Pedomicrobium Americanum Sp

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. 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%. VOL.38, 1988 PEDOMICROBIUM SPP. 305

TABLE 3. Conditions used for DNA-DNA hybridization reactions T,, temp Radio- Sp act after Sp act for Amt of labeled Ratio of Labeled DNA in o.17 active labeling hybridization DNA per labeled DNA HL:g-Iwa&a;gn ~~~1~~~ Incubation Filter from strain: NaCl (oc) (cp;~~,) of (cpm/pg of hybridization to unlabeled (u) temp K) type DNA) reaction (pg) DNA temp rC) IFAM S-1290T 95.5 3H 19,348 19,348 0.12 or 0.19 1:1,250 or 1:790 65 16 1,250' 51 GFIC' IFAM G-1381T 95.0 '''I 2.40 X lo6 2.40 X lo6 0.10 1:1,500 70 20 100d 56 BA83' IFAM BA-869 95.1 '*'I 7.44 x lo6 0.77 x lo6 0.10 1:1,500 70 20 50d 56 BA83' IFAM ST-1306T 95.4 *''I 2.99 x lo6 2.99 x lo6 0.10 1:1,500 10 20 100" 56 BA83' IFAM E-1129T 95.2 '''1 2.73 x lo6 0.79 x lo6 0.10 1:1,500 70 20 SO@ 56 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 same time. Mature buds either separate from the hyphae as hybridization reactions are shown in Table 3. uniflagellated swarmers or remain attached. Buds may arise in an intercalary fashion by localized hyphal swelling. Oc- RESULTS AND DISCUSSION casionally, direct budding of mother cells or division of single mother cells is observed (1, 3). The description of the genus Pedomicrobium (7) was (ii) Cellular structure and composition. Cells stain gram based on six strains of P. ferrugineum and one strain of P. negative, although older cells may stain gram variable. manganicum. Five new strains from aquatic habitats exhibit Extracellular polymers stain with ruthenium red. Oxidized the major characteristics of the genus; i.e., the cells form iron compounds or manganese compounds or both are threadlike outgrowths (hyphae), multiply by budding, and deposited primarily on mother cells and also on hyphae. produce extracellular polymers that serve as matrices for Intracytoplasmic membranes are observed in cells of man- deposition of iron oxides or manganese oxides or both. ganese-depositing species. Up to three granules of poly-p- However, these strains are unlike the previously described hydroxybutyric acid are stored per cell. Small, dense gran- species P. ferrugineum and P, manganicum, as well as "P. podsolicum" (2, 3) and "P.manganicum subsp. sacchalini- ules stainable with Loefffer methylene blue suggest the cum" (18, 19). presence of polyphosphates. Liquid cultures grown in the Our data (see below) allow emendation of the genus dark and in synthetic light may be yellowish white and description, an emended description of the type species, P. reddish orange, respectively. ferrugineum, and descriptions of two new species. (iii) Colonial morphology. Two colony types may develop Pedomicrobium Aristovskaya 1961 (Gebers 1981) genus on solid media. Type 1 colonies are round and convex and emend. (i) Morphology. Cells are spherical, oval, tetrahedral, have entire or rhizoid edges; they are yellowish red to dark or rod, pear, bean, or spindle shaped. Up to five or more brown, sometimes with concentric rings. These colonies prosthecae (i.e., cellular outgrowths [hyphae] of constant have a soft consistency. Type 2 colonies are round and flat; diameter) are formed per cell; these are 0.15 to 0.3 pm in they pit the agar so that their surfaces remain near the agar diameter and vary in length with cultural conditions. At least level. Their edges are entire or rhizoid. Type 2 colonies are one hypha originates laterally; other hyphae may appear yellowish red to dark brown, and the cells in the center of the polarly or subpolarly. True branching of the hyphae occurs. colonies are often lysed, giving a granular appearance to the Multiplication is primarily by budding at the hyphal tips, colonies. These colonies have a cartilaginous consistency where local swellings form young buds which elongate and may be removed from the agar intact. perpendicularly to the hyphal axis and enlarge up to the size After restreaking on solid media, type 1 or 2 colonies give of a mother cell. Hyphal insertion of the buds is lateral in all rise to both types of colonies. With periodic transfers to cases. Up to three buds per mother cell may occur at the fresh medium type 1 colonies tend to predominate.

