INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1991, p. 82-87 Vol. 41, No. 1 0020-7713/91/010082-06$02.OO/O Copyright 0 1991, International Union of Microbiological Societies

Sarcobium Zyticum gen. nov., sp. nov., an Obligate Intracellular Bacterial Parasite of Small Free-Living Amoebae? WINCENTY J. DROZANSKI Department of General , Maria Curie-Skiodowska University, 20-033 Lublin, Poland

A new genus and species of obligate intracellular bacterial parasite of small free-living amoebae is described. This bacterium causes fatal infections in amoebae belonging to the Acanthamoeba-NaegZeria group. It does not grow on any artificial substrate deprived of living cells. The entry of the bacterium into a host occurs by , but growth occurs in the , not in phagosomes. This parasite is readily distinguished from other kinds of previously recognized bacteria that live within amoeba cells on the basis of its host cell lytic activity. The bacterium is a gram-negative, short rod with tapered ends. It multiplies intracellularly by a transverse central pinching-off process. Cells are motile by means of a polar tuft of flagella. The bacterium is surrounded by a distinct multilayered cell envelope with a chemtype A,y peptidoglycan. The peptidoglycan is unique in its high content of glucosamine residues with free amino groups. The DNA base composition of this organism is 43 mol% guanine plus cytosine. The name Sarcobium Zyticum gen. nov., sp. nov. is proposed for this bacterium. The type strain of S. Zyticum is strain L2 (= PCM 2298).

More than 30 years ago a new obligate intracellular nized as being unusual, and therefore I decided to attempt to bacterial parasite (OIBP) which causes fatal infections of classify it. hosts was found in a raw culture of amoebae that were freshly isolated from soil (7). This OIBP was isolated from MATERIALS AND METHODS soil from the Lublin area, Poland. In previous reports I have described the infection processes for Acanthamoeba castel- Organisms and isolation procedure. The OIBP, the caus- lanii, Hartmannella rhysodes, Hartmannella astronyxis, ative organism of fatal infections of small free-living amoe- Mayorella palestinensis, Didasculus thorntoni, Schizopyre- bae belonging to the Acanthamoeba-Naegleria group, was nus ruselli, and Naegleria gruberii, as well as 41 other found in a raw culture of amoebae that were isolated from unclassified amoebae isolated from soil and water reservoirs soil from the Lublin area, Poland (7)’ by using the enrich- (8, 10). The OIBP did not grow on any medium when living ment method. Crumbs of soil were placed onto the surface of amoebae were not present. Even a thick suspension of a non-nutrient agar plate covered with a thin film of a thick disrupted amoeba cells was ineffective. It has been shown suspension of Aerobacter aerogenes and incubated at room that the OIBP cannot multiply if after infection the mixed temperature for 8 to 10 days. In this way the growth of all culture is heated at 43°C for 10 min; this treatment is microorganisms except those that feed on bacteria or para- sufficient to kill trophic forms of the amoeba, but the sitize bacterium feeders was checked. When this procedure bacterium survives. Microscopy has shown (8, 11). that the was used, several different isolates which could infect amoe- parasites are readily phagocytized by amoebae and that their bae were obtained. The isolate described in this paper was immediate place of residence is within phagosomes. The designated strain L2T (T = type strain). Strain L2T was neighboring phagosomes fuse extensively with parasite-con- purified from contaminating bacteria by allowing infected taining ; however, even dead bacteria evade lyso- amoebae to migrate on agar surface as described previously soma1 attack because the is resistant to the host’s (8). A two-member (host-parasite) culture was established bacteriolytic enzymes. Later, living bacteria escape from by growing infected amoebae in PYG medium (8). phagosomes into the cytoplasm, where multiplication oc- Acanthamoeba casteltanii, which was obtained from W. curs. The final effect of the infection is total lysis of the host Balamuth, Department of Zoology, University of California, cells. Although the morphology of the OIBP and the pres- was used as a host for maintenance of the OIBP. ence of peptidoglycan in its cell wall suggest that this Propagation of the OIBP. The OIBP was propagated by organism is a bacterium, it has not been possible to classify transferring a small amount of lysate from an ipfected culture it previously because of a lack of nutritional tests; the OIBP into a 2-day-old culture of amoebae grown on PYG medium does not correspond to any bacterium described in Bergey’s as described previously (8, 24). Bacteria were also propa- Manual of Determinative Bacteriology (3). Most of the many gated on amoeba cells harvested from an axenic culture at kinds of previously recognized bacteria that live within the exponential phase of growth and suspended in saline amoeba cells behave as temperate endosymbionts, and some prepared as described by Band (2). The OIBP propagated on appear to provide benefit to the hosts (1,4,15-18,23,25). To amoebae suspended in saline produced more uniform cells my knowledge, the only exception is the OIBP, which and survived for a longer period of time than the OIBP causes lysis of the amoeba cells. This organism was recog- propagated on amoebae in PYG medium (9). Microscopic observations. Observations on the course of infection were made with living materials by using phase- contrast microscopy and fixed preparations stained by the t Dedicated to W. J. H. Kunicki-Goldfinger on the occasion of his methods of Macchiavello (5) and Gimenez (15). The stains 70th birthday in friendship and admiration. used for bacteria liberated from their hosts were the stains

