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1979

Characterization and identification of an isolate of halobacterium from Soda Springs Mojave Desert

Abayomi Odubela

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Recommended Citation Odubela, Abayomi, "Characterization and identification of an isolate of halobacterium from Soda Springs Mojave Desert" (1979). Theses Digitization Project. 67. https://scholarworks.lib.csusb.edu/etd-project/67

This Thesis is brought to you for free and open access by the John M. Pfau Library at CSUSB ScholarWorks. It has been accepted for inclusion in Theses Digitization Project by an authorized administrator of CSUSB ScholarWorks. For more information, please contact [email protected]. CHARAG'liERIZATION AND IDENT'IFICATION GF AN ISOLATl OF HALOBACTERIUM FROM SODA:SPRINGS. MOJAVE DESERT —,

A Thesis

Presented to the

Faculty of

California State

Cblleges San Bernardino

in Partial FulfilIment of the.Requirements for the .Degree

Master of Science in: • .

Biology

hy .

Abayomi Qdubela

April 1979 CHARACTERIZATION AND IDENTIFICATION OF AN ISOLATE OF . HALOBACTERIUM FROM SODA SPRINGS MO J AYE 'DESERT

A Thesis ..

Preisented to the

Faculty of

California State

College, San Bernardino

by

Abayomi Odubela

April 1979

Approved bys

Gh^i»arson Biol _ Date Graduate Committee

Corama^tee Member

Co mi itt Member

Mayor Professor ■ .

Representative of^he Graduate^ Dean ABSTRACT : : A strai|i of Halobacterium . isolated from a vond at the California State Universities and Colleges Desert . Studies Center |at, Soda Springs, Mojave Desert, California „ was studied in order to define its taxonomic placeuin the genus Halohactelrium. ■ \

To defihe the characteristics, of this 'bacterium, ' , standard physiological activities, as listed in Bergey's Manual of Determinative Bacteriology, 8^ Ed. (Gibbons 197^) were studied, lAdditionally, the red uifTient produced, by ; the ■organis.m wa|s ' charaterised, , and,.,the, molar 'percent 1; ; .; guanine plus cytosine determined, ; 1 The orgianism is aerobic, non-motile, Gram*", and pleomorphic, exhibiting both swollen rods and coeooid .cell ' : form. It grows: well at a temperature between 3?^ "to 33°C, and requires at:, least 18?! NaCl for growth, v Optimuxn occurs at a NaCl ooncentratioh between 25^ and. 30%. It ' V ; ' does not liquifif gelatin, indols is not produced and.the . ' organism doeS' not. reduce .nitrate,- nor produce■ nitroge.n. There is no une-fuivocable evidence that;:the' organism ,uti.Iizas . .. carhohydrates, The red pigment in the organism is ■ ; bacterioruberin, as supported, by spectral and TLG mobility data. The DNA Of the organism consists of two components having'59 and 6|8 molar percent guanine plus cytosine : respectively,.

The organism is,classified as a strain, of Halobacterium salinarium, on the basis of the various characteribties indic8.te,d. above:-. . ^ ' ACKNOWLEDGEMENT

I would like to thank Dr. Dalton Harrington, my major professor, for his guidance, trust and help throughout my graduate studies. Thanks to Drs. Miller and Egge for reading the first draft of my thesis and their suggestions. Special thanks are due Mr. Richard Fehn of Loma Linda University for his assistance with the DNA studies and Dr. George Lessard of Loma Linda University for the use of his laboratory for the DNA studies and for his help in the analysis of my data.

My deepest thanks go to my mother and late father for their love, financial and moral support up to this point of my education.

Finally my sincere thanks to my wife, Bola, for her understanding and encouragement throughout my graduate studies and for her assistance in the preparation of this thesis.