TABLE 4. Growth in PYVM medium or in PSM with or without buffers added" P. nmerictinum strains P. austrcllicum strains P. ferrugineum IFAM s-1290T IFAM G-1381T IFAM BA-868 IFAM BA-869h IFAM ST-130bTh IFAM WD-1355 Buffer added to broth Relative Relative Relative Relative Relative Relative shiftPH growth growth g:t growth g:t growth shiftPH growth i:t gr,ooth (%) (%I (%I (%I (%I None 6.618.7 100 6.618.8 100 6.118.5 100 6.918.8 97 6.918.9 loo 6.718.3 50 10 mM HEPES + NaOH 7.017.5 71 1.017.5 44 1.017.2 18 6.818.6 100 6.818.3 53 7.017.1 8 35 mM Tris + HCl' 7.017.7 52 7.018.0 57 6.817.0 3 6.917.4 49 6.917.1 23 6.817.0 2 None 7.518.5 67 7.518.9 100 7.518.5 52 7.619.0 90 7.619.0 97 7.5J8.5 100

(I 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. 306 GEBERS AND BEESE INT. J. SYST. BACTERIOL.

1501 1

h cn c .-c 3 cb -Q Y Y

.IL. 2 =8

I *-8 b

5' 260 3001 400I 500I 6001 % 0 m time(h) 0 I

5' I I I FIG. 2. Growth of P. ferrugineum IFAM S-1290T in PSM sup- 0 100 200 plemented with aromatic compounds. Symbols: x , no supplement, time (h) pH 8.7 at the end of growth; 0,lO mM phenol added; pH 8.6; 0,lO mM benzoic acid, pH 8.9; B, 10 mM toluene, pH 8.8. 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. yeast extract, peptone, Casamino Acids (Difco), or Soytone (Difco) at a concentration of 0.05%. Vitamin mixtures stimulate growth; lack of vitamins pro- duces pleomorphic cells. Good growth occurs in the pres- (iv) Physiological properties. Cells are microaerophilic to ence of 0.1% NaC1; no growth, but survival, occurs in the aerobic and heterotrophic. Slow, poor growth occurs on 0.1 presence of 1 or 2.5% NaCl; 5% NaCl is bactericidal for the to 1% fulvic acid iron sesquioxide complexes as sole carbon species that have been described (7). and nitrogen sources; good growth occurs with 10 mM Gelatin and starch are not hydrolyzed. Neither acid nor acetate, 10 mM malate, 10 mM succinate, or 10 mM gluco- gas is produced anaerobically from glucose. Anaerobic nate as the carbon source and a nitrogen source such as growth is not observed in stab cultures. Catalase is pro-

TABLE 5. Growth on carbon sources" Growth index for: Carbon source (10 mM)' P. ferrugineurn P. umericunum strains P. uustrulicum strains IFAM S-1290T IFAM G-1381' IFAM BA-868 IFAM BA-869 IFAM ST-1306T IFAM WD-1355

~ ControlC 21 24 17 61 49 6 Acetate 100 100 100 100 100 100 Caproate 44 5id 15 14i 1% 24 Citrate 3i Oi Oi 92 42i Oi Ethanol 1Oi 5i 16 44i 44i 18 Formate 14i 30 30 75 51 15 Gluconate 18 35 33 84 109 25 D-Glucose 1li 6i 13 4% 56 4 Glycerol Oi 30 43 69 63 Oi D-Lactate 8i 20 7i 23i 20i 7 Lactose 15i 7i 21 41i 74 23 DL-Malate 22 23 102 121 156 30 D-Mannitol 24 21 36 52i 89 4 Methanol 19 21 17 37i 34 12 Phenol Oi 1Oi 12i NT' NT Oi Propanol 8i 8i 29 58 1Oi 11 Pyruvate 70 60 41 39i 32i 56 D-Ribose lli 5i 8i 48i 52 2i Succinate 37 66 28 108 NT 18

a 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. VOL. 38, 1988 PEDOMICROBIUM SPP. 307

