Family Nitro Bacteraceae Buchanan Requests for 0Pin I O Nsl

INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY Vol. 21, No. 3 July 1971, pp. 254-270 Printed in U.S.A. Copyright 0 1971 International Association of Microbiological Societies Taxonomic Considerations of the Family Nitro bacteraceae Buchanan Requests for 0pin i o nsl

STANLEY W. WATSON

Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02.543

A revision of the family Nitrobacteraceae Buchanan is proposed. It is requested that the Judicial Commission issue an Opinion placing the generic names Nitrosocystis Winogradsky, Nitrosogloea Winogradsky, and Nitrocystis Winogradsky on the list of rejected names as nomina dubia. Organisms currently placed in these genera are to be transferred to other genera or their names are to be regarded as later, subjective synonyms of other names or as nomina dubia. Thus Nitrosocystis oceanus Watson will be transferred to Nitrosococcus as Nitrosococcus oceanus (Watson) comb. nov.; Nitrosocystis coccoides Starkey is to be regarded as a synonym of Nitrosococcus nitrosus (Migula) Buchanan, Nitrosomonas monocella Nelson, a synonym of Nitrosomonas europaea Winogradsky, Nitrosospira antarctica Winogradsky and Winogradsky, a synonym of Nitrosospira briensis Winogradsky and Winogradsky, and Nitrocystis sarcinoides Winogradsky, Nitrocystis micropunctata (Winogradsky) Winogradsky, and Nitrobacter agilis Nelson as later, subjective synonyms of Nitrobacter winogradskyi Winslow et al. ; Nitrosogloea merismoides Winogradsky, Nitrosogloea mem branacea Winogradsky, Nitrosogloea schizo bacteroides Winogradsky, and Nitrosocystis javanensis (Winogradsky) Starkey are to be regarded as nomina dubia. The Judicial Commission is requested, therefore, to issue an Opinion placing these nomina dubia (names whose application is uncertain) on the list of rejected names. The type strain of Nitrosolobus multiformis, ATCC 25 196, is described. The following strains are described and are here designated as type strains: Nitrosococcus oceanus ATCC 19707, Nitrococcus mobilis ATCC 25380, and Nitrospina gracilis ATCC 25379.The following strains are described and are here designated as neotypes: Nitrosomonas europaea ATCC 25978, Nitro- sospira briensis ATCC 25961, and Nitrobacter winogradskyi ATCC 2539 1.

The family Nitrobacteraceae Buchanan con- gradsky. All of these were originally described tains those chemoautrophic bacteria which oxi- by Helene or Sergei Winogradsky (21, 22, 24, dize either ammonia to nitrite or nitrite to 26, 27). Many of the original descriptions were nitrate to fulfill their primary energy needs. based on mixed cultures and were so brief that Bergey 's Manual of Determinative Bacteriology it is impossible to identify an organism from (1) lists the genera of ammonia-oxidizing bac- them. Some of the characteristics used for the teria as Nitrosomonas Winogradsky, Nitro- separation of these bacteria into genera are so cystis Winogradsky, Nitrosogloea Wino- variable and have resulted in the grouping of gradsky, Nitrosospira Winogradsky and Wino- several morphologically unrelated strains into gradsky, and Nitrosococcus Winogradsky, and one genus. Moreover, none of the original the genera of nitrite-oxidizing bacteria as Nitro- strains were preserved in culture. The intent of bacter Winogradsky and Nitrocystis Wino- this study was to update the specific and generic descriptions of the nitrifying bacteria 'Contribution no. 2434 from the Woods Hole and, where necessary, to designate type and Oceanographic Institution, Woods Hole, Mass. 02 543. neotype strains and to make taxonomic and 254 VOL 21,1971 FAMILY NITROBACTERACEAE BUCHANAN 255 nomenclatural recommendations concerning Waterbury (20), when grown in pure culture in these organisms. a chemostat, grew as single cells freely suspended in the medium for 1 to 2 months but then MATERIALS AND METHODS started to grow in small aggregates suspended in Bacterial strains. The sources of the organ- the medium. Once a culture started to clump, it isms used in this study are given below. could not be induced to grow again as single Methods. The methods used have been cells even by varying the cultural conditions. described previously ( 18, 19). The ability of cells to grow in clumps forming zoogloeas or cysts is a characteristic shared by RESULTS AND DISCUSSION several morphological types of ammonia- and Subsequent to the work of the Wino- nitrite-oxidizing bacteria. Thus zoogloea and gradskys, most investigators were able to isolate cyst formations are not reliable taxonomic only members of the genus Nitrosomonas or criteria, and it is recommended that they Nitrobacter, and doubts were expressed as to should no longer be used to separate genera. the validity of the other genera (6, 7). Until If the recommendation not to place organ- recently, less than a dozen strains of nitrifying isms into taxa solely on the basis of their bacteria were preserved in culture, and all of proclivity to aggregate is accepted, then disposi- these belonged either to the genus Nitro- tion of those bacteria in the genera Nitro- somonas or Nitro bacter. These organisms have a sogloea, Nitrosocystis, and Nitrocystis must be generation time of 8 hr or more, making it arranged. Recommendations concerning the difficult to isolate them in pure culture, thus organisms currently placed in all the ammonia- few pure cultures existed. Although the taxo- oxidizing genera will be discussed first. The nomy of the bacteria in the family Nitrobac- disposition of the organisms in the genus teraceae has long been in need of revision, the Nitrocystis will be reserved until the nitrite lack of cultures has made such a task impossi- oxidizers are reviewed. ble. The organisms in the genus Nitrosogloea Studies on nitrifying bacteria were initiated include N. merismoides Winogradsky (2 1-23), at the Woods Hole Oceanographic Institution in N. schizobacteroides Winogradsky (2 l), and N. 1957 and are continuing at present. During this membranacea Winogradsky (2 1). The first of period, thousands of enrichment cultures were these three organisms is described as ellipsoidal made, and over 200 nitrifying bacteria were rods, 0.5 to 1.5 pm, the second as elongated isolated in pure culture. These studies showed rods, 3 to 4pm in length, and the last as that several morphologically different types of ellipsoidal rods which often occur in pairs. The nitrifying bacteria existed and indicated that original descriptions of these three organisms not all of the genera of nitrifying bacteria listed consist of only a few short sentences, and in Bergey’s Manual were valid. The purpose of except for the size and shape of these organisms this present publication is to make specific no other information is available on the mor- recommendations concerning the taxonomy of phology of these cells. It is impossible to the family Nitrobacteraceae. identify these organisms or to assign them to The three genera of most questionable other genera from their original descriptions. validity are Nitrosogloea, Nitrosocystis, and Therefore, it is proposed to consider each of Nitrocystis. The first two genera are comprised these three names as well as the generic name of ammonia oxidizers and the latter of nitrite Nitrosogloea as nomina dubia (i.e., names oxidizers. All of these genera are reserved for whose applications are uncertain), and it is those bacteria which grow in aggregates forming proposed that they should be included on the either zoogloeas or cysts. Based on these list of nomina rejicienda according to Rule 24f aggregation characteristics, several morpholog- of the International Code of Nomenclature of ically unrelated strains have been placed in the Bacteria (8). genus Nitrosogloea. Studies in this laboratory Since cyst formation can be observed in have demonstrated that most ammonia- and several morphologically unrelated strains in nitrite-oxidizing bacteria in mixed culture have mixed but not in pure cultures, it is proposed a proclivity to grow in aggregates forming either that the organisms in the genus Nitrosocystis zoogloeas or cysts. Some organisms, e.g. Nitro- should be reassigned to other genera or that socystis oceanus Watson ( 17), no longer formed their names be considered as nomina dubia. cysts when isolated in pure culture whereas Only one member of the genus Nitrosocystis, others tended to aggregate only when cultural N. oceanus, is preserved in culture. This organ- conditions were unfavorable. Other nitrifying ism is a peritrichously flagellated large coccus bacteria, e.g. Nitrococcus mobilis Watson and (Fig. 1 and 19) and is characterized by exten- 256 WATSON INT. J. SYST. BACTERIOL.