This thesis is dedicated to the memory of my late father Mr. Gilbert 0. Odubela. TABLE OP /CONTENTS

Page

Introduction f: . . . ■ 1

Materials and Methods™— 5;

Results —--r-­ 16

Discussion --- 23

Literature cited 28 VI

LIST OP TABLES

Table 1. Growbli of Halobacterium sp. from Soda Springs in BSIvlK , suppriiiented with various sugars, (initial pH 7»05 incubated for 96 hrs. at 37 C) ■"— ­ 19 vil

LIST OF FIGURES ­

Fig. 1. Structure ofs^-Baeterioruberin 4 Fig, 2. Gompa:rison of the growth of Halobacterium . sp, in; CM and SM. . IS;: Fig. 3. Spectrjum in acetone of the pigment isolated, from the halophilic bacterium from Soda Siprings. 20; Pig, 4. AbsorbanGe/Temperature Plot of the DHA of .the Halobacterium sp. heated in 0..iM " ■ salinercitrate, 22' Fig. 5» Blosynjfchetic pathways of CeQ-bacterioruberin INTRODUCTIONi Halophiles are organisms that require, or are able to grow in, high salt concentrations. They are aerobic and grow at a temperature between 37'^G-45°C (Gibbons 1969» Gibbons and Payne 196I). There are moderate and extreme halophiles, so differentiated on the basis of the salt concentration required for their growth. The moderate halophiles require 3-15?^ salt concentrations for growth, while extremej halophiles require 155^ or higher (Gibbons 1969)« Halophilic bacteria are divided into two groups, Halobacterium, which is rod-shaped, and HalococcusgWhich is spherical. Most physiological studies have been done with Halobacterium. However, this organism is more sensitive than Halococcus to changes in salt concentration, lysing of the cells occuring at lower salt concentrations (Kushner I968). The economic impact of the fact that halophilic bacteria spoil fish, bacon and hides stimulated early research on these organisms (Browne 19^7» Lochhead 193^)• Later interest in halophiles was directed to the basic physiology of these organisms because of their high salt requirement (3/^-25/^» salt) as compared to the normal physiological salt requirement of 0.9% for most organisms

(Gibbons 1969). The requirement for this level of salts in the medium is considered the result of adaptation to a severe environment and involves many specialized biochemical events relative to growth. Relatively few strains of these organisms have hjeen studied., Gomparedjl to other bacteria, halophilic bacteria are much more difficult to culture because of their high . salt requirementi,, Also it taices longer to overcome the lag period (Buchanan 1918) before they grow, if at all they grow (Gochnauer iand Kushner 1969» Weber 19^-9)« While some attempts have been made to find a cheadically-defined medium for halophilic bacteria (Weber 19^9? Katznelson and Lochhead 1952, dnishi et al 1965» Grey and Pitt 1975) generally satisfjactory medium for all strains has not been

.developed. - ^ . To be able to study the metabolism of this red and extremely halophilic bacterium, a suitable chemically-

defined growth medium wasnecessary. For this study, the synthetic medium of Grey and Pitt (1975) was ;used, which is a modification of the synthetic medium of Onishi et ai (1965)« The synthetic medium of Grey and Pitt (1975) differs from that of Onishi et al (1965) in that it contains three times higher phosphate concentration. Only L-araino acids are used, and ammonium chloi'ide and nudleotides are omitted from the medium. The synthetic medium; of Grey and Pitt (1975) has been used for this study because it is the least complex medium available from the compared literature, "Grey and Pitt (1975) showed

that the nucleotide fraction is not needed in the synthetic

medium since it 'only serves as an additional source of phosphate in a medium deficient in phosphate, The nucleotides are degraded to nucleosides in the culture Riedia and

nucleosides havd'no effect on growth.