TABLE 6. Utilization of amino acids"

% Remaining after 6 days of growth

Amino acid P. P. americanum strains P. austrulicum strains ferrugineum Avg IFAM S-1290T IFAM G-1381T IFAM BA-868 IFAM BA-869 IFAM ST-1306T IFAM WD-1355 DL- Alanine 18 14 21 64 68 23 35 Ammonia 62 57 67 55 62 67 62 L-Arginine 50' 26' 37' 69 82 84' 58 L-Aspartic acid 36 34 36 56 32 42 39 L-Glutamic acid 24 21 25 49 49 45 36 Glycine 46 47 52 19 22 47 39 L-Histidine 44 40 71 28 42 75 50 L-Isoleucine 22 19 22 29 82 106 47 L-Leucine 18 17 20 37 75 88 43 L-Lysine 54 50 51 55 86 102 66 L-Pheny lalanine 27 14 22 30 36 92 37 L-Proline 56 99 42 76 72 65 68 L-Serine 35 32 36 59 57 73 49 L-Threonine 39 38 41 53 57 71 50 L-Tyrosine 55 41 78' ND' ND 56 58 D-Valine 13 10 13 25 56 91 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 to be 100%. Approximate values. ND, Not determined.

TABLE 7. Inhibition of growth by antibiotics

~~ % Inhibition of growth of" Concn P. uustrulicum strains Antibiotic (&ml) P. ferrugineum P. americunum strains IFAM S-1290T IFAM G-1381T IFAM BA-868 IFAM BA-869 IFAM ST-1306' IFAM WD-1355 Ampicillin 1 22 19 3 17 26 61 10 65 91 78 74 43 91 100 82 95 93 91 91 91 Penicillin G 1 3 0 73 10 23 69 10 82 0 73 59 98 78 100 87 93 97 90 97 95 Cephalothin 1 18 0 4 19 0 76 10 81 0 60 45 41 75 100 +b 77 68 + + 84 C ycloserine 1 12 16 63 59 0 71 10 73 89 90 59 17 77 100 82 95 96 92 94 95 Polymyxin B 1 5 80 75 33 18 55 10 82 + + + + 89 100 + + + + + + Neomy cin 1 82 86 84 77 80 96 10 81 85 85 90 85 95 100 84 84 82 84 83 95 Chloramphenicol 1 74 43 74 5 76 81 10 83 90 94 47 78 85 100 79 92 98 88 98 82 Streptomycin 1 85 84 73 78 84 85 10 76 88 69 83 77 82 100 + + + + + + Sulfanilamide 1 5 0 16 48 13 15 10 3 0 20 49 13 19 100 16 3 22 47 8 33 " 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%). 308 GEBERS AND BEESE INT. J. SYST.BACTERLOL.