82 VOL. 41, 1991 SARCOBIUM LYTICUM GEN. NOV., SP. NOV. 83

FIG. 1. Phase-contrast micrograph of Acantharnoeba castellanii 2 h after infection with the OIBP, showing enlarged parasitophorous vacuoles containing a few parasites. Bar = 10 pm.

described by Norris and Swain (22). For transmission elec- tron microscopy, infected amoebae were fixed in amoeba saline supplemented with 1.5% glutaraldehyde and postfixed in Veronal-acetate-buffered OsO,. Following dehydration in ethanol, pellets packed by centrifugation were embedded in Vestopal W (Serva). Thin sections of embedded specimens FIG. 3. Electron micrograph of a section through an Acan- were cut with a Tesla model BS490A ultramicrotome and tharnoeba castellhi cell infected with the OIBP, showing an stained with uranyl acetate and ~~~~~ldlead citrate. sam- enlarged parasitophorous containing a few bacteria and ples for negative staining with sodium phosphotungstate structures. Bar = pm.

were prepared as described previously (12). Grids were examined with Zeiss model DZ and Tesla model BS613 electron microscopes that were operated at 50 or 80 kV. Oxygen consumption measurements. A conventional War- burg respirometer was used for oxygen consumption mea- surements. Each flask usually received 4 x lo1' OIBP cells suspended in saline and substrate to bring the total volume to 2.0 ml. The bacteria were prepared by the procedure de- scribed below. A 2-day-old axenic culture of Acanthamoeba castellanii, which had reached a concentration of 3 X lo6 cells per ml, was infected with the OIBP. The ratio of bacteria to amoebae at the time of infection was approxi- mately 7:l. The infected amoebae were incubated on a shaker at 28°C. Bacteria liberated from the infected amoebae were pelleted and freed from cysts and unbroken host cells by fractional centrifugation at 120 and 3,000 x g, resus- pended in saline, and starved for 2 h on a shaking water bath. Respiration measurements were carried out at pH 7.4 and 25°C for 2 h with a gas phase of air. CO, was trapped with 0.2 ml of 20% KOH. DNA extraction and estimation of base composition. DNA was extracted from bacteria frozen in saline (0.15 M NaC1, 0.1 M EDTA, pH 8.0) after disruption of the cells in 2% sodium dodecyl sulfate at 60°C and then purified by using the FIG. 2. Phase-contrast micrograph of Acantharnoeba castellanii method Of Marmur (20)*The purified DNA were 8 h after infection, showing one large lacuna (long arrow) filled with dissolved in ssc (0.15 M NaCl Plus 0.015 M sodium citrate) the bacteria and several enlarged parasitophorous vacuoles (short and stored at 2°C Over chloroform. Base ComPosition was arrows). Bar = 10 pm. determined by performing a melting point analysis, using the 84 DROZANSKI INT. J. SYST.BACTERIOL.

FIG. 5. Micrograph of Acantharnoeba castellanii, showing bac- teria surrounded by electron-transparent zones. Bar = 1 pm.