These "bacteria obtain their energy requirements

from amino acid metabolism. They do not utili2se carbohydrates

as a carbon source, for the most part. However, low level utilization has ibeen reported for some strains (Gibbons 197^)•

One species, H. ^marismortui, is reported to metabolize yarious carbdhydrates as energy sources and to produce gas from carbohydrates (Breed et al 1957)» Several new strains of rod'-shaped hdlophilic bacteria that utilize carbohydrates in nutrition and produce acidic compounds during growth have recently been idolated (Tomlinson and Hochstein 1972 a, b).^ Cells of;extremely halophilic bacteria in various : media are red. This red color is due to the presence of fat-Soluble carotenoid pigments (Baxter i960), fetter

(1931) ahovifed that the red color in Halobaeterium is due to the presence bf two carotenpids which she designatedi^­ and^-bacterioruberin. The^-bacterioruberin pfedominates with absorption taaxima at tj.67, 490, and 522 nM in methanol. •bacterioruberih exihibits maxima at 453, 482, and 504 nld, Baxter (i960) confirmed Fetter's findings. Based on similarities in absorption spectra i^-bacterioruberin is considered to bei identical with spirilloxanthin (Lederer

1938), a carotenoid pigment isolated from Rhodospirillum rubrum (Van Neil and Smith 1935)• Lederer (1936) suggested .^-bacterioruberin as a demethylated derivative of spirillo* xanthin. However, Kelly et al (1970) provided evidence that '^~bacterioruberin differs from spirilloxanthin., They (Kelly £t al 1970) found that«^-bacterioruberxn (see Fig.1) is a C^Q-tetraol with a molecualr formula (G2qH^2^0H)jij,)s ^nd spii'illoxanthin a C^Q~diol.

I i

FlgT™!I Structure of «2<"Bact8rioruberIn ' In this jStudy the physiological requirements and identification oif a Halobaeterium species isolated from a pond located ajt Soda Springs, Mojave Desert, are considered. Included are grojivth studies in complex and defined media, ; effects of carboiiydrates on growth in defined medium, delineation of standard physiological activities in , consistence with those included in BergeyVs. Manual of

Determinative Bacteriology 8 Ed. (Gibbons 197^}-) for the genus in general, and isolation and characterisation of the red pigment in the organism. With the data accrued in the study, it was possible to define this strain as to its taxonomic place in the Halobaeterium. MTERIALS AND METHODS; , . , , ; .

Source of Organism and Habitat Characteristic^t ,

The organism was isolated from salt crystals

obtained from the surface of a pond at the California State

Universities and Colleges Desert Studies Center at Soda

Springs, iVlojave Desert, California. This pond forms a thick surface crust of orange-red salts with an orange-red

solution under it during the summer season. The bacteria

are found primarily inside the salt crystals and not in

the water. In spring and winter seasons the pond is orange-red with no crystals on the surface. The bacterium,

in this case, is found, primarily in the mud at the bottom of the pond rather than suspended in the solution.

Growth Mediag a,} Modified Complex Medium of Sehgal and Gibbons (Gochnauer et ^ 1972), referred to as CM for the rest of this report,, was used for initial plating of the organism. All ingredients are expressed as^ gm/liter excepf for iron, - and includes ' t.: Difco Casamino Acids (vitamin free) 7.5

Difco yeast extract 10.0 MgSOi^.yHgO; 20.0 Sodium citrate 3,0

, KCl , 2.0­ NaCl ' 250,0. Ferrous ion; (Fe**"^) was supplied by adding 1 ml of stock solution of Uf.9/^ FeS02^.7H20 (lO.OOOppm in .OOIN HGl to each liter of CM to provide a filial concentration of 10 ppm Fe"^^, The pH was adjusted to 6.6 with 1.0 N NaOH. The medium was sterilized at 122°G and 18 lb pressure for 15 minutes. Using aseptic techniques, agar (1.5%) was added to the sterile solution to provide the final plating medium. b.) Fish Broth-Peptone-Agar (Lochhead 193^)t referred to in the rest of this report as FPA, was used as a second plating medium. A fish broth was prepared by steaming 500 gm of minced fresh cod fish in 1000 ml of tap water for one hour. The steamed preparation was then filtered through several layers of cheese cloth, and the pH of the filtrate adjusted to 8.2 by addition of 1.0 N NaQH. The broth was diluted It 1 before use. To 1.0 liter of diluted broth was added the following substances in gra/liter«

Peptone 1.0 ■ ■

Glyceroi 5.Q ' ■ .NaCl' . 250.0 ■ , 1 .