FIG. 3. Electron micrograph of p. americanum IFAM G-1381* FIG. 5. Electron micrograph of P. arnericanurn IFAM G-1381T negatively stained with 0.5% phosphotungstic acid: oval-shaped negatively stained with 0.5% phosphotungstic acid: mother cell with imother cell with lateral hypha and bud. The arrows indicate extra- polar hypha and bud. Note beginning outgrowth of second, subpolar cellular polymers incrusted with iron oxides or manganese oxides or hypha (arrow). Bar = 1 wn. both. Bar = 1 bm. duced. On PSM agar supplemented with sheep blood (100 manganese compounds. Depositions occur primarily on mlAiter), all strains grow well but are not hemolytic. mother cells and later on hyphae. Intracytoplasrnic mem- (v) Occurrence. These organisms are widely distributed in branes have not been found in ultrathin sections of strain podzolic and other soils, freshwater habitats, iron springs, IFAM S-1290T. The temperature for optimum growth is 29 to and seawater; they are probably ubiquitous. 30°C; the temperature range is 18 to 43°C. The pH range for The DNA base composition (four species, 11 strains) growth is 6.6 to 8.7 (Table 4). P. ferrugineum IFAM S-1290T ranges from 63 to 66 mol% guanine plus cytosine (G+C) as grows in 50 mM HEPES-buffered PSM (Fig. 1); however, determined by the thermal denaturation (T,) method. 100 and 200 mM HEPES prevent growth. Growth in unbuf- The levels of DNA-DNA homology of P. ferrugineum fered PSM at pH 6.6 gives higher protein yields than growth IFAM S-1290Tto representative strains of other genera of in PSM buffered with 10 mM HEPES or 35 rnM tris(hydroxy- hyphal, budding bacteria are less than 7% (Table 2). methy1)aminomethane hydrochloride (Table 4). Growth in The type species is P. ferrugineum Aristovskaya 1961. PSM containing 10 mM HEPES is best at an initial pH of 8.1. P. ferrugineum Aristovskaya 1961 emend. Cells are spher- During growth, the pH of all cultures increases. ical, oval, tetrahedral, or rod or bean shaped and are 0.6 to The generation time of strain IFAM S-1290T in PSM is 10 2 by 0.6 to 2.5 pm. At least one hypha originates laterally; h at 30°C. The concentration of acetate in modified PSM less frequently, other hyphae appear polarly or subpolarly. (containing 0.1% yeast extract) influences the generation Multiplication is by budding at the tips of the hyphae which time and maximum optical density of P. ferrugineum are 0.2 pm or less in diameter. Initially, buds are spherical to cultures; 20 mM acetate results in a minimum generation oval; they are motile by a single polar to subpolar flagellum. time of 15.5 h and an optical density of 210 Klett units. With Occasionally, intercalary buds are observed. Deposition of 30 mM acetate, the generation time is 20.5 h, and a value of oxidized iron compounds occurs when cells are grown with 240 Klett units is reached; however, cells of the latter fulvic acid iron sesquioxide complexes, elemental iron pow- cultures produce large granules of poly-P-hydroxybutyric der, FeS, or iron paper clips; there is no accumulation of acid.

FIG. 6. Electron micrograph of P. arnericanurn IFAM G-1381T negatively stained with 0.5% phosphotungstic acid: separated FIG. 4. Electron micrograph of P. americanum IFAM BA-869 daughter cell in the flagellated swarmer stage. Note the subpolarly negatively stained with 0.5% phosphotungstic acid: tetrahedral inserted flagellum and the separation nose (arrow), where the former mother cell with branching hypha. Bar = 1 pm. mother cell hypha was attached. Bar = 1 pm. 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 transfer to PSM supplemented with 10 mM phenol com- glutamate. Slow, poor growth occurs with 10 mM oxalate, 10 pletely prevented growth (Table 5). mM tartrate, 10 mM cholesterol, 10 mM aspartate, 10 mM Good growth occurs with 0.05% yeast extract or 0.05% tryptophan, 10 mM alanine, or 0.3% paraffin. No growth peptone as a nitrogen source. Within 6 days, strain IFAM occurs with 10 mM galactose, 10 mM fructose, 10 mM S-1290T removes 44 to 87% of the amino acids from PSM maltose, 10 mM sucrose, 10 mM phenylalanine, 10 mM culture broth (Table 6). Slow, poor growth occurs with serine, or 10 mM lysine (7). The utilization of other sub- 0.05% Soytone, 0.05% Casamino Acids, 10 mM 2-aminoben- strates is shown in Table 5. Amino acids are utilized simul- zoic acid, 10 mM NaNO,, 10 mM acetamide, 10 mM urea, 10 taneously as nitrogen sources (7). mM N-acetylglucosamine, or 10 mM methylamine hydro- The growth of strain IFAM S-1290T in PSM supplemented chloride; and no growth occurs with 0.01 M NaNO,, 0.58 M with 10 mM phenol, 10 mM benzoic acid, or 10 mM toluene (4%) NaNO,, or 1.06 M (9%) NaNO, (7). was monitored by determining optical density values. In A vitamin mixture (24) or 1 pg of cyanocobalamin per liter these cultures, the lag phase was increased compared with is required for good growth. The presence of 0.1% NaCl cultures in pure PSM (Fig. 2). Benzoic acid and toluene stimulates growth. reduced the growth of strain IFAM S-1290T only to a small The cells are positive for cytochrome oxidase and for extent, whereas phenol caused a strong inhibition. A second reduction of nitrate to nitrite.