FIG. 1. Phase-contrast photomicrograph of Nitrosococcus oceanus (formerly Nitrosocystis oceanus). X2,SOO; FIG. 2. Electron micrograph of Nitrosococcus oceanus showing centrally arranged cytomembranes. X31,900. FIG. 3. Phase-contrast photomicrograph of Nitrosomonas europaea. X2,500. FIG. 4. Electron micrograph of Nitrosomonas europaea showing peripheral cytomembranes. X47,800. FIG. 5. Electron micrograph of marine Nitrosomonas sp. showing rounded ends and peripheral cytomembranes. X34,800. FIG. 6. Frozen-etched preparation showing extra cell wall layer of a marine Nitrosomonas sp. X95,700. VOL 21,1971 FAMILY NITROBACTERACEAE BUCHANAN 257 sive cytomembranes arranged as flattened Rule 9d (l), Note 2(c) of the Bacteriological vesicles in the central region of the cell (Fig. 2). Code (8). Neither flagella nor motility has The size and shape of the vegetative cells of been observed in this designated neotype strain. Nitrosocystis oceanus are similar to those of In all other respects the characters of this Nitrosocystis coccoides Starkey 1948 (1 5) and designated neotype strain (see Table 1) agree Nitrosococcus nitrosus (Migula 1900) Buchanan with those recorded in the original species 1925 (4, 10). It is suggested that all three of description (26). Nitrosomonas europaea was these organisms belong to the genus Nitro- originally described as having a single, polar sococcus and that this genus include spherical flagellum. Although we have not observed organisms having a diameter of 1.5 pm or flagella in the designated neotype strain just greater. It is proposed that Nitrosocystis mentioned, other strains of Nitrosomonas coccoides be regarded as a later, subjective species were found to have one or two flagella synonym of Nitrosococcus nitrosus. with subterminal insertion (Fig. 20). The cells It is also proposed that Nitrosocystis of the neotype strain are either ellipsoidal or oceanus be transferred to the genus Nitro- short rods (0.8 by 1 to 2 pm) with pointed ends sococcus as Nitrosococcus oceanus (Watson) (Fig. 3), and their fine structure is characterized comb. nov. Although Nitrosococcus oceanus is by cytomembranes (1 1) which are arranged in similar in size and shape to Nitrosococcus flattened vesicles in the peripheral regions (Fig. nitrosus, the former is an obligate marine 4). organism and the latter is not. Since the fine Although only one species of Nitrosomonas, structure of Nitrosococcus nitrosus has not N. europaea, will be listed in this taxonomic been detailed, it is impossible to state whether revision, other bacteria exist which should be the two organisms are similar, To avoid further placed in this genus. If this genus is to include taxonomic confusion, we recommend keeping rod-shaped organisms which have peripheral Nitrosococcus oceanus as a separate species. cytomembranes, then organisms which we have Although no cultures of Nitrosococcus nitrosus isolated from the marine environment and from are available, this organism must still be con- the Chicago sewer system should also be placed sidered the type species of the genus. in it as new species. For example, obligate The species Nitrosocystis jauanensis (Wino- marine ammonia oxidizers were isolated which gradsky) Starkey (15, 26) is described as a were rod-shaped, with rounded ends, 1.0 to 1.3 small, ellipsoidal cell 0.5 to 0.6 pm in diameter. pm wide and 2.0 to 2.5 pm long. All of these Since it is proposed that the genus Nitro- had membranes arranged as flattened lamellae sococcus contains only large, spherical, ammon- in the peripheral regions of the cell (Fig. 5) and ia-oxidizing bacteria, N. jauanensis cannot be an additional cell wall layer (Fig. 6) not found transferred to this genus. This organism is in the strains of Nitrosomonas europaea that we poorly defined in the literature, and it is examined. recommended that this species name as well as A different rod-shaped, ammonia-oxidizing the generic name Nitrosocystis Winogradsky be bacterium was isolated from the Chicago sewer considered as nomina dubia and placed on the system. The cells of this organism were 1.2 list of nomina rejicienda. pm wide and 2.0 to 3.5 pm long with pointed The genus Nitrosomonas contains rod- ends, and there were prominent connections shaped organisms which oxidize ammonia to between dividing cells (Fig. 7). Electron micro- nitrite, and it is recommended that this genus scopic observations (Fig. 8) showed that, al- remain as described. Currently two species are though two or more cells were often intercon- listed in Bergey’s Manual as belonging to this nected, no separating cross wall was visible. I genus, N. europaea Winogradsky 1892 (26) and would like to postpone judgment of the species N. monocella Nelson 1931 (1 2). Comparison of designation of these strains until they are the original descriptions of these two organisms studied in greater detail. These strains are reveals that they are morphologically similar. mentioned only to illustrate that not all strains For this reason it is recommended that Nitro- of Nitrosomonas are morphologically identical. somonas monocella be considered as a later, Another genus of ammonia-oxidizing bac- subjective synonym of Nitrosomonas europaea. teria listed in Bergey’s Manual is Nitrosospira Since none of the original strains of Nitro- (27), which includes two species, N. briensis somonas europaea appear to be extant, it is Winogradsky and Winogradsky 1933 (28) and recommended that an existing strain, filed with N. antarctica Winogradsky and Winogradsky the American Type Culture Collection (ATCC) 1933 (28). Both organisms have spiral-shaped and assigned ATCC no. 25978, should be cells and apparently have a similar morphology. designated as the neotype strain according to The only difference noted was that the cells of 258 WATSON INT. J. SYST. BACTERIOL.