Agar 15jO The FPA medium was autoclaved at 122°G and 18 lb pressure for 15 minutes and then poured into petri plates. c.) Synthetic Medium (Grey and Fitt 1975)» referred to as

SM in the rest of this report, was used to grow standard broth cultUz-es. Amino acids to prepare 1.0 liter of the complete SM includes

L-alanine 43 mg

L-arginine 40 mg Xj-cysteine,HC1,.H20 5 mg

L-glutamic acid 130 .mg

Glycine 6 mg

L-isoleucine hh rag

L~leucine 80 mg

L-proline 5 rag

L-serine. 61 rag

L-lysine 85 rag.

L-methionine- 37 rag

L-phenylalanine 26 mg

L-threonine 50 .rag

L-tyrosine 20 rag

L-valine 100 rag other components added to the 1.0 liter volume include

NaCl 250.0 t?m ; ; MgS0i|,.7H20 2.0.gm Glycerol 0.10 gm CaGlg.EH^O 0,0? tng FeOl^.tegO . 0.23 rag ,

100,0 mg K2HPO11 15'0 ing KNO3 . 10.0 mg. Sodium citrate 50.0 mg

15.0 mg 2nS0i^.7H20 M.O^g OuSO/,^.5H2^ 5.oytg MnS02^.H20 30,0 /4g 8

The ingiredients were obtained from separate stoclc solutions (l?ow/V) of each amino acid and. of the minor elements and combined as required. The pH was adjusted to 6.6 with 1.0 K KOH before dilution to final volume, and the' medium was autoclaved at i21°G .and 18 lb pressure for 15 minutes. The synthetic medium (SM) was modified into Basal Synthetic Medium Potassium (BSMK) which was prepared by incorporating KCl in the amount of 200 mg/lOO ml in the Basal Synthetic iMediura {BSM) which is SM less the glycerol. . BSM,, supplemented with various sugars, was used to determine if the isolatedihalophilic bacterium metabolises carbohydrates.

I , ■ ■ ■ . ■ ■ ■ . ■ ■ ■ ■ ■ ■ Isolation Procedures> Salt crj/^stals (2.75 gm) from the pond were placed in 10.0 ml CM of FP broth Containing 25% NaCl. The culture was incubated at 37°G in a shaking water bath until the liquid turned red (in about seven days). The culture was centrifuged -for 10 minutes at 5000 to 7000 x g, then resuspended'in 5.0 ml sterile saline (25%) solution. FPA plates were streaked with 0.2 ml aliquots of the centrifuged culture and incubated at 37°G in sealed plastic bags.

After tvro weeks colonies with variable intensity of pigmentation

appeared. These colonies were subcultured on fresh FPA plates which wepe streaked for isolation and then incubated

as described' aboye. After two weeks, uniformly pinkish-red colonies were folrmed. A representative colony was maintained

on a FPA slant. Physiological T^stss Inocula |for various physiological tests were "l ' ' ^ ' .■ ' ■ -o' prepared by growing the organism in GM incubated at 37 G for ^8-72 hoursJ The CM cultures were centrifuged for 10 itiinutes at 0°|G, at 5000 to 6000 x g. The lowered temperature was necessary to prevent lysis of cells during ■ ■ ■ ■ ! , , ■ ■ ■ ■■ . centrifugation, , The pellet was suspended in 25% NaGl and 0.2 ml inocula w|ere used for various tests in which the

final solution volume was 10 ml. For some tests* 0.^ ml of inoculum was hade up to a final culture vplume of 20 ml.

Nitrate reduction, indole formation and gelatin riquification v/ere determined, in CM supplemented with 1% KNOj, 1% t,rypton^ (Difco) and 12% gelatin respectively. In the case of nitrate reduction and indole formation, 20 ml of medium was dispersed into 2.5 x 15 cm test tubes and 0,^ ml of inoculum added. To test for nitrogen evolution, Durham tubes were placed into the hitrate-containing cultures. After autoclaving the apparatus, it was allowed to cool to room temperature. Int^culation of the medium with the organism was follov/ed by incubation at 37°G for* two weeks. The p extended length of incubation was consistent with the relatively slow pate of growth of the organism.

