrow). Bar = 1 wn. duced. On PSM agar supplemented with sheep blood (100 mlAiter), all strains grow well but are not hemolytic. (v) Occurrence. These organisms are widely distributed in podzolic and other soils, freshwater habitats, iron springs, and seawater; they are probably ubiquitous. The DNA base composition (four species, 11 strains) ranges from 63 to 66 mol% guanine plus cytosine (G+C) as determined by the thermal denaturation (T,) method. The levels of DNA-DNA homology of P . ferrugineum IFAM S-1290Tto representative strains of other genera of hyphal, budding bacteria are less than 7% (Table 2). The type species is P . ferrugineum Aristovskaya 1961. P . ferrugineum Aristovskaya 1961 emend. Cells are spherical, oval, tetrahedral, or rod or bean shaped and are 0.6 to 2 by 0.6 to 2.5 pm. At least one hypha originates laterally; less frequently, other hyphae appear polarly or subpolarly. Multiplication is by budding at the tips of the hyphae which are 0.2 pm or less in diameter. Initially, buds are spherical to oval; they are motile by a single polar to subpolar flagellum. Occasionally, intercalary buds are observed. Deposition of oxidized iron compounds occurs when cells are grown with fulvic acid iron sesquioxide complexes, elemental iron powder, FeS, or iron paper clips; there is no accumulation of manganese compounds. Depositions occur primarily on mother cells and later on hyphae. Intracytoplasrnic membranes have not been found in ultrathin sections of strain IFAM S-1290T.The temperature for optimum growth is 29 to 30°C; the temperature range is 18 to 43°C. The pH range for growth is 6.6 to 8.7 (Table 4). P. ferrugineum IFAM S-1290T grows in 50 mM HEPES-buffered PSM (Fig. 1); however, 100 and 200 mM HEPES prevent growth. Growth in unbuffered PSM at pH 6.6 gives higher protein yields than growth in PSM buffered with 10 mM HEPES or 35 rnM tris(hydroxymethy1)aminomethane hydrochloride (Table 4). Growth in PSM containing 10 mM HEPES is best at an initial pH of 8.1. During growth, the pH of all cultures increases. The generation time of strain IFAM S-1290T in PSM is 10 h at 30°C. The concentration of acetate in modified PSM (containing 0.1% yeast extract) influences the generation time and maximum optical density of P. ferrugineum cultures; 20 mM acetate results in a minimum generation time of 15.5 h and an optical density of 210 Klett units. With 30 mM acetate, the generation time is 20.5 h, and a value of 240 Klett units is reached; however, cells of the latter cultures produce large granules of poly-P-hydroxybutyric acid. FIG. 4. Electron micrograph of P. americanum IFAM BA-869 negatively stained with 0.5% phosphotungstic acid: tetrahedral mother cell with branching hypha. Bar = 1 pm. FIG. 6. Electron micrograph of P. arnericanurn IFAM G-1381T negatively stained with 0.5% phosphotungstic acid: separated daughter cell in the flagellated swarmer stage. Note the subpolarly inserted flagellum and the separation nose (arrow), where the former mother cell hypha was attached. Bar = 1 pm. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 VOL. 38, 1988 PEDOMICROBIUM SPP. 309 FIG. 7. Thin section of P.arnericanurn IFAM G-1381T. Note the large granule of poly-P-hydroxybutyric acid (PHB) and the extracellular polymers incrusted with iron oxides or manganese oxides or both (arrow), as well as the section through the branching hypha. Bar = 0.5 pm. Good growth occurs with 10 mM fumarate or 10 mM glutamate. Slow, poor growth occurs with 10 mM oxalate, 10 mM tartrate, 10 mM cholesterol, 10 mM aspartate, 10 mM tryptophan, 10 mM alanine, or 0.3% paraffin. No growth occurs with 10 mM galactose, 10 mM fructose, 10 mM maltose, 10 mM sucrose, 10 mM phenylalanine, 