FIG. I. Phase-contrast photomicrograph of a Nitrosomonas sp. isolated from the Chicago sewer system, showing pointed ends of cells and interconnections between cells. X25,OOO. FIG. 8. Electron micrograph of Nitrosomonas sp. in Fig. 7, showing the lack of cross walls between cells. X18,800. FIG. 9.Phase-contrast photomicrograph of Nitrosospira briensis showing that the spiral nature of cells is barely visible. X2,500. FIG. 10. Negatively stained preparation of Nitrosospira briensis showing spiral nature of cells and flagella. X28,700. FJG.11. Phase-contrast photomicrograph of Nitrosolobus multiformis showing lobular nature of cells. X2,500. FIG. 12. Electron micrograph of Nitrosolobus multiformis showing how the lobular cells are partially compartmentalized by cytomembranes. X26,600. VOL 21,1971 FAMILY NITROBACTERA CEAE BUCHANAN 259

Nitrosospira antarctica form more compact here, Nitrosomonas would contain the rod- spirals than do the cells of Nitrosospira briensis. shaped organisms, Nitrosococcus the large, Nitrosospira briensis was isolated recently in spherical organisms, Nitrosospira the spiral- this laboratory and described in detail (18). It shaped organisms, and Nitrosolobus the lobu- was observed that the degree of compactness of lar-shaped organisms. the spirals depended upon the mounting tech- The genera of nitrite-oxidizing bacteria listed nique. If heated, the cells would often partially in the 7th edition of Bergey’s Manual include uncoil. It does not seem justified to use this Nitro bacter and Nitrocystis. Winogradsky (2 6) criterion for the separation of these organisms described the first nitrite-oxidizing bacterium into two species. It is recommended that the and proposed the genus Nitrobacter for it. This name Nitrosospira an tarctica be considered a generic name, with Nitro bacter winogradskyi later, subjective synonym of Nitrosospira Winslow et al. 1917 (31) as the type species, briensis. was conserved by the Judicial Commission (9). It is further recommended that the genus The cells of this species were described as short Nitrosospira contain the ammonia-oxidizing rods, 0.6 to 0.8 pm wide and 1.0 to 1.2 pm bacteria whose cells are tightly wound (Fig. 9), long, often pear- or wedge-shaped, and so much so that the spiral nature may not be reproducing by budding (5, 13, 15, 30). The obvious under a phase-contrast microscope. typical morphology of Nitrobacter winograd- However, the spiral nature of these cells should skyi cells is illustrated in Fig. 13. The fine be clearly evident when negatively stained structure of Nitrobacter species is characterized preparations (Fig. 10) are examined with an by a polar cap of cytomembranes arranged as electron microscope. In thin sections, several flattened lamellae (Fig. 14). sections of a single cell will be seen lying In the present studies two strains of Nitro- side-by-side due to the spiral nature of the bacter winogradskyi received from August Van organism. N. briensis does not possess a well- Gool, who designated these strains Engel strain developed cytomembrane system that is char- No. 1 and Lees strain No. 2, were examined. acteristic of the other ammonia oxidizers. Since Strains of Nitrobacter agilis were obtained the original strain of N. briensis was not from Helen Funk and Alvin Nason. In addition, preserved in culture, ATCC 25961 is here over 15 other strains of Nitrobacter species designated as the neotype strain of this species were isolated and studied in our own labora- (Table 1). This designated neotype strain of tory . Nitrosospira briensis is peritrichous (Fig. 2 l), The second species of lvitrobacter, N. agilis, but in all other respects the characters of this was described by Nelson (12). The chief differ- strain agree with those recorded in the original ence between N. agilis and N. winogradskyi was species description (28). that the former organism was observed to be Recently a peritrichous, lobular-shaped, motile and the latter was not. In studies ammonia-oxidizing bacterium (Fig. 11 and conducted in our laboratory, all strains were 22) was isolated in this laboratory and found to be motile when cultivated in a named Nitrosolobus multiformis Watson et al. chemostat, but none of the strains were motile (19). It was proposed that the genus Nitro- when grown in batch culture. Separation of N. solobus contain the lobular-shaped organisms winogradskyi and N. agilis into two species with cytomembranes which partially compart- based on their motility or lack of motility does mentalize the cells (Fig. 12). The type strain of not appear to be justified. this species is ATCC 25196 (reference 19; The cells of all strains were similar in size incorrectly cited herein as ATCC 251 9), and its and shape and all appeared to reproduce by characters are summarized in Table 1. budding. The fine structure of all the strains Watson and Mandel (19a) analyzed the was similar and all had a polar cap of cytomem- d eoxyribonucleic acid (DNA) base com- branes (1 1, 16). The GC compositions of five positions of 19 strains of ammonia-oxidizing strains of Nitrobacter sp. varied from 61.7 to bacteria; these were found to form 60.2 moles per cent. These strains formed a two homogenous groups with respect to their homogenous group with respect to their GC guanine plus cytosine (GC) compositions. The compositions. Since these two species seem to GC compositions of members of Nitrosomonas be similar in all respects, it is recommended and Nitrosocystis ranged from 47.7 to 51.0 that N. agilis be considered a synonym of N. moles per cent and those of the members of winogradskyi. Nitrosolobus and Nitrosospira from 53.6 to 5 5.1 moles per cent. The genus Nitrocystis (21) was proposed for According to the recommendations made ellipsoidal to rod-shaped cells which grew em- E3 0

TABLE1. Descriptionsof type and neotype strains of nitrzfiing bacteria

Type strains Neotype strains Characters ~ ~~ ~ ~ ~ Nitrobacter winogmdskyr~- Nitrosolobus multiformis Nitrosococcus oceanus Nitroococcusmobilis Nitrospina gracilis Nitrosospira briensis Nitrosomomseuropaea - ATCC 25196a ATCC 19707 ATCC 25380 ATCC 25379 ATCC 25961 ATCC 25391 ATCC 25978 Shape Lobular Spherical Spherical Slcnder rod Spiral Pearlike iod

Size bm) 1.0 x 1.5 1.8 X 2.2 1.5 X 1.8 D.3-0.4 X 2.7-6.5 1.3-0.4 X 1.0-5.0 3.6-0.8 X 1.0-2.0 1.8-0.9 X 1.0-2.0

Method of reproduction Binary fission Binary fission Binary fission Binary fission Binary fission Budding 3inary fission

Nature of cytomembranes Internal, compartrnentaliz- Internal, lamellar Internal, tubular Cy tomembrancs Zytomembranes Peripheral, lamellar 'eripheral, lamellar ingthe cell absent absent

Flagellation Peritrichous Peritrichous Twosubpolar None observed Peritrichous Single subpolar gone observed

Cram reaction Endosporcs 0 z Oxidation of nitrite to + t + nitrate

Oxidation of ammonia to + + + + nitrite + Obligately aerobic + + + + + +

Optimum remp (C) 25-30 25-30 25-30 25-30 25-30 25-30 25-30

OptimumpH 75-8.0 7.5-8.0 7.5-8.0 7.5-8.0 7.5-8.0 7.5-8.0 7.5-8.0

~o~esZGC~ in DNA 54.6 50.5 61.2 57.7 54.1 60.7 51.0 aATCC, American Type CultureCollection, Rockville, Md. b%GC, Per cent guanine plus cytosine. VOL 21,1971 FAMILY NITROBACTERACEAE BUCHANAN 26 1