P To test for indole formation, 0.8 ml of .Kovac's Reagent (pifco 1^53) was added to a 24-hour culture of the organism in the tryptone-supplemented medium. The culture 10 had heen incubajted at 37°G until addition of the reagent. Qelatini liquification was tested by inoculating petri plates ofj CM plus 12?^ gelatin, incubating the cultures at room temperature (approximately 31°C) for seven days.

The incubation temperature was sufficient to cause the gelatin to initially solidify. The eff'ects of different sugars (glucose, galaetose, sucrose, lactosb* trehalose) on the growth of the organism Were determined by adding the sugars to a concentration in separate BSMK culture flasks equipped with side arms.

The side arms the culture flasks are of the dimensions

necessary for insertion into the sample holder of the Klett-Summerson, colorimeter. Thus, turbidity measurements were made directly of the culture, with dimihished chance of error that cbuld arise if a culture sample was transferred

into a separate cuvette. Turbidity was measured with a

number h2 blue filter in the colorimeter,.with changes in

turbidity with time indicative of growth rates of the organism. Turbidity readings were made at 2h-hour intervals for 96 hours.

Concoinmitant with the measurements of turbidity

in these cultures, changes in pii were monitored with a

Corning low-sodium-error combination microelectrode to

determine whether acidic substan.ces were being produced by

■the organism. ; '

Additionally, motility of the organism was tested 11 in CM using thei hanging-drop technique. This technique allows ready determination of motility of a bacterium in a drop of culture fluid Buspended beneath a cover slip and viewed under the compound microscope. To test the effect of varying NaCl concentration on cell morphologys the cells were viewed in condition of 15% and 10% NaCl strength as■well. ■ Staining^ ■ ' ­ For the study of cellular forms the Dussault method of staining halophilic bacteria was used (Dussault 1955)• One large loopful of the broth culture, or suspension of the organism in!25^ NaCl solution, is spread on a clean microscope slide and allowed to air dry. It is then simultaneously fixed and desalted by immersing in 2?^ acetio acid for five minutes. The slide is then removed and dried unwashed. After drying, the smear is covered with a 25?^ aqueous solution of crystal violet and allowed to stand for three minutes. This is followed by rinsing excess crystal violet from the slide with water. After drying, the slide is ready for microscopic study.

Iso3.ation- of Deoxyribonucleic Acid in Biologically-Active

Forms ■ Following the procedures of Hotchlciss (1957)»' fbe organism was cultured in 500 ml of CM for 72 hours. The culture was centpifuged for 10 minutes at 2500 to 5000 x g at 5^C, and the supernatant fluid removed by decantation.' 12

The cells were resuspended without washing in O.IM standard salihe-citrate (SSC), using about 0,3% to 1% of the original culture volume. The cells were then lysed by adding 1.0 ml of 5% sodium deoxycholate per 25 ml of the culture suspension and left at room temperature for one to two minutes after which time the solution became viscous. An equal volume of chloroform and 1/40 volume of isoamyl alcohol were added, and the mixture mechanically shaken in a wellrstoppered centrifuge tube for 15 to 20 minutes. This treatment resulted in protein interfacial denaturation. The centrifuged mixture separated into three layerss a lower chloroform layer, an intermediate layer of denatured protein-chloroform emulsion^ and a cloudy, yellowish, viscous supernatant layer containing the DNA, The top layer Was pipetted into another centrifuge tube and precipated with 1,5 volumes of 95% ethanol. This resulted in formation of a stringy precipitate. The precipitate was promptly oollected by winding on a glass rod. Residual stringy precipitate was collected by slow centrifugation. The collected precipitates were returned separately to saline'­ citrate (10 ml SSC per 1.5 liters of original culture volumej.