FIG. 4. Micrograph of Acanthamoeba castellanii, showing a parasitophorous vacuole with a stream of cytoplasm inside (arrow- heads). Bar = 1 pm. plasma membrane liberated about 2 x lo3 bacteria into the environment. An examination of ultrathin sections of Acanthamoeba method of Marmur and Doty (21) and DNA from Escherichia castellanii by transmission electron microscopy revealed coli (51 mol% guanine plus cytosine) as a reference. that soon after infection bacteria engulfed by host cells were tightly surrounded by membranes of endocytic vacuoles. Later in infection the parasitophorous vacuoles increased to RESULTS abnormal sizes because of the fusion of neighboring en- Microscopic observations. (i) Microscopy of infected amoe- docytic vacuoles containing bacteria with each other and bae. Typical Acanthamoeba castellanii cells which were with (Fig. 3). At this stage of infection no division infecied with the OIBP are shown in Fig. 1and 2. At 2 h after figures of bacteria were observed, and parasitophorous infection the cells remained remarkably intact. When phase- vacuoles were filled with membranelike structures scattered contrast microscopy was used, differentiation of the cyto- in an electron-translucent background, as well as with a few plasm into ecto- and endoplasmic layers was clearly ob- bacteria. Later in infection the membranes of bacterium- served. Also, the nuclei and other cell structures appeared to bearing parasitophorous vacuoles underwent lysis, and par- be physically undamaged. However, the food vacuoles bear- asites escaped into the cytoplasm (Fig. 4 and 5). The ing bacteria became enlarged, yet they were not filled with electron-translucent space surrounding bacteria gradually OIBP cells. Within 6 to 9 h after infection, although the underwent resorption, and as a consequence bacteria came bacteria were present in almost all of the food vacuoles, no into direct contact with the host cytoplasm, where they were distinct morbid changes in host cells were observed. The clearly distinquished from other subcellular components differentiation of ectoplasm and endoplasm was still main- (Fig. 6). Once the bacteria were inside the cytoplasm, tained, and the host nuclei were prominent and appeared to multiplication started. In the last stage of infection lacunae be physiologically intact. The amoebae moved normally, filled with dividing bacteria increased in size, and the prolif- collected food, and even reproduced; however, the invading erating parasites disarranged the host cell structure, causing bacteria had increased in numbers and occupied a large area autolysis of mitochondria and disruption of the plasma of the host endoplasm. In the later stages of infection some membrane (Fig. 7). Division of the parasites was not ob- bacteria remained in the parasitophorous vacuoles, but most served outside the amoebae cells; however, the liberated of them were found in the cytoplasm, where lacunae filled bacteria remained alive for several months after being re- with parasites were formed. In heavily infected cells the leased from their hosts. were displaced by bacteria. In the last stage of (ii) Morphology and fine structure of the OIBP. The bacte- infection the host cells took on a ball-shaped form and were ria liberated from host cells were straight rods with tapered filled with rapidly moving bacteria. The rupture of each ends. The mean size of the OIBP was calculated from VOL. 41, 1991 SARCOBIUM LYTICUM GEN. NOV., SP. NOV. 85

FIG. 6. Micrograph of Acanthamoeba castellanii, showing bac- teria scattered in the host cytoplasm (arrows). Bar = 1 km. FIG. 7. Micrograph of Acanthamoeba castellanii, showing lacu- nae filled with bacteria. Bar = 2 Fm. organisms stained with phosphotungstic acid, was 0.6 pm wide by 1.9 pm long. Cells more than 5.0 pm long generally occurred in poorly aerated cultures. The surfaces of nega- content of glucosamine residues with free amino groups in tively stained bacteria were electron opaque and smooth. On the glucan strands of the peptidoglycan (13). cells immediately liberated from their hosts tufts of flagella Oxygen consumption. Purified suspensions of the OIBP on one pole were observed (Fig. 8). The maximum number exhibited metabolic activity, as shown by consumption of of flagella observed at a single tuft was five. Phase-contrast oxygen and production of carbon dioxide in the presence of microscopic observations of bacteria freshly liberated from disrupted amoeba cells or PYG medium. Freshly collected hosts revealed the presence of motile rods. Motility occurred bacteria in buffered saline had an endogenous Qo, of 24 k 3 within the hosts and decreased rapidly after the bacteria Fl per lolo cells (Q~?(~lolo cells) = pl of O2 taken up per 1 were liberated into the ambient medium. The bacterium x lo1' cells per h). A homogenate of Acanthamoeba castef- were gram negative. Neither capsules nor spores were lanii prepared from a suspension containing 2 x lo7 cells per observed. Thin sections showed that the OIBP was essen- ml did not consume 0, but stimulated respiration of the tially a gram-negative bacterium with a trilaminar outer OIBP fivefold compared with endogenous respiration. Glu- envelope and a cytoplasmic membrane (Fig. 9). With the cose and related sugars, as well as glutamate and amino technique used no definite structures were observed in the acids synthesized by Acanthamoeba castellanii or included cytoplasm that might be interpreted as spores, beta-hydroxy- in the synthetic medium to support the growth of amoebae butyric acid, or starch granules. The cytoplasm contained an (6), were not metabolized by the OIBP. KCN at a final obvious nucleoplasm which appeared to be a filamentous concentration of 5 X M invariably inhibited all respira- skein distributed along the entire length of the bacterium. tory activities of the OIBP. The part of the cytoplasm that was not occupied by the DNA base composition. The DNA of the OIBP contained nucleoplasm contained numerous small electron-dense gran- 43 mol% guanine plus cytosine as determined by thermal ules that probably represented ribosomes. melting point analysis. Peptidoglycan. The procaryotic nature of this organism was established by the discovery in its cell wall of com- DISCUSSION pounds that are regarded as specific to bacteria (e.g., the bag-shaped peptidoglycan composed of glucosamine, mu- Certain species of free-living amoebae harbor microorgan- ramic acid, alanine, glutamic acid, and diaminopimelic acid isms. Many of these microorganisms are the same size as in a molar ratio of 0.4:0.4:1.4:1:1)(12).The peptidoglycan ordinary bacteria, and some have been identified as archaeo- was completely insensitive to lysozyme and bacteriolytic bacteria. Unfortunately, because of their specialized ecolog- N,O-diacetylmuramidase of amoeba1 origin. The resistance ical niche, none of these organisms can be cultivated apart to the bacteriolytic enzymes was presumably due to the large from the eucaryotic cells which they inhabit. Consequently, 86 DROZANSKI INT. J. SYST.BACTERIOL.