FIG. 13. Phase-contrast photomicrograph of Nitrobacter winogradskyi showing wedge-like shape of cells. X2,500. FIG. 14. Electron micrograph of Nitrobacter winogradskyi showing polar cap of cytomembranes. X51,300. FIG. 15. Phase-contrast photomicrograph of Nitrospina gracilis showing the long and slender morphology of the cells. X2,500. FIG. 16. Electron micrograph showing longitudinal and cross sections of Nitrospina gracilis. XI5,900. FIG. 17. Phase-contrast photomicrograph showing spherical nature of Nitrococcus mobilis. X2,500. FIG. 18. Electron micrograph of Nitrococcus mobilis showing tubular cytomembrane system. X31,900. 262 WATSON INT. J. SYST. BACTERIOL.

bedded in a slime and united to form compact (Fig. 17). When motile, the cells exhibited a zoogloeal aggregates. The two species described rolling, spinning action but rarely moved in a were N. sarcinoides Winogradsky 1937 (23), given direction for more than a few microm- whose cells were small rods, 0.5 by 1 .O pm, and eters. The cells were propelled by one or two N. micropunctata (Winogradsky 1935) Wino- flagella which were inserted close together and gradsky 1937 (21), whose cells were described subterminally in dividing cells (Fig. 24). The as ellipsoidal rods about 0.5 pm in diameter. A flagella were 12.5 to 15.0 nm in diameter and 3 clear-cut morphological distinction between to 4 pm long. The fine structure of Nitrococcus these two species is not obvious from their mobilis is unique in that this organism has a original descriptions. tubular cytomembrane system (Fig. 18). N. Studies in this laboratory of over 200 strains mobilis was isolated from a marine environment of nitrite-oxidizing bacteria revealed that most and has an obligate salt requirement. The GC of these strains grew in compact clumps in composition of this bacterium was 6 1.2 moles enrichment cultures. The clumps were com- per cent. The type strain (20) of N. mobilis is posed of cells similar in shape and size to those ATCC 25380. of Nitrobacter winogradskyi as illustrated in Thus it is recommended that only three Fig. 13, and the fine structure of all strains was genera of nitrite-oxidizing bacteria, Nitrobacter, similar to or identical with that of strains of Nitrospina, and Nitrococcus, be considered as Nitrobacter winogradskyi (Fig. 14). When these valid genera. Nitrobacter should include the strains were isolated in pure culture, aggregates short, ellipsoidal to pear-shaped rods which were no longer formed. For these reasons it is reproduce by budding, Nitrospina the long, recommended that Nitrocystis sarcinoides and slender rods, and Nitrococcus the large, spheri- Nitrocystis micropunctata be considered as cal organisms. Although morphologically three later, subjective synonyms of Nitrobacter wino- different types of nitrite oxidizers exist, they gradskyi Winslow et al. 19 17 and that the name could be segregated into only two groups with Nitrocystis be declared a nomen dubium and respect to their GC compositions. placed on the list of nomina rejicienda. Since the original strain of Nitrobacter winogradskyi was not preserved in culture, ATCC Emended description of the family 25391 is here designated as the neotype strain. Nitrobacteraceae Buchanan 19 17 This strain was obtained from August Van Goo1 and was designated as Lees strain No. 1 of Ni.tro.bac.te.ra 'ce.ae. M.L. Nitrobacter name Nitrobacter winogradskyi. The characters of type genus of the family; -aceae ending to (Table 1) of this designated neotype strain denote a family; M.L. plural noun Nitrobac- agree with those recorded in the original species teraceae the Nitrobacter family. description (31). The cells of this strain were Rod-shaped, ellipsoidal, spherical, spirillar, rarely motile, and electron micrographs of their and lobular cells without endospores. Flagella flagella were not obtained. In another strain a subpolar or peritrichous and often absent. single, subterminally inserted flagellum was Gram-negative. Cells derive energy from the observed (Fig. 23). oxidation of ammonia or nitrite and satisfy Two additional nitrite-oxidizing bacteria, Ni- their carbon needs by the fixation of COz. trospina gracilis Watson and Waterbury and Only one species, Nitrobacter winogradskyi, has Nitrococcus mobilis Watson and Waterbury, been shown to be facultatively autotrophic. were described recently (20). Nitrospina gracilis Not parasitic. Commonly found in soil, fresh is a long, slender, nonmotile rod, 0.3 to 0.4 pm water, and seawater. wide and 2.7 to 6.5 pm long (Fig. 15), and re- The type genus of the family is Nitrobacter produces by binary fission. Its fine structure is Winogradsky. characterized by the absence of the extensive cytomembrane system found in most other ammonia- and nitrite-oxidizing bacteria (Fig. 16). N. gracilis was isolated from a marine environ- Key to the genera of the family ment and has an obligate salt requirement. The Nitrobacteraceae GC composition of this organism was found to be 57.7 moles per cent. The type strain (20) of Nitrospina gracilis is ATCC 25379. I. Nitrite oxidized to nitrate. Nitrococcus mobilis is a large coccus which A. Cells rod-shaped. measures 1.5 to 1.8 pm in diameter just after 1. Short rods, often wedge- or pear-shaped, division and 1.8 by 3.5 pm just prior to division with a polar cap of cytomembranes. VOL 21,1971 FAMILY NITROBACTERA CEAE BUCHANAN 263

FIG. 19. Shadowed preparation of Nitrosococcus oceanus showing peritrichous cells. X 10,000. FIG. 20. Negatively stained preparation of Nitrosomoms europaea showing paired flagella inserted subpolarly. X 14,500. FIG. 21. Negatively stained preparation of Nitrosospira briensis showing peritrichous cells. X22,300. FIG. 22. Negatively stained preparation of Nitrosolobus multiformis showing peritrichous cells. FIG. 23. Negatively stained preparation of Nitrobacter winogradskyi showing single flagellum with subpolar insertion. FIG. 24. Negatively stained preparation of Nitrococcus mobilis showing paired flagella. XI7,500. 264 WATSON INT. J. SYST. BACTERIOL.