This was accomplished within 30 minutes in order for the precipitates to dissolve easily,

To remove ribonucleic acid, 0.05 mg of crystalline ribonuclease v/as added per 10 ml of suspension. The solution was incubated at 37°0 for 15 minutes. A second, 0,05 mg 13

portion of the enzyme was then added and incubation continued for another 15 minutes. One and one-half volumes of 95?^ ethanol were added to the solution and mixed with a glass rod moved in a circular path, always in one direction. The fibrous precipitate was again wound around the glass rod ; and transferred to 0.8 ml of fresh SSC in v/hich it dissolved. The solution was shaken with chloroform and isoamyl alcohol using the same relative amounts as before, for 15 winutes and then centrifuged. The nearly clear suid colorless upper layer (approximately 2.5 ml) was pipetted into a clean centrifuge tube and two alcohol precipitations (addition of 1.2 to 1.5 volumes of alcohol) were used to remove the chloroform, isoamyl alcohp1 and other low molecular v/eight impurities. The. fibrous precipitate was wound around the glass rod as previously described and

dissolved in fresh SSC solution. The clear, nearly colorless

solution contained PNA in biologically active form.

Determination of the Thermal Denaturation Temperature of the. DKAi - (Marmur and Doty 1962) , . . Fifty microliters of the DM solution were diluted to 9 ,ml with fresh SSC to . yield a DM concentration of 6^g/ml One ml aliquots were placed in glass stoppered quartz

cuvettes. The samples were placed in the Beckman DU spectrophotoraeter chamber, and blanked against fresh SSC.

Chamber temperature v/as controlled by circulating hot water through the two thermal spacers on either side of the 14­

chamber. The,photocell spacers received circulating cold tap water to protect the photocell from high temperature. A thermocouple was placed in the cuvette containing the

blank to measure temperature changes. The spectrophotometer

was attached to two recorders, one to record the temperature

curve, and the other absorbance at 260 nM, Temperature corresponding to half the increase in absorbance in two appropriate parts of the absorbance/temperature curve were

determined as the thermal denaturation temperatures of the DNA. From these data, two g/C ratios were calculated.

Sxtraction of red pigment t

The red pigment was isolated after the procedures of Gochnauer eb ^ (1972). One ml of cell Suspension was diluted with 3«0 ml of acetone/methahol (l»lv/v) and the

mixture shaken vigorously. After standing at room tempei'ature

for one hour, the mixture was centrifuged, and the aupernatant

fluid decanted,. The residue vms extracted twice with ■ 2.0 ml portions of acetone/raethanol (Islv/v), The combined

extracts then v/ere made up to 10.0 ml volume with fresh acetone/raethanol Clslv/v). This solution was used for ­

spectrophotometric cha-racterization of the pigment,

Chromatographie separations ■

To determine whether there was only a single red

pigment, the chromatographic procedures of Gouchnauer et al

(1972) were followed, A portion of the extracted pigment was

concentrated under a stream of nitrogen to near dryness. 15

The residue was dissolved in a very siiiall volume of chloroform, and the pink solution was analyzed on silica gel chromatography paper previously washed in chloroform/ methanol (isl v/v) and activated at 100®G. .Chloroform/ methanol (93s7) was used as the developer to ascertain whether isomers of 'bacterioruherin were presents 16

RESULTS 8

Based on culture conditions, and microscopic observations of stained cells and of fresh cells suspended in CM, the.Halobacterium isolate used in this study is aerobic, Gram", variably rod- and copcoid-shaped, and non-motile. VJhile this organism grows well at 37^C in CM, in consistence with reported optimum temperatures for other strains of Halobacterium (Gochnauer and Kushner 1969)/ its best growth temperature on FPA plates was somewhat lower at 32-33°C. On FPA plates there is no growth at temperatures above 35°C, Additionally, fluctuations in culture temperature apparently prevented growth of the organism on FPA plates. y According to Gochnauer and Kushner (1969) and Eirahjellen (I965), Halobacterium strains require at least iBfi NaCl in the culture medium for grov/th to occur, with optimal growth produced at NaCl concentration of 25~30?»»