FIG. 8. Electron micrograph of P. australicitrn IFAM ST-1306=, FIG. 9. Electron micrograph of P. austraficitm IFAM WD-1355, Pt-C shadowed: oval mother cell with polar hypha and spindle- Pt-C shadowed: spindle-shaped mother cell with lateral hypha and shaped bud. Bar = 5 pm. bud. Bar = 1 pm. 310 GEBERS AND BEESE INT. J. SYST.BACTERIOL.

FIG. 10. Electron micrograph of P. australicum IFAM ST-1306T FIG, 12. Electron micrograph of P. australicum IFAM ST-1306T negatively stained with 0.5% phosphotungstic acid: mother cell with negatively stained with 0.5% phosphotungstic acid: mother cell with one lateral bpha and one polar hypha. Note the terminal swelling of hypha branching twice and with three buds at different developmen- the polar hypha. Bar = 1 pm. tal stages. Bar = 1 pm.

P.ferrugineum strains are not pathogenic for guinea pigs are up to 1.3 by 1.8 km. Up to three hyphae of various after intraperitoneal injection of lo8 cells (7). lengths originate laterally or polarly with similar frequency In addition to antibiotic susceptibilities in liquid cultures (Fig. 5). True branching of the hyphae occurs (Fig. 4). (Table 7), cells of strain IFAM S-1290T are susceptible in Multiplication is by budding at the hyphal tips (Fig. 3 and 5). agar diffusion tests (7) to rifampin, tetracycline, gentamicin, Occasionally, intercalary buds are observed. Mature buds and nitrofurazone, separate from the hyphae as motile swarmers with one The DNA base composition of the type strain is 65 mol% subpolar flagellum (Fig. 6). Direct budding or division of G+C (TnImethod) (7, 9). The average DNA base composi- single mother cells has not been observed. tion of four additional strains is 65 mol% G+C (T, method). The cells have gram-negative cell walls (Fig. 7). Extracel- These strains exhibit levels of DNA-DNA homology of 97 to lular structures appear primarily on cell surfaces opposite 104% to P. ferrugineum IFAM S-1290T (11). The levels of the lateral hyphae (Fig. 3 and 7). These structures have been DNA-DNA homology to the other Pedomicrobium species identified as polyanionic, ruthenium red- and Alcian blue- are listed in Table 1. staining polymers which form fine filaments or arrays of All strains were isolated from podzolic soils in northern particles (7, 14, 15) and provide the matrix for incrustations Germany (8). of iron oxides and manganese oxides. Deposition of iron The type strain is strain IFAM S-1290 (= ATCC 33119 = compounds occurs when cells are grown on fulvic acid iron DSM 1540). Other strains include strains IFAM P-1196 (= sesquioxide complexes or on PSM agar containing 5 mg of ATCC 33116 = DSM 1541), IFAM Q-1197 (= ATCC 33117 FeSO, . 7H,O per liter. Deposition of manganese(1V) oxides = DSM 1542), IFAM R-1198 (= ATCC 33118 = DSM 1543), occurs on PSM agar containing 1.54 mg of MnSO, . H,O per and IFAM T-1130 (= ATCC 33120 = DSM 1544). liter. Intracytoplasmic membranes are present. Granules of Pedomicrobium americanum sp. nov. Cells are oval (Fig. 3), poly-P-hydroxybutyric acid appear frequently in the cyto- tetrahedral (Fig. 4), or short rod, bean, or spindle shaped and plasm (Fig. 7). Polyphosphate granules are present.