Genus I. Nitrobacter. Optimum growth temperature, 25 to 30 C. 2. Long, slender rods with no extensive Range of DNA base ratios: 60.7 to 61.7 cytomembrane system, moles % GC (buoyant density; one species, six Genus 11. Nitrospina. strains). B. Cells spherical (diameter 1.54 pm or more) Habitat: Soils, fresh water, and seawater. with cytomembranes forming a branched, Type species: N. winogradskyi Winslow et al. tubular network in the cytoplasm. Genus 111. Nitrococcus. Emended description of Nitro bac ter 11. Ammonia oxidized to nitrite. winogradskyi Winslow et al. 19 17 A.Cells are straight rods with peripheral membranes occurring as flattened wi.no.grad'sky.i. Named for S. Winogradsky, lamellae. the microbiologist who first isolated these bac- Genus IV. Nitrosomonas. teria; M.L. mas. gen. n. winogradskyi of B.Cells are not straight rods and do not Win ogradsky . contain flattened lamellae in the Short rods, often pear- or wedge-shaped, 0.6 peripheral regions. to 0.8 by 1.0 to 2.0 pm, gram-negative. Cells 1. Cells are spirals with no evident have a polar cap of cytomembranes arranged as cyt omembranes. flattened vesicles. Motile or non motile. Cells Genus V. Nitrosospira. reproduce by budding. Grow either free in a 2. Cells are not spirals but do contain liquid medium or in small clumps of 100 or cyt omembranes. more cells embedded in a slime matrix. a. Cells spherical (diameter 1.5 pm or Cells are obligate chemoautotrophs or facu- more) with cyt omem branes forming lative autot ro p hs . When grown aut otro phicall y , flattened lamellae in center of cells. cells oxidize nitrite to nitrate and fix C02 to Genus VI. Nitrosococcus. fulfill their energy and carbon needs. Strictly b. Cells lobular and partially compart- aerobic, using oxygen as a terminal electron mentalized by cytomembranes. acceptor. Temperature range of growth, 14 to 40 C; optimum temperature for growth, 25 to Genus VII. Nitrosolobus. 30 C; optimum pH for growth, 7.5 to 8.0. Many strains will grow either in a fresh water or Emended description of the genus Nitrobacter a seawater medium. Marine strains are morpho- Winogradsky 1892 logically indistinguishable from terrestrial strains. Ni.tro.bac'ter. Gr. noun nitrum nitre, M.L. Cell suspensions show characteristic oxidized nitrate; M.L. noun bacter the masculine form of minus dithionite reduced absorption peaks at the Gr. neut. n. bactrum a rod; M.L. mas. n. 420, 440, 522, 550, 587, and 600 nm. These Nitrobacter nitrate rod. absorption peaks represent cytochromes which Cells are short rods, often wedge- or impart a brownish-yellow color to cell suspen- pear-shaped, 0.6 to 1.0 by 1.0 to 2.5 pm. sions and a reddish-brown color to a pellet of Reproduce by budding. Gram-negative. Cells cells. possess a polar cap of cytomembranes arranged Storage materials: Glycogen, poly-p- to form flattened vesicles. Usually not motile hydroxybutyrate, and polyphosphates. unless grown in continuous culture; when Range of DNA base ratios: 60.7 to 61.7 motile, cells are propelled by a single, moles 7% GC (buoyant density; six strains). subterminal flagellum. Cells are rich in Habitat : Widely distributed in soils. cytochromes, imparting a yellowish color to Designated neotype strain: ATCC 25391 cell suspensions; void of other pigments. (isolated from soil; see Table 1 for description). Some strains are obligate chemoautotrophs which oxidize nitrite to nitrate and fix C02 to Description of the genus Nitrospina fulfill energy and carbon needs. The auto- Watson and Waterbury 1971 trophic medium consists of fresh water or seawater enriched with nitrite and other inor- Ni.tro.spi'na. Gr. noun nitrum nitre, M.L. ganic salts, no organic growth factor being nitrate; L. noun spina spine; M.L. mas. n. required. Some strains can be grown hetero- Nitrospina nitrate spine. trophically, but the growth rate is slower than Cells are straight, slender rods, 0.3 to 0.4 pm that when the cells are grown autotrophically. by 2.7 to 6.5 pm; spherical forms, 1.35 to Strictly aerobic, using oxygen as a terminal 1.45 pm in diameter, are found in senescent electron acceptor. Optimum pH, 7.5 to 8.0. cultures. There is no extensive cytomembrane VOL 21,1971 FAMILY NITROBACTERACEAE BUCHANAN 265 system. Cell division is by binary fission. Gram- Description of the genus Nitrococcus negative. Nonmotile. Cells have cytochromes Watson and Waterbury 1971 but no other pigments. Obligat ely chemoaut otrophic bacteria which Ni.tro.coc'cus. Gr. noun nitrum nitre, M.L. oxidize nitrite to nitrate and fix C02 to fulfill nitrate; Gr. coccus berry; M.L. mas. n. Nitro- energy and carbon needs. Grow in seawater coccus nitrate sphere. enriched with nitrite and inorganic salts; no Cells spherical, 1.5 pm or larger. Gram-nega- organic growth factors are required. Strictly tive. Motile by means of one or two subter- aerobic, using oxygen as a terminal electron minally inserted flagella. Division by binary acceptor. Optimum growth temperature, 25 to fission. Cells are rich in cytochromes imparting 30 C. Optimum pH range, 7.5 to 8.0. Opti- a yellowish to reddish color to cell suspensions; mum growth in 70 to 100% seawater; no void of other pigments. growth in distilled water mineral salts medium Obligately chemoautotrophic bacteria which even if NaCl is included. oxidize nitrite to nitrate and fix C02 to fulfill DNA base ratio: 57.7 moles % GC (buoyant energy and carbon needs. Growth medium is density; one species, one strain). seawater enriched with nitrite and other inor- Habitat: South Atlantic Ocean. ganic salts, no organic growth factor being Type species: N. gracilis Watson and Water- required. Strictly aerobic, using oxygen as a bury. terminal electron acceptor. Optimum pH, 7.5 to 8.0. Optimum growth temperature, 25 to 30 C. Description of Nitrospina gracilis DNA base ratio: 6 1.2 moles % GC (buoyant Watson and Waterbury 1971 density; one species, one strain). Habitat: South Pacific Ocean, Type species: N. mobilis Watson and Water- gra'ci.lis. L. adj. gracilis slender. bury. Cells are long, slender rods, 0.30 to 0.40 pm by 2.7 to 6.5 pm; spherical forms 1.35 to 1.45 pm in diameter are found in old cultures. Cells Emended description of Nitrococcus lack an extensive cytomembrane system, but mobilis Watson and Waterbury 1971 occasional bleb-like invaginations of the plasma membrane occur. Occur singly or in pairs. mo'bi.lis. L. adj. mobilis moveable, motile. Multiplication is by binary fission. Gram-nega- Cells spherical, 1.5 to 1.8 pm in diameter tive. Nonmotile. Grow free in liquid media but after division, but elongating with dimensions will adhere to culture vessel walls in old of 1.8 by 3.5 pm just before division. Cells cultures. occur singly or in pairs and divide by binary Cells are obligate chemoautotrophs using fission. Gram-negative. Cells have an extensive nitrite and carbon dioxide as major energy and cytomembrane system which forms tubes ran- carbon sources. Will not grow on organic media, domly arranged in the cytoplasm. Motile by and no organic growth factors are required; means of one or two flagella inserted subpolarly some organic compounds inhibit growth. Grow in elongated cells; the flagella are 12.5 nm in 70 to 100% seawater enriched with nitrite wide and 3 to 4 pm long. When motile, cells and other mineral salts. Strictly aerobic, using have a turning, twisting, erratic motion. Grow oxygen as a terminal electron acceptor. Opti- either free in liquid media or in small clumps of mum growth temperature, 25 to 30 c. Opti- 100 or more cells embedded in a slime matrix; mum pH, 7.5 to 8.0. this aggregate of cells is not surrounded by any Cell suspensions show characteristic oxidized type of limiting membrane. minus dithionite reduced absorption peaks at Cells are obligate chemoautotrophs oxidizing 425, 532, and 553 nm. These absorption peaks nitrite to nitrate and fixing C02 to fulfill their represent cytochromes which impart a brown- energy and carbon needs. Strict aerobes using ish-yellow color to cell suspensions and to oxygen as a terminal electron acceptor. Tem- pellets of cells. perature range of growth, 14 to 40 C; opti- Storage material: Glycogen. mum temperature for growth, 25 to 30 c. DNA base ratio: 57.7 moles % GC (buoyant Optimum pH for growth, 7.5 to 8.0. Optimum density). growth in 70 to 100% seawater; will not grow Designated type strain: ATCC 25379 (iso- in fresh water even if NaCl is included. lated from the South Atlantic Ocean; see Table Cell suspensions show characteristic oxidized 1 for description). minus dithionite reduced absorption peaks at 266 WATSON INT. J. SYST. BACTERIOL.