The standard NaCl concentration used in this study was

25%• , However, when NaCl concentration was reduced to 15?'^» all cells viewed in the microscope field became spherical. When NaCl concentration was reduced further (to lOi^Ol the cells lysed. . ■ This bacterium does not produce nitrogen gas nor

reduce nitrate when it is cultured in CM supplemented with

1% KNOo, even after two weeks. There was no evidence of

indole production in cultures supplemented with tryptone. 1?

Gelatin was not liquified after seven days incubation at approximately 31°C'

Nutritional Studies;

Growth characteristics of the bacterium in CM and in 3m are compared in Fig, 3« Optical density values for CM are higher than for 3iVi due to the more highly colored nature of CM, although the initial inoculum was equal in each. With optical density plotted against time, the rate of growth is apprGximately equivalent in both media, fhis conclusion is based upon the ciose similarity in slope of the growth curves in both media between and 72"^hours incubation after inoculation, fhere is an apparent l2-hQUr shortening of the lag phase of growth in SM in comparison to that in CM.

The extremely halophilic bacterium isolated in the present study from a pond at Soda Springs, Mojave Desert has been tested to see if it utilize carbohydrates, i The organism studied does not appear to utilize carbohydrates because there was no appreciable change in pH during a 96-.hour growth period (Table 1). The O.D. data (Table 1) also indicate no stimulation of growth in the presence of the five carbohydrates used. 18

085 SM

CM 0.80 210

OS o

> H W

Ui Q 0.70*7 2.09 . < O

H­ O. o

060 "I 2-08 Ksss-f=^r O 12 24 48 72 96 O' 12 24 48 72 96 TIME(HOURS)

Fig. 2s Comparison of the growth of Ha.lobacterium sp, in Complex Medium and: Synthetic'hyiedium. T^plicate cultures) . , , 19

■ ■ ."i] » 0.D. (Klett Units)

iviediura 2^■ hrs 48 hrs 72 hrs 96 hrs Final pH

BSiviC . 7 4 3 2 7.1

BSivK-Glu 2 ■ ' ' 3//'; . 4 0 6.9

BSMR-Gal 0 2 4 ■ 2 6,9

BSM-Suc 3; -12 -5 ' ■ ~3 7.1

BSMK-Lac 2 3 ■ ■ ■ 2, ■ 2^' 7.3

7.2 BSfi/lK-Trehalose 4 ' ■ 1 . ."3 i in BSMK'Supplemented with Various Sugans. Initial, pH 7.0; ■incubated for 96 hours at 37°G}.

CharaGteri'^ation i and Identification of the Red Pigments s

Spectra of acetone^methanol extracts of cells grown for 5 days:in both CM and SM were recorded on a

Perlcin-Elraer Dual Beam Recording Spectrophotometer. The ; resulting spectra each showed absorption maxima (Fig. 3) at 370» 389. ^+95», and 528 nMI with a shoulder at 466-^78 nivi, with these maxima corresponding to those of^-bacterioruberin (Jensen 1962, Kelly et ^ 1970). There were no pigment isomers present in the exiract, as determined by thin- layer chromatography. The mobility of the pigment on the chromatograra (Rf O.I3) is identical to that found by Kelly _et^ (1970} for ^-bacterioruberin.

The base composition of DNA isolated from the orgaiTism, expressed in terms of mole percent of guanine 20

■ '45 495

•40 466-478 528

m o ■ c (0 30

t-' 389 o 370 «3

25

.20

350 400 450 500 550 600 Waveiength (nM) , Pig, 3s Spectrum in acetone of the pigment isolated 'from ■the halophilic hacterium from Soda Springs. 21

plus cytosine, was determined from its thermal denaturation tiemperature:.(Tm)i The ahsorbance/temperature plot (Pig, 4) I , '" ' ■ ■ ■ ' ' • ijndicates that the organism: contains DNA which displays r t'wo components corresponding to said 68% GG content

respectively. The GO content was calculated from Marmur ajid Doty's equation* Tra = 69,3 "»• 0.>1 x (GG) (Marmun & Dbty 1962). Tm is the temperature at the midpoint of the a|Dsor"bance rise in °G measured at.260 nisi, and GC is the mole percent guaiiine cytosine. 22