10 mM serine, or 10 mM lysine (7). The utilization of other substrates is shown in Table 5. Amino acids are utilized simultaneously as nitrogen sources (7). The growth of strain IFAM S-1290Tin PSM supplemented with 10 mM phenol, 10 mM benzoic acid, or 10 mM toluene was monitored by determining optical density values. In these cultures, the lag phase was increased compared with cultures in pure PSM (Fig. 2). Benzoic acid and toluene reduced the growth of strain IFAM S-1290T only to a small extent, whereas phenol caused a strong inhibition. A second transfer to PSM supplemented with 10 mM phenol completely prevented growth (Table 5 ) . Good growth occurs with 0.05% yeast extract or 0.05% peptone as a nitrogen source. Within 6 days, strain IFAM S-1290T removes 44 to 87% of the amino acids from PSM culture broth (Table 6). Slow, poor growth occurs with 0.05% Soytone, 0.05% Casamino Acids, 10 mM 2-aminobenzoic acid, 10 mM NaNO,, 10 mM acetamide, 10 mM urea, 10 mM N-acetylglucosamine, or 10 mM methylamine hydrochloride; and no growth occurs with 0.01 M NaNO,, 0.58 M (4%) NaNO,, or 1.06 M (9%) NaNO, (7). A vitamin mixture (24) or 1 pg of cyanocobalamin per liter is required for good growth. The presence of 0.1% NaCl stimulates growth. The cells are positive for cytochrome oxidase and for reduction of nitrate to nitrite. FIG. 8. Electron micrograph of P . australicitrn IFAM ST-1306=, Pt-C shadowed: oval mother cell with polar hypha and spindleshaped bud. Bar = 5 pm. FIG. 9. Electron micrograph of P . austraficitm IFAM WD-1355, Pt-C shadowed: spindle-shaped mother cell with lateral hypha and bud. Bar = 1 pm. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 310 INT. J . SYST.BACTERIOL. GEBERS AND BEESE FIG. 10. Electron micrograph of P. australicum IFAM ST-1306T negatively stained with 0.5% phosphotungstic acid: mother cell with one lateral b p h a and one polar hypha. Note the terminal swelling of the polar hypha. Bar = 1 pm. FIG, 12. Electron micrograph of P . australicum IFAM ST-1306T negatively stained with 0.5% phosphotungstic acid: mother cell with hypha branching twice and with three buds at different developmental stages. Bar = 1 pm. P.ferrugineum strains are not pathogenic for guinea pigs after intraperitoneal injection of lo8 cells (7). In addition to antibiotic susceptibilities in liquid cultures (Table 7), cells of strain IFAM S-1290T are susceptible in agar diffusion tests (7) to rifampin, tetracycline, gentamicin, and nitrofurazone, The DNA base composition of the type strain is 65 mol% G+C (TnI method) (7, 9). The average DNA base composition of four additional strains is 65 mol% G+C (T, method). These strains exhibit levels of DNA-DNA homology of 97 to 104% to P. ferrugineum IFAM S-1290T (11). The levels of DNA-DNA homology to the other Pedomicrobium species are listed in Table 1. All strains were isolated from podzolic soils in northern Germany (8). The type strain is strain IFAM S-1290 (= ATCC 33119 = DSM 1540). Other strains include strains IFAM P-1196 (= ATCC 33116 = DSM 1541), IFAM Q-1197 (= ATCC 33117 = DSM 1542), IFAM R-1198 (= ATCC 33118 = DSM 1543), and IFAM T-1130 (= ATCC 33120 = DSM 1544). Pedomicrobium americanum sp. nov. Cells are oval (Fig. 3), tetrahedral (Fig. 4), or short rod, bean, or spindle shaped and are up to 1.3 by 1.8 km. Up to three hyphae of various lengths originate laterally or polarly with similar frequency (Fig. 5). True branching of the hyphae occurs (Fig. 4). Multiplication is by budding at the hyphal tips (Fig. 3 and 5). Occasionally, intercalary buds are observed. Mature buds separate from the hyphae as motile swarmers with one subpolar flagellum (Fig. 6). Direct budding or division of single mother