420, 440, 522, 550, 587, and 600 nm. These ammonia and hydroxylamine to nitrite. Growth absorption peaks represent cytochromes which occurs with ammonia but not with hydroxyla- impart a brownish-yellow color to cell suspen- mine. Fix COz to fulfill their carbon needs. sions and a reddish-brown color to a pellet of Grow in particulate-free liquid salts media as cells. individual cells or in small aggregates embedded Storage materials: Glycogen and poly$- in a slime. Strict aerobes which use oxygen as a hydroxybutyrate. terminal electron acceptor but which can oxi- DNA base ratio: 61.2 moles % GC (buoyant dize ammonia at reduced oxygen tensions. density). Temperature growth range, 15 to 30 C; opti- Designated type strain: ATCC 25380 (iso- mum temperature for growth, 25 to 30 c; lated from South Pacific Ocean; (see Table 1 for optimum pH for growth, 7.5. No sodium description). chloride requirement. Cannot be grown in seawater . Emended description of the genus Cell suspensions show characteristic oxidized Nitrosomonas Winogradsky 1890 minus dithionite reduced absorption peaks at 423, 465, 522, 552, and 605 nm. These Ni.tro.so.mo’ nas. M.L. nitrosus nitrous; Gr. absorption peaks represent cytochromes which monas, monadis a unit, monad; M.L. fem. n. impart a brownish to straw color in cultures Nitrosomonas nitrous monad, i.e., the monad and a reddish color to a pellet of cells. producing nitrite. Storage materials : Poly phosp hates. Cells ellipsoidal or short rods, 0.8 to 1.0 by Range of DNA base ratios: 50.5 to 51.0 1.0 to 3.0 pm, motile or nonmotile, occurring moles % GC (buoyant density; three strains). singly, in pairs, or as short chains. Gram-nega- Habitat: Widely distributed in soils. tive. Possess cytomembranes which occur as Designated neotype strain: ATCC 25978 flattened vesicles in the peripheral regions of (isolated from soils in the United States; see the cytoplasm. Cells grow free in the medium Table 1 for description). or are embedded in a slime matrix. Cells are rich in cytochromes, which impart a yellowish to reddish color to cell suspensions; void of Emended description of the genus other pigments. Nitrosospira Winogradsky 19 3 1 0bligat ely chemoau t otrophic bacteria which oxidize ammonia to nitrite and fix C02 to Ni.tro.so.spi’ra. M.L. nitrosus nitrous; Gr. fulfill energy and carbon needs. Grow in fresh spira a coil, spiral; M.L. fem. n. Nitrosospira water or seawater enriched with ammonia and nitrous spiral. inorganic salts, no organic growth factors being required. Strictly aerobic, using oxygen as a Cells spiral shaped. Gram-negative. Cells lack cytomembranes. Motile, the cells being per- terminal electron acceptor. Optimum pH range, 7.5 to 8.0. Optimum growth temperature, 25 to itrichous. Grow free in liquid medium. 30 C. Cells are rich in cytochromes, which impart a Range of DNA base ratios: 47.4 to 51.0 yellowish to reddish color to cell suspensions; moles % GC (buoyant density; thirteen strains). void of other pigments. Habitat: Soils, fresh water, and seawater. Obligately chemoautotrophic bacteria which Type species : N. europaea Winogradsky. oxidize ammonia to nitrite and fix C02 to fulfill energy and carbon needs. Grow in fresh water enriched with ammonia and inorganic Emended description of Nitrosomonas salts; no organic growth factors are required. europaea Winogradsky 1892 Strictly aerobic using oxygen as a terminal electron acceptor. Optimum pH range, 7.5 to Eu.ro.pae’a. Gr. adj. europaeus of Eur- 8.0. Optimum growth temperature, 25 to ope, European. 30 C. Rods, 0.8 to 0.9 by 1.0 to 2.0 pm, occurring DNA base ratio: 54.1 moles % GC (buoyant singly, rarely in chains. Gram-negative. When motile, possess one to two subpolar flagella density; one species, one strain). which measure three to four times the length of Habitat: Soils from Brie, France; Island of the rod. Cells divide by binary fission and have Crete; the summit of Mt. Pilatus in Switzerland; cytomembranes which form flattened lamellae and the Parthenon in Athens, Greece. in the peripheral regions of the cytoplasm. Type species : N. briensis Winogradsky and Cells are obligate chemoautotrophs oxidizing Winogradsky. VOL 21,1971 FAMILY NITROBACTERACEAE BUCHANAN 267