FIG. 13. Electron micrograph of P. uustralicum IFAM ST-1306T FIG. 11. Electron micrograph of P. uustralicum IFAM ST-1306T negatively stained with 0.5% phosphotungstic acid: hypha showing negatively stained with 0.5% phosphotungstic acid: mother cell with local swellings, one of which formed an intercalary bud (arrow). Bar branching hypha and two buds. Bar = 1 pm. = 1 pm. VOL.38. 1988 PEDOMICROBIUM SPP. 311

FIG. 14. Electron micrograph of P. australicum IFAM ST-1306T FIG. 16. Thin section of P. australicum IFAM ST-1306T. Note negatively stained with 0.5% phosphotungstic acid: motile swarmer the intracytoplasmic membranes in the transverse section of the left cell with subpolarly inserted flagellum. The arrow indicates the basal cell. Bar = 0.5 pm. hook of the flagellum. Bar = 1 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. 312 GEBERS AND BEESE INT. J. SYST.BACTERIOL.

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 temper- BA-868 and stimulates the growth of strain IFAM BA-869. ature range is 15 to 41°C. The pH range for optimal growth in The presence of 0.1% NaCl stimulates the growth of strain unbuffered media is 6.6 to 7.5; during growth, the pH IFAM BA-868. increases (Table 4). The optimal pH of buffered media Catalase is produced by strains IFAM G-1381T and IFAM (containing 10 mM HEPES) is 7.6 to 8.4. BA-868, but not by strain IFAM BA-869. All strains are The generation times in PSM or PYVM medium are 10 to positive for cytochrome oxidase and for reduction of nitrate 20 h. to nitrite. Stimulation or inhibition of growth by various carbon The strains of P. americanum are susceptible to several sources differs for individual strains (Table 5). Good growth antibiotics (Table 7); only strain IFAM G-1381T is resistant occurs with 0.05% yeast extract or 0.025% yeast extract plus to sulfanilamide. 0.025% peptone; nitrate does not serve as a sole nitrogen The DNA base compositions of strains IFAM G-1381T, source. Within 6 days, the strains of P. americanurn remove IFAM BA-868, and IFAM BA-869 are 64,65, and 64 mol% 1 to 90% of the amino acids from PSM or PYVM culture G+C, (Tnzmethod) (12), respectively. The DNA relatedness broth (Table 6). of the three strains is shown in Table 1. A vitamin mixture (24) or 1 pg of cyanocobalamin per liter All strains are from freshwater habitats in North America is required for growth of strains IFAM G-1381T and IFAM (12); strain IFAM G-1381T was isolated from pond water in

TABLE 8. Differential characteristics of Pedomicrobium species Intracytoplasmic Spherical Deposition Of iron ('I1) Deposition of manganese (IV) Spindle-shaped Species during growth on membranes cells during growth on PSM agar cells occur humic gel agar present occur P.ferrugineum" + - - P. americanum + + + P. australicum + + + - P. manganicum" - + + + '' Data from reference 7. VOL.38, 1988 PEDOMICROBIUM SPP. 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 fla- gellum (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). Depo- sition 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 con- tains 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 polyphos- phate occur in the cytoplasm (Fig. 19). The optimal growth temperature is 29 to 32°C; the temper- ature 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 11 to 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 FIG. 18. Thin section of P. australicum IFAM WD-1355. Note PYVM culture broth (Table 6). Strain IFAM WD-1355 does the invagination of the cytoplasmic membrane and the mesosome- not use isoleucine and lysine. Good growth of strain IFAM like cell compartment. Bar = 0.2 pm. WD-1355 depends on the presence of 1 pg of cyanocobala- min per liter. The presence of 0.1% NaCl stimulates the growth of strain IFAM WD-1355. Both strains are positive Sapsucker Woods, Ithaca, N.Y., by E. Dale and strains for cytochrome oxidase and for reduction of nitrate to IFAM BA-868 and IFAM BA-869 were isolated from a nitrite. temporary puddle by J. A. Babinchak in Ann Arbor, Mich. The strains are susceptible to several antibiotics (Table 7); The type strain is strain IFAM G-1381 (= ATCC 43612). sulfanilamide causes only little inhibition of growth. Other strains include strains IFAM BA-868 (= ATCC 43613) The DNA base compositions of strains IFAM ST-1306T and IFAM BA-869 (= ATCC 43615). and IFAM WD-1355 are 65 and 63 mol% G+C (T, method) Pedomicrobium australicum sp. nov. Cells are oval (Fig. 8), (12), respectively. The DNA relatedness of the strains is tetrahedral, or short rod, bean, or spindle shaped (Fig. 9) and 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. 314 GEBERS AND BEESE INT. J. SYST. BACTERIOL.