cells has not been observed. The cells have gram-negative cell walls (Fig. 7). Extracellular structures appear primarily on cell surfaces opposite the lateral hyphae (Fig. 3 and 7). These structures have been identified as polyanionic, ruthenium red- and Alcian bluestaining polymers which form fine filaments or arrays of particles (7, 14, 15) and provide the matrix for incrustations of iron oxides and manganese oxides. Deposition of iron compounds occurs when cells are grown on fulvic acid iron sesquioxide complexes or on PSM agar containing 5 mg of FeSO, . 7H,O per liter. Deposition of manganese(1V) oxides occurs on PSM agar containing 1.54 mg of MnSO, . H,O per liter. Intracytoplasmic membranes are present. Granules of poly-P-hydroxybutyric acid appear frequently in the cytoplasm (Fig. 7). Polyphosphate granules are present. FIG. 11. Electron micrograph of P . uustralicum IFAM ST-1306T negatively stained with 0.5% phosphotungstic acid: mother cell with branching hypha and two buds. Bar = 1 pm. FIG. 13. Electron micrograph of P. uustralicum IFAM ST-1306T negatively stained with 0.5% phosphotungstic acid: hypha showing local swellings, one of which formed an intercalary bud (arrow). Bar = 1 pm. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 VOL.38. 1988 PEDOMICROBIUM SPP. FIG. 14. Electron micrograph of P . australicum IFAM ST-1306T negatively stained with 0.5% phosphotungstic acid: motile swarmer cell with subpolarly inserted flagellum. The arrow indicates the basal hook of the flagellum. Bar = 1 pm. 311 FIG. 16. Thin section of P . australicum IFAM ST-1306T. Note the intracytoplasmic membranes in the transverse section of the left cell. Bar = 0.5 pm. FIG. 15. Thin section of P . australicum IFAM ST-1306T.Note the intracytoplasmic membranes and extracellular polymers incrusted with iron oxides or manganese oxides or both (arrows). Bar = 0.5 pm. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 312 INT. J. SYST.BACTERIOL. GEBERS AND BEESE FIG. 17. Thin section of P . australicum IFAM WD-1355. Note the intracytoplasmic membranes which formed cell compartments;note the section through the separation nose and the extracellular polymers incrusted with iron oxides or manganese oxides or both (arrows). Bar = 0.5 pm. The optimal growth temperature is 32 to 38°C; the temperature range is 15 to 41°C. The pH range for optimal growth in unbuffered media is 6.6 to 7.5; during growth, the pH increases (Table 4). The optimal pH of buffered media (containing 10 mM HEPES) is 7.6 to 8.4. The generation times in PSM or PYVM medium are 10 to 20 h. Stimulation or inhibition of growth by various carbon sources differs for individual strains (Table 5). Good growth occurs with 0.05% yeast extract or 0.025% yeast extract plus 0.025% peptone; nitrate does not serve as a sole nitrogen source. Within 6 days, the strains of P . americanurn remove 1 to 90% of the amino acids from PSM or PYVM culture broth (Table 6). A vitamin mixture (24) or 1 pg of cyanocobalamin per liter is required for growth of strains IFAM G-1381Tand IFAM BA-868 and stimulates the growth of strain IFAM BA-869. The presence of 0.1% NaCl stimulates the growth of strain IFAM BA-868. Catalase is produced by strains IFAM G-1381T and IFAM BA-868, but not by strain IFAM BA-869. All strains are positive for cytochrome oxidase and for reduction of nitrate to nitrite. The strains of P . americanum are susceptible to several antibiotics (Table 7); only strain IFAM G-1381T is resistant to sulfanilamide. The DNA base compositions of strains IFAM G-1381T, IFAM BA-868, and IFAM BA-869 are 64,65, and 64 mol% G+C, (Tnzmethod) (12), respectively. The DNA relatedness of the three strains is shown in Table 1. All strains are from freshwater habitats in North America (12); strain IFAM G-1381Twas isolated from pond water in TABLE 8. Differential characteristics of Pedomicrobium species Species P . ferrugineum" P . americanum P . australicum P . manganicum" Deposition Of iron ('I1) during growth on humic gel agar + + + - Deposition of manganese (IV) during growth on PSM agar Intracytoplasmic membranes present - - + + + + + + '' Data from reference 7. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 Spindle-shaped cells occur Spherical cells occur - + PEDOMICROBIUM SPP. VOL.38, 1988 FIG. 18. Thin section of P. australicum IFAM WD-1355. Note the invagination of the cytoplasmic membrane and the mesosomelike cell compartment. Bar = 0.2 pm. Sapsucker Woods, Ithaca, N.Y., by E. Dale and strains IFAM BA-868 and IFAM BA-869 were isolated from a temporary puddle by J. A. Babinchak in Ann Arbor, Mich. The type strain is strain IFAM G-1381 (= ATCC 43612). Other strains include strains IFAM BA-868 (= ATCC 43613) and IFAM BA-869 (= ATCC 43615). Pedomicrobium australicum sp. nov. Cells are oval (Fig. 8), tetrahedral, or short rod, bean, or spindle shaped (Fig. 9) and 313 are up to 1.2 by 1.8 bm. Up to three hyphae of varying lengths originate laterally or polarly with similar frequency (Fig. 10). True branching of the hyphae occurs (Fig. 11 and 12). Multiplication is by budding at the hyphal tips (Fig. 8 through 10). Up to three buds per mother cell may occur at the same time (Fig. 11 and 12). Occasionally, intercalary buds are formed (Fig. 13). Mature buds separate from the mother hyphae as motile swarmers with one subpolar flagellum (Fig. 14). The cells have a gram-negative cell wall structure (Fig. 15 and 16). Extracellular polymers appear primarily on cell surfaces opposite the lateral hyphae (Fig. 15 and 17). Deposition of iron compounds occurs when cells are grown on fulvic acid iron sesquioxide complexes or on PSM agar which contains 5 mg of FeSO, . 7H,O per liter. Deposition of manganese(1V) oxides occurs on PSM agar which contains 1.54 mg of MnSO, . H,O per liter. Intracytoplasmic membranes appear to form compartments within the cells (Fig. 17 and 18). Involvement of these membranes in the oxidation or deposition of manganese remains questionable (13). Granules of poly-P-hydroxybutyric acid and polyphosphate occur in the cytoplasm (Fig. 19). The optimal growth temperature is 29 to 32°C; the temperature range is 15 to 36°C. The pH for optimal growth in unbuffered media is 6.9 to 7.5; during growth, the pH increases (Table 4). The optimal pH of buffered media is 7.3 to 7.6. The generation times in PSM or PYVM medium are 11to 26 h. Stimulation or inhibition of growth by various carbon sources differs between strains (Table 5). Good growth occurs with 0.05% yeast extract or 0.025% yeast extract plus 0.025% peptone; nitrate does not serve as a sole nitrogen source. Within 6 days, strains IFAM ST-1306T and IFAM WD-1355 utilize 8 to 78% of the amino acids from PSM or PYVM culture broth (Table 6). Strain IFAM WD-1355 does not use isoleucine and lysine. Good growth of strain IFAM WD-1355 depends on the presence of 1 pg of cyanocobalamin per liter. The presence of 0.1% NaCl stimulates the growth of strain IFAM WD-1355. Both strains are positive for cytochrome oxidase and for reduction of nitrate to nitrite. The strains are susceptible to several antibiotics (Table 7); sulfanilamide causes only little inhibition of growth. The DNA base compositions of strains IFAM ST-1306T and IFAM WD-1355 are 65 and 63 mol% G + C (T, method) (12), respectively. The DNA relatedness of the strains is shown in Table 1. FIG. 19. Thin section of P. australicum IFAM WD-1355. Note the transparent granule of poly-p-hydroxybutyric acid (PHB) (arrow) and the dark, electron-dense granule of polyphosphate (PP) (arrow) within the mother cell, as well as the dark genome within the bud. Bar = 0.5 CLm. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 314 INT. J. SYST. BACTERIOL. GEBERS AND BEESE TABLE 9. Compositions of optimal nutrient media for Pedomicrobium strains Strain IIFAM S-1290T IFAM G-1381T lFAM BA-868 Carbon source (10 mM) lFAM BA-869 Acetate Acetate Acetate or malate Malate IFAM ST-1306T Malate [FAM WD-1355 Acetate Nitrogen source Vitamin source" Mineral salts sourceb Sodium chloride concn (g/liter) medium pH of 10 mM HEPESbuffered medium pH of Yeast extract (0.5 g/liter) Yeast extract (0.5 g/liter) Yeast extract (0.5 g/liter) Mixture (10 ml) Mixture (10 ml) Mixture (10 ml) Metals 44 (1ml) Metals 44 (1 ml) Metals 44 (1ml) 1 0 1 6.6 6.6 or 7.5 6.7 8.1 8.4 7.7 Yeast extract (0.25 g/liter) + peptone (0.25 g/liter) Yeast extract (0.25 g/liter) + peptone (0.25 g/liter) Yeast extract (0.5 g/liter) Mixture (10 ml) HBM (20 ml) 0 6.9 7.6 Mixture (10 ml) HBM (20 ml) 0 6.9 7.6 Cyanocobalamin (1 Fg/liter) Metals 44 (1 ml) 1 7.5 7.3 a The vitamin mixture (24) contained (per liter) 2 mg of D-( +)-biotin, 2 mg of folic acid, 5 mg of thiamine hydrochloride, 5 mg of calcium-D-(+)-panthothenate, 0.1 mg of cyanocobalamin, 5 mg of riboflavin, 5 mg of nicotinic acid, 5 mg of para-aminobenzoic acid, and 10 mg of pyridoxin hydrochloride. Metals 44 was a mineral salts solution (4) containing (per liter) 10.95 g of ZnSO, . 7H20, 5 g of FeSO, . 7H20, 1.54 g of MnSO, . H,O, 392 mg of CuSO, . 5H20, 248 mg of C O ( N O ~.)6H20, ~ 177 mg of Na2B40, . 10H,O, and 2.5 g of ethylenediaminetetraaceticacid. HBM was a mineral salts solution (4) containing (per liter) 29.7 g of MgSO, . 7H20, 3.34 g of CaCI, . 2H,O, 99 mg of FeS04 . 7H,O, 9.25 mg of (NH4)6M07024 . 4H20, 10 g of nitrilotriacetic acid, and 50 ml of metals 44 solution. The pH was adjusted with KOH to 6.8. Both strains were isolated from a freshwater reservoir in New South Wales, Australia, by J. T. Staley (12, 25). The type strain is strain IFAM ST-1306 (= ATCC 43611). The other strain which has been identified is strain IFAM WD-1355 (= ATCC 43614). Differentiation of Pedomicrobiurn species is based primarily on morphological characteristics (Table 8) and on DNADNA homologies (Table 1). All of the Pedomicrobiurn strains that have been described so far have similar morphologies, biochemical capabilities, and antibiotic susceptibilities. With regard to carbon source preference, amino acid utilization, optimal pH, and incubation temperature, each of the strains exhibits its individual pattern, so differentiation among species is unreliable on these bases. Suggested media for the species are shown in Table 9. ACKNOWLEDGMENTS We are grateful to P. Hirsch and J. L. Johnson for advice and fruitful discussions and to U. Wehmeyer for technical assistance. We thank W. Liesack for performing the amino acid analyses. Without the donation of bacterial strains by J. A. Babinchak, W. C, Ghiorse, P. Hirsch, and J. T. Staley, this study would not have been possible. LITERATURE CITED 1. Aristovskaya, T. V. 1961. Accumulation of iron in breakdown of organomineral humus complexes by microorganisms. Dokl. Akad. Nauk SSSR 136:954-957. (In Russian.) 2. Aristovskaya, T. V. 1963. On the decomposition of organic mineral compounds in podzolic soils. Pochvoved. Akad. Nauk SSSR 1:3042. (In Russian.) 3. Aristovskaya, T. V., and P. Hirsch. 1974. Genus Pedomicrobium Aristovskay 1961, p. 151-153. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. 4. Cohen-Bazire, G., W. R. Sistrom, and R. Y. Stanier. 