Emended description of Nitrosospira briensis fulfill energy and carbon needs. Grow in fresh Winogradsky and Winogradsky 1933 water or seawater enriched with ammonia and inorganic salts, no organic growth factors being bri.en'sis. French Brie, place name; M.L. adj. required. Strictly aerobic, using oxygen as a briensis of Brie. terminal electron acceptor. Optimum pH range, Cells are tightly wound spirals with 3 to 20 7.5 to 8.0. Optimum growth temperature, 25 to turns. Short spirals have the appearance of 30 C. short rods and ellipsoidal cells; the spiral nature Range of DNA base ratios: 50.5 to 51.0 of the cells may not be evident when examined moles % GC (buoyant density; one species, with a light-field or a phase-contrast micro- three strains). scope. Width of spiral filament, 0.3 to 0.4pm; Habitat: Soils and seawater. amplitude of the spiral, 0.8 to 1.0pm. Small Type species: N. nitrosus (Migula) Bu- pseudococci are observed in senescent cultures. chanan. Gram-negative. Motile or nonmotile; when motile, cells are peritrichous, containing 1 to 6 Emended description of Nitrosococcus flagella 3 to 5 Prn in length. Grow in particu- nitrosus (Migula) Buchanan late-free liquid mineral salts medium as individual cells. ni.tro 'sus. M.L. adj. nitrosus nitrous. Cells are obligate chemoautotrophs oxidizing Large spheres, 1.5 to 1.7pm in diameter, ammonia and hydroxylamine to nitrite and with thick cell membranes. Motility has not fixing C02. Growth occurs with ammonia but been demonstrated. Stain readily with aniline not with hydroxylamine. Strict aerobes, but dyes. Zoogloea formation not observed. ammonia can be oxidized at reduced oxygen Growth medium consists of fresh water, tensions. Temperature range of growth, 15 to inorganic salts and ammonia. Aerobic cells use 30 C; optimum temperature for growth, 25 to oxygen as a terminal electron acceptor. Opti- 30 C; optimum pH for growth, 7.5. No sodium mum temperature, 20 to 25 c. chloride requirement; cannot be grown in sea- Source: Soils from Quito, Ecuador; water. Campinas, Brazil; and Melbourne, Australia. Cell suspensions show characteristic oxidized Habitat : Presumably widely distributed in minus dithionite reduced absorption peaks at soil. 423, 465, 522, 552, and 605 nm. These Emended description of Nitrosococcus absorption peaks represent cytochromes which impart a brownish straw like color to cultures oceanus (Watson) comb. nov. and a reddish color to a pellet. o.ce.an'us. L. mas. n. oceanus ocean. DNA base ratio: 54.1 moles % GC (buoyant Cells spherical to ellipsoidal, 1.8 to 2.2 pm density). in size. Gram-negative. Occur singly, in pairs, Habitat: Widely distributed in soils. and occasionally as tetrads either growing free Designated neotype strain: ATCC 2596 1 (isolated from soil from the Island of Crete; see in a liquid medium or as aggregates suspended Table 1 for description). in the medium. Cells divide by binary fission. Cysts were occasionally observed in mixed cultures but have not been found in pure Emended description of the genus cultures. Motile or nonmotile; when motile, Nitrosococcus Winogradsky 1892 the cells are peritrichous, with a single flagellum or a small tuft. Cells have cytomembranes Ni.tro.so.coc bus. M.L. nitrosus nitrous; Gr. which are arranged as flattened lamellae in the coccus grain, berry; M.L. mas.n. Nitrosococcus central region of the cell. nitrous sphere. Cells are obligate chemoautotrophs oxidizing Cells spherical, 1.5 pm or larger in diameter. ammonia and hydroxylamine to nitrite and Motile or nonmotile. When motile, the cells are fixing C02 to fulfill their energy and carbon peritrichous with 1 to 12 flagella. Gram- needs. Growth occurs with ammonia but not negative. Grow singly, in pairs or in tetrads with hydroxylamine. Strict aerobes, using oxy- either suspended free in liquid medium or gen as a terminal electron acceptor. Tempera- embedded in slime to form aggregates which are ture range of growth, 2 to 30 C; optimum either attached to vessel walls or are suspended temperature for growth, 25 to 30 C; optimum in the liquid medium. pH for growth, 7.5 to 8.0; optimum growth in Obligately chemoautotrophic bacteria which 70 to 100% seawater; will not grow in fresh oxidize ammonia to nitrite and fix C02 to water even when NaCl is included. 268 WATSON INT. J. SYST. BACTERIOL.

Cell suspensions show characteristic oxidized Cells composed of multiple ellipsoidal units, minus dithionite reduced absorption peaks at randomly arranged, forming lobular, pleo- 423, 465, 522, 552, and 605 nm. These morphic cells which are internally compart- absorption peaks represent cytochromes which mentalized, having one to five membrane- impart a brownish color to cultures and a bounded central areas surrounded by numerous reddish color to a pellet of cells. No other smaller membrane-bounded areas rich in glyco- pigments are present. gen deposits. Cells brain-like in appearance and Storage materials: Glycogen and poly- 1.O to 1.5 pm by 1 .O to 2.5 pm. Gram-negative. phosphates. Motile by means of 1 to 20 flagella randomly Range of DNA base ratios: 50.5 to 51.0 arranged; the flagella are 15.0 nm wide and 2.2 moles % GC (buoyant density; three strains). to 5.0 pm long. Grow in particulate-free liquid Habitat: Atlantic and Pacific Oceans. mineral salts medium as individual cells. Designated type strain: ATCC 19707 (iso- Cells are obligate chemoautotrophs oxidizing lated from North Atlantic seawater; see Table 1 ammonia, urea, hydroxylamine, and biuret to for description). nitrite and fixing COz. Growth occurs with ammonia and urea but not with hydroxylamine Description of the genus Nitrosolobus or biuret. Strict aerobes, although cells can Watson et al. 1971 oxidize ammonia at reduced oxygen tensions. Temperature range of growth, 15 to 30 C; Ni.tro.so.lob'us. M.L. nitrosus nitrous; M.L. optimal temperature for growth, 25 to 30 C; noun lobus a lobe; M.L. mas. noun Nitro- optimum pH for growth, 7.5. No sodium solobus nitrous lobe, i.e., a lobe producing chloride requirement; cannot be grown in sea- nitrite. water. Cells pleomorphic and lobate, 1.0 to 1.5 pm Cell suspensions show characteristic oxidized in diameter; division by constriction. Gram- minus dithionite reduced absorption peaks at negative. Cells partially compartmentalized by 423, 465, 522, 552, and 605 nm. These the invagination of the plasma membrane and absorption peaks represent cytochromes, which other segments of the cell envelope into the impart a brownish color to cultures and a cytoplasm forming vesicular regions. Motile, the reddish color to a pellet of cells. cells being peritrichous. Grow free in Storage materials: Glycogen and poly- liquid medium. Cells are rich in cytochromes, phosphates. which impart yellowish to reddish color to cell DNA base ratio: 54.6 moles % GC (buoyant suspensions; void of other pigments. density). Obligat ely chemoaut otrophic bacteria which Habitat: Widely distributed in soils. oxidize ammonia to nitrite and fix C02 to Type strain (original designation; originally fulfill energy and carbon needs. Grow in fresh cited in reference 19 as ATCC 25 19): ATCC water enriched with ammonia and inorganic 25 196 (isolated from soils from Surinam, South salts; no organic growth factors are required. America; see Table 1 for description). Strictly aerobic, using oxygen as a terminal electron acceptor. Optimum pH range, 7.5 to 8.0. Optimum growth temperature, 25 to REQUESTS FOR OPINIONS 30 C.

moides Winogradsky, Nitrosogloea membranacea Winogradsky, Nitrosogloea schizobac- Description of Nitrosolobus multiformis teroides Winogradsky, and Nitrosocystis java- Watson et al. 1971 nensis (Winogradsky) Starkey are regarded as mul.ti.for.'mis. L. adj. multus many; L. noun nomina dubia, and-the Judicial Commission is forma shape. M.L. adj. multiformis many asked to issue an Opinion placing these names shapes. on the list of nomina rejicienda. VOL 21,1971 FAMILY NITROBACTERA CEAE BUCHANAN 269