TABLE 9. Compositions of optimal nutrient media for Pedomicrobium strains Sodium pH of pH of 10 mM Carbon Mineral salts chloride HEPES- Strain source (10 Nitrogen source Vitamin source" sourceb concn buffered mM) (g/liter) medium medium IIFAM S-1290T Acetate Yeast extract (0.5 g/liter) Mixture (10 ml) Metals 44 (1 ml) 1 6.6 8.1 IFAM G-1381T Acetate Yeast extract (0.5 g/liter) Mixture (10 ml) Metals 44 (1 ml) 0 6.6 or 7.5 8.4 lFAM BA-868 Acetate or Yeast extract (0.5 g/liter) Mixture (10 ml) Metals 44 (1 ml) 1 6.7 7.7 malate lFAM BA-869 Malate Yeast extract (0.25 g/liter) Mixture (10 ml) HBM (20 ml) 0 6.9 7.6 + peptone (0.25 g/liter) IFAM ST-1306T Malate Yeast extract (0.25 g/liter) Mixture (10 ml) HBM (20 ml) 0 6.9 7.6 + peptone (0.25 g/liter) [FAM WD-1355 Acetate Yeast extract (0.5 g/liter) Cyanocobalamin Metals 44 (1 ml) 1 7.5 7.3 (1 Fg/liter)