1957. Kinetic studies of pigment synthesis by nonsulfur purple bacteria. J. Cell. Comp. Physiol. 49:25-68. 5. Duchow, E., and H. C. Douglas. 1949. Rhodomicrobium vannielii, a new photoheterotrophic bacterium. J. Bacteriol. 58:409416. 6. Feigl, F. 1960. Tupfelanalyse, vol. 1. Akademische VerlagsGesellschaft, Frankfurt. 7. Gebers, R. 1981. Enrichment, isolation, and emended description of Pedomicrobium ferrugineuw Aristovskaya and Pedomicrobium rnunganicum Aristovskaya. Int. J. Syst. Bacteriol. 31: 302-316. 8. Gebers, R., and P. Hirsch. 1978. Isolation and investigation of Pedomicrobiurn spp., heavy metal-depositing bacteria from soil habitats, p. 911-922. In W. Krumbein (ed.), Environmental biogeochemistry and geomicrobiology, vol. 3. Ann Arbor Science Publishers, Ann Arbor, Mich. 9. Gebers, R., M. Mandel, and P. Hirsch. 1981. Deoxyribonucleic acid base composition and nucleotide distribution of Pedornicrobium spp. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe C 2:332-338. 10. Gebers, R., B. Martens, U. Wehmeyer, and P. Hirsch. 1986. Deoxyribonucleic acid homologies of Hyphomicrobium spp., Hyphomonas spp., and other hyphal, budding bacteria. Int. J. Syst. Bacteriol. 36:241-245. 11. Gebers, R., R. L. Moore, and P. Hirsch. 1981. DNA-DNA reassociation studies on the genus Pedomicrobium. FEMS Microbiol. Lett. 11:283-286. 12. Gebers, R., U. Wehmeyer, T. Roggentin, H. Schlesner, J. Kolbel-Boelke, and P. Hirsch. 1985. Deoxyribonucleic acid base compositions and nucleotide distributions of 65 strains of budding bacteria. Int. J. Syst. Bacteriol. 35260-269. 13. Ghiorse, W. C. 1984. Biology of iron- and manganese-depositing bacteria. Annu. Rev. Microbiol. 38:515-550. 14. Ghiorse, W. C., and P. Hirsch. 1979. An ultrastructural study of iron and manganese deposition associated with extracellular polymers of Pedornicrobium-like budding bacteria. Arch. Microbiol. 123:213-226. 15. Ghiorse, W. C., and P. Hirsch. 1982. Isolation and properties of ferromanganese-depositing budding bacteria from Baltic Sea ferromanaganese concretions. Appl. Environ. Microbiol. 43: 1464-1472. 16. Hirsch, P. 1968. Biology of budding bacteria. IV. Epicellular deposition of iron by aquatic budding bacteria. Arch. Mikrobiol. 60:201-216. 17. Hirsch, P., and G. Rheinheimer. 1968. Biology of budding bacteria. V. Budding bacteria in aquatic habitats: occurrence, enrichment, and isolation. Arch. Mikrobiol. 62:289-306. 18. Khak-mun, T. 1967. Iron- and manganese-oxidizing microorganisms in soils of South Sakhalin. Mikrobiologiya 36:337-344. (In Russian. ) 19. Khak-mun, T. 1968. The biological nature of iron-manganese Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54 VOL. 38, 1988 PEDOMICROBIUM SPP. crusts of soil-forming rocks in Sakhalin mountain soils. Mikrobiologiya 37:749-753. (In Russian.) 20. Kolbel-Boelke, J., R. Gebers, and P. Hirsch. 1985. Genome size determinations for 33 strains of budding bacteria. Int. J. Syst. Bacteriol. 35270-273. 21. Kutuzova, R. S. 1972. Electron microscopic studies of oozedwelling microorganisms. Mikrobiologiya 41:859-861. (In Russian.) 22. Skerman, V. B. D. 1967. A guide to the identification of the genera of bacteria, 2nd ed. The Williams & Wilkins Co., 315 Baltimore. 23. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath (ed.). 1980. Approved lists of bacterial names. Int. J. Syst. Bacteriol. 30:225420. 24. Staley, J. T. 1968. Prosthecamicrobium and Anculomicrobium, new prosthecate freshwater bacteria. J. Bacteriol. 9519211942. 25. Staley, J. T., K. C. Marshall, and V. B. D. Skerman. 1980. Budding and prosthecate bacteria from freshwater habitats of various trophic states. Microb. Ecol. 5245-251. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 01 Aug 2017 18:54:54