SPECIFIC PROPOSALS 8. International Code of Nomenclature of Bacteria. 1966. Int. J. Syst. Bacteriol. 16:459490. 1. The genus Nitrosococcus Winogradsky 9. Judicial Commission. 1958. Opinion 23. Rejec- will contain the large spherical organisms which tion of the generic names Nitromonas Wino- gradsky 1890 and Nitromonas Orla-Jensen oxidize ammonia to nitrite; the fact that the 1909, conservation of the generic names Nitro- type species, N. nitrosus, has not been culti- somonas Winogradsky 1892, Nitrosococcus vated does not affect the validity of this genus. Winogradsky 1892, and Nitrobacter Winograd- 2. Nitrosocystis coccoides Starkey IS sky 1892, and the designation of the type regarded as a later, subjective synonym of species of these genera. Int. Bull. Bacteriol. Nitrosococcus nitrosus (Migula) Buchanan. Nomencl. Taxon. 8:169-170. 3. Nitrosocystic oceanus Watson is trans- 10. Migula, W. 1900. System der Bakterien. 2 Gustav ferred to the genus Nitrosococcus; the name of Fisher, Jena. 11. Murray, R. G. E., and S. W. Watson. 1965. t lus organism therefore is Nitrosococcus Structure of Nitrosocystis oceanus and compari- oceanus (Watson) comb. nov. son with Nitrosomonas and Nitrobacter. J. 4. Nitrosomonas monocella Nelson is a later, Bacteriol. 89:1594-1609. subjective synonym of Nitrosomonas europaea 12. Nelson, D. H. 1931. Isolation and characterization Win ograd s ky. of Nitrosomonas and Nitrobacter. Zentralbl. 5.Nitrosospira antarctica Winogradsky and Bakteriol. Parasitenk. Abt. 2. 83:280-311. Winogradsky is a later, subjective synonym of 13. Pope, L. M., D. S. Hoare, and A. J. Smith. 1969. Nitrosospira briensis Winogradsky and Wino- Ultrastructure of Nitrobacter agih grown under gradsky. autotrophic and heterotrophic conditions. J. Bac teriol. 9 7 :9 36-93 9. 6. Nitrocystis sarcinoides Winogradsky, 14. Sack, J. 1924. Nitrobildende Bakterien. Zentralbl. Nitrocystis micropunctata (Winogradsky) Wino- Bakteriol. Parasitenk. Abt. 2.62: 15-24. gradsky, and Nitrobacter agi2is Nelson are later, 15. Starkey, R. L. 1948. Family I. Nitrobacteriaceae subjective synonyms of Nitrobacter winograd- Buchanan, p. 69-81. In R. S. Breed, E. G. D. skyi Winslow et al. Murray, and A.P. Hitchens (ed.); Bergey’s manual of determinative bacteriology, 6th ed. The Williams & Wilkins Co., Baltimore. ACKNOWLEDGMENTS 16. VanGool, A. P., R. Lambed, and H. Laudelout. 1969. The fine structure of frozen etched This investigation was supported by contract AT Nitrobacter cells. Arch. Mikrobiol. 69:28 1-293. (30-1)4138 reference no. NY04138-12 of the 17. Watson, S. W. 1965. Characteristics of a marine Atomic Energy Commission and by Public Health nitrifying bacterium, Nitrosocystis Oceanus sp. Service grant GM-11214 from the National Institute of n. Limnol. Oceanogr. 10:274-289. General Medical Sciences. 18. Watson, S. W. 1971. Reisolation of Nitrosospira bnensis. Arch. Mikrobiol. 75:179-188. LITERATURE CITED 19. Watson, S. W., L. B. Graham, C. C. Remsen, and F. W. Valois. 1971. A lobular, ammonia- oxidiz- ing bacterium, Nitrosolobus multiformis Nov. 1. Breed, R. S., E. G. D. Murray, and N. R. Smith. Gen. Nov. Sp. Arch. Mikrobiol. 76:183-203. 1957. Bergey’s manual of determinative bacteri- 19a. Watson, S. W. and M. Mandel. 1971. Compdson ology, p. 68-73, 7th ed. The Williams & Wilkins of the morphology and deoxyribonucleic acid Co., Baltimore. composition of 27 strains of nitrifying bacteria. 2. Buchanan, R. E. 1917. Studies on the nomencla- J. Bacteriol. 107:563-569. ture and classification of the bacteria. 111. The families of the Eubactenales. J. Bacteriol. 20. Watson, S. W., and J. B. Waterbury. 1971. Characteristics of two marine nitrite oxidizing 2 347-3 50. bacteria, Nitrospira gracilis nov. gen. nov. sp. 3. Buchanan, R. E. 1918. Studies in the nomencla- and Nitrococcus mobilis nov. gen. nov. sp. Arch. ture and classification of the bacteria. VI. Mikrobiol. 77: 203-230. Subdivisions and genera of the Spirillaceae and Nitrobacteriaceae. J. Bacteriol. 3 :175-1 8 1. 21. Winogradsky, H. 1935A. On the number and 4. Buchanan, R. E. 1925. General systematic bacteri- variety of nitrifying organisms. Int. Congr. Soil ology. The Williams & Wilkins Co., Baltimore. Sci. 1:138-140. 5. Gibbs, W. M. 1919. The isolation and study of 22. Winogradsky, H. 1935B. Sur la microflore nitrifi- nitrifying bacteria. Soil Sci. 8:427481. catrice des boues activkes de Paris. C. R. Acad. 6. Grace, J. B. 1951. Myxobacteria mistaken for Sci. 200: 1886-1888. nitrifying bacteria. Nature (London) 168: 117. 23. Winogradsky, H. 1937. Contribution a l’dtude de 7. Imsenecki, A. 1946. Symbiosis between myxobac- la microflore nitrificatrice des boues activdes de teria and nitrifying bacteria. Nature (London) Paris. Ann. Inst. Pasteur 58:326-340. 157:877. 24. Winogradsky, S. 1890. Recherches sur les organ- 2 70 WATSON INT. J. SYST. BACTERIOL.

ismes de la nitrification, Ann. Inst. Pasteur velles recherches sur les organismes de la nitrifi- 4 1257-275. cation. Ann. Inst. Pasteur 50:350432. 25' Winogradsky, s. 1891* les Organ- ismes de la nitrification. Ann. Inst. Pasteur 29. Winslow,'', -E. A*,J. Broadhurst, R. E. Buchanan, 5:577616. C. Krumwiede, Jr., L. A. Rogers, and G. H. 26. Winogradsky, S. 1892. Contributions i la mor- Smith. 1917. The families and genera of the phologie des organismes de la nitrification. bacteria. Preliminary report of the Committee Arch. Sci. Biol. St. Petersb. 1:86-137. of the Society of American Bacteriologists on 27. Winogradsky, S. 1931. Nouvelles recherches sur characterization and classification of bacterial les microbes de la nitrification. C. R. Acad. Sci. types. J. Bacteriol. 2:505-566. 192:1000-1004. 30. Zavarzin, G., and R. Legunkova. 1959. The 28. Wtnogradsky, S., and H. Winogradsky. 1933. morphology of Nitrobacter winogradskyi. J. Etudes sur la microbiologie du sol. VII. Nou- Gen. Microbiol. 21 :186-190.