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 CO(NO~)~. 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 6. Feigl, F. 1960. Tupfelanalyse, vol. 1. Akademische Verlags- New South Wales, Australia, by J. T. Staley (12, 25). Gesellschaft, Frankfurt. The type strain is strain IFAM ST-1306 (= ATCC 43611). 7. Gebers, R. 1981. Enrichment, isolation, and emended descrip- The other strain which has been identified is strain IFAM tion of Pedomicrobium ferrugineuw Aristovskaya and Pedomi- WD-1355 (= ATCC 43614). crobium rnunganicum Aristovskaya. Int. J. Syst. Bacteriol. 31: Differentiation of Pedomicrobiurn species is based primar- 302-316. ily on morphological characteristics (Table 8) and on DNA- 8. Gebers, R., and P. Hirsch. 1978. Isolation and investigation of Pedomicrobiurn spp., heavy metal-depositing bacteria from soil DNA homologies (Table 1). All of the Pedomicrobiurn habitats, p. 911-922. In W. Krumbein (ed.), Environmental strains that have been described so far have similar morphol- biogeochemistry and geomicrobiology, vol. 3. Ann Arbor Sci- ogies, biochemical capabilities, and antibiotic susceptibili- ence Publishers, Ann Arbor, Mich. ties. With regard to carbon source preference, amino acid 9. Gebers, R., M. Mandel, and P. Hirsch. 1981. Deoxyribonucleic utilization, optimal pH, and incubation temperature, each of acid base composition and nucleotide distribution of Pedorni- the strains exhibits its individual pattern, so differentiation crobium spp. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. among species is unreliable on these bases. Suggested media Hyg. Abt. 1 Orig. Reihe C 2:332-338. for the species are shown in Table 9. 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. ACKNOWLEDGMENTS 11. Gebers, R., R. L. Moore, and P. Hirsch. 1981. DNA-DNA reassociation studies on the genus Pedomicrobium. FEMS We are grateful to P. Hirsch and J. L. Johnson for advice and Microbiol. Lett. 11:283-286. fruitful discussions and to U. Wehmeyer for technical assistance. 12. Gebers, R., U. Wehmeyer, T. Roggentin, H. Schlesner, J. We thank W. Liesack for performing the amino acid analyses. Kolbel-Boelke, and P. Hirsch. 1985. Deoxyribonucleic acid base Without the donation of bacterial strains by J. A. Babinchak, W. C, compositions and nucleotide distributions of 65 strains of bud- Ghiorse, P. Hirsch, and J. T. Staley, this study would not have been ding bacteria. Int. J. Syst. Bacteriol. 35260-269. possible. 13. Ghiorse, W. C. 1984. Biology of iron- and manganese-depositing bacteria. Annu. Rev. Microbiol. 38:515-550. LITERATURE CITED 14. Ghiorse, W. C., and P. Hirsch. 1979. An ultrastructural study of iron and manganese deposition associated with extracellular 1. Aristovskaya, T. V. 1961. Accumulation of iron in breakdown of polymers of Pedornicrobium-like budding bacteria. Arch. Mi- organomineral humus complexes by microorganisms. Dokl. crobiol. 123:213-226. Akad. Nauk SSSR 136:954-957. (In Russian.) 15. Ghiorse, W. C., and P. Hirsch. 1982. Isolation and properties of 2. Aristovskaya, T. V. 1963. On the decomposition of organic ferromanganese-depositing budding bacteria from Baltic Sea mineral compounds in podzolic soils. Pochvoved. Akad. Nauk ferromanaganese concretions. Appl. Environ. Microbiol. 43: SSSR 1:3042. (In Russian.) 1464-1472. 3. Aristovskaya, T. V., and P. Hirsch. 1974. Genus Pedomicrobium 16. Hirsch, P. 1968. Biology of budding bacteria. IV. Epicellular Aristovskay 1961, p. 151-153. In R. E. Buchanan and N. E. deposition of iron by aquatic budding bacteria. Arch. Mikrobiol. Gibbons (ed.), Bergey's manual of determinative bacteriology, 60:201-216. 8th ed. The Williams & Wilkins Co., Baltimore. 17. Hirsch, P., and G. Rheinheimer. 1968. Biology of budding 4. Cohen-Bazire, G., W. R. Sistrom, and R. Y. Stanier. 1957. bacteria. V. Budding bacteria in aquatic habitats: occurrence, Kinetic studies of pigment synthesis by nonsulfur purple bacte- enrichment, and isolation. Arch. Mikrobiol. 62:289-306. ria. J. Cell. Comp. Physiol. 49:25-68. 18. Khak-mun, T. 1967. Iron- and manganese-oxidizing microorga- 5. Duchow, E., and H. C. Douglas. 1949. Rhodomicrobium vannie- nisms in soils of South Sakhalin. Mikrobiologiya 36:337-344. (In lii, a new photoheterotrophic bacterium. J. Bacteriol. 58:409- Russian. ) 416. 19. Khak-mun, T. 1968. The biological nature of iron-manganese VOL. 38, 1988 PEDOMICROBIUM SPP. 315

crusts of soil-forming rocks in Sakhalin mountain soils. Mikro- Baltimore. biologiya 37:749-753. (In Russian.) 23. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath (ed.). 20. Kolbel-Boelke, J., R. Gebers, and P. Hirsch. 1985. Genome size 1980. Approved lists of bacterial names. Int. J. Syst. Bacteriol. determinations for 33 strains of budding bacteria. Int. J. Syst. 30:225420. Bacteriol. 35270-273. 24. Staley, J. T. 1968. Prosthecamicrobium and Anculomicrobium, 21. Kutuzova, R. S. 1972. Electron microscopic studies of ooze- new prosthecate freshwater bacteria. J. Bacteriol. 951921- dwelling microorganisms. Mikrobiologiya 41:859-861. (In Rus- 1942. sian.) 25. Staley, J. T., K. C. Marshall, and V. B. D. Skerman. 1980. 22. Skerman, V. B. D. 1967. A guide to the identification of the Budding and prosthecate bacteria from freshwater habitats of genera of bacteria, 2nd ed. The Williams & Wilkins Co., various trophic states. Microb. Ecol. 5245-251.