FIG. 8. Electron micrograph of negatively stained OIBP with a tuft of flagella at one pole. Bar = 1 pm.

FIG. 9. Electron micrograph of a section through cells of the little information that permits assessment of the taxonomic OIBP. The ultrastructure of this bacterium resembles that of gram- affinities of these organisms has been obtained. However, negative bacteria. Bar = 1 pm. the assignment of names to organisms which have not been cultivated in vitro is feasible partly because of our increasing knowledge of the bacteria living inside eucaryotic cells and Description of Sarcobium lyticum sp. nov. Sarcobium lyti- partly because of the fact that the most reliable taxonomic cum (ly’ ti. cum. M. L. neut. adj. lyticum, that which causes characteristics are DNA base ratio and the chemical corn- lysis). Description as for the genus. Type species of the position of cell walls. Most of the previously described genus Sarcobium. The type strain of Sarcobium lyticum is bacteria which inhabit amoeba cells have one feature in strain L2,T which has been deposited in a two member common; namely, they behave as temperate parasites. Some culture with the Polish Culture Collection of Microorganisms even provide benefit to the host. The only exception to my as culture PCM 2298. The description of the type strain is the knowledge is the OIBP described in this paper. The uncon- same as that given above for the genus and species, and, in trolled growth pattern of this organism in the cytoplasm of the absence of cultivation in pure culture, this description amoebae belonging to Acanthamoeba-Naegleria group, its forms the nomenclatural type Rule 18a of the International DNA base composition, and the chemical makeup of its cell Code of Nomenclature of Bacteria [19]). wall peptidoglycan are sufficient to warrant placing it in a new genus. The proposed new genus Sarcobium does not ACKNOWLEDGMENT appear to belong to any of the currently recognized families This work was supported in part by a grant from the Committee of of bacteria. Microbiology of the Polish Academy of Sciences. Description of Sarcobium gen. nov. Sarcobium (Sar. co’ bi. urn. Gr. n. sarcos, flesh; Gr. n. bios, life; M. L. neut. n. REFERENCES Sarcobium, that which lives in the sarcode or flesh [cyto- 1. Ball, G. H. 1969. Organisms living on and in , p. plasm]). Rods that are 0.6 by 1.9 pm. Gram negative. Motile 565-718. In T.-T. Chen (ed.), Research in protozoology, vol. 3. by means of three to five polar flagella. Cell division occurs Pergamon Press, London. by a transverse central pinching-off process. The DNA base 2. Band, R. N. 1959. Nutritional and related biological studies on composition is 43 mol% guanine plus cytosine, as deter- the free-living soil amoeba Hartmnnnefla rhysodes. J. Gen. mined by the thermal denaturation method. Chemotype A,? Microbiol. 20:80-95. peptidoglycan with a free amino group on the glucosamine 3. Buchanan, R. E., and N. E. Gibbons (ed.), 1974. Bergey’s manual of determinative bacteriology, 8th ed. The Williams & residues. Obligate intracellular parasite of amoebae belong- Wilkins Co., Baltimore. ing to the Acanthamoeba-Naegferiugroup and related pro- 4. Chapman-Andresen, C. 1971. Biology of the large amoebae. tozoa (e.g., Dictiostelium discoideum). The bacterium is Annu. Rev. Microbiol. 252748. able to escape from parasitophorous vacuoles into the cyto- 5. Conn, H. J., B. Cohen, M. W. Fennison, L. S. McClung, and plasm, where it proliferates. A. J. Riker. 1951. Staining methods, p. IV,,-3-IV5,-24. In VOL.41, 1991 SARCOBIUM LYTICUM GEN. NOV., SP. NOV. 87

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