.067­

.065­

.06

.05­

.04

.03

•02

01­

41 61 71 81 Tempe ratu re(^C) Fig. k: AbsorbanceAemperature plot of DNA of the Halobacterium sp. heated lln 0.1 M saline-citrate. 23

DISCUSSION;

Microscolpic observation of the cells of the bacterium i3olat||ld from the pond in Soda Springs showed, that they are red, short rods and cocci, and are Gram". The requirement iif to 30/^ NaCl for growth confirms that the bacterilm is halophilic. These are common characteristics If Halobacterium. During the isolation procedures for tJis halophilic bacterium, it was observed

that the. organisir did not grow in GMA while the PPA

supported growth. On the other hand the CM broth supported growth. The probable low oxygen solubility in a medium such as CMA, withj such high salt concentration, may be-^ a reason v/hy there jias no growth, since Halobacterium is ; an obligate aerob|L On the other hand, Gochnauer and Kushner (1969) havie shown that glycerol stimulates growth

of halophilic bacteria. Hence, the presence of glycerol in the FPA may havie stimulated the growth of the organism,

k'hile the mechanism for this effect is unknown, it is suggested that thej presence of glycerol provides inore favorable conditions relative to maintenance of optimum

surface tension ch racteristics of the bacterial cells, However, this hypotnesis has not been tested in the present

study.

Halophlies equire high concentrations of protein

or their derivatives for growth (Katanelson and. Lochhead

1952). Halophilic Bacteria studied require the majority 24

ol" amino acids riorraally found in protein for good growth in SM (Grey and [.Pitt 1975)• This suggests that such organisms do not readily synthesize amino acids. Mhatever ■ the case may he,I SM used in this study provides a suitable h' ■ ^ ■ chemically-defined medium for growth of this organism, Isolatioi^ of extremely halophilic bacteria that utilize carbohydfetes (Torolinson and Hochstein 1972a, b; 1976) supported lihe findings of Gochnauer and Kushner (I969).

However, the conclusion of Tomlinson and Hochstein (1972) that utilization pf carbohydrate by the bacterium can b© determined by monitoring pH decreases is in conflict with the findings of' Gbchnauer and Kushner (I969) who determined ' ■ • i . - ' ' ■ ■ ■ that glucose and galactose stimulated growth of Halobacterium halobium in SM without a decrease in pH of the medium. They (Gochnauer aijtd Kushner 1969) suggested that measurement ofeoxild carbohydrate lead to failjure utilization to detect solely carbohydrate by means of use acid at all production by extremely haloplbilic bacteria. Tomlinson and Hochstein (1976) in turn measured residua.l carbohydrate present in the culture medium during growth to detex^raine whether carbohydrate was being used even when acidic products v/ere not being formed. |ln the present study, elucidation of the possible influe^nce of selected carbohydrates on growth of the organism was!:'attempted by monitoring turbidity changes as a measure of growth and pH changes in the medium. As the datk show (Table 1), turbidity 25 measurements ar| equivocable in the carbohydrate -supplemented cultures j and tijiere is no appreciable decrease in pH of the medii^ra. Injfact, in the case of some of the sugars tested, the pH ©;f the medium shows a slight increase, Gochnauer and Kilshner (1969) also reported an increase in

pH of culture meidiura containing glucose with growth of

Kalobacterium ha^iobium. Interestingly, such an increase inpH could be indicative of possible photosynthetic activity by the organism,Ialthough this would not be expected because of the alfrobie requirements exhibited by the organism. Whate-|er the case, the data accrued in this study are such tlat it is impossible to state whether this

strain of Halobaciterium utilise carbohydrates or not.

Further study is needed. Petter (ibil) has shown that the red color in Halobacterium grojm in CM or SM is due to the presence of bacterioruberih. The spectrum of the pigment isolated in: ■ this■ ■■■ ■ . study■ ■ ■ ■ shoe's.'. ir absorption' , ■ - maxima corresponding■ ' ■ ■■ ■ to ■ ■ those■ ■■ ■ of •s^-bacterioruberin lias measured by Jensen (I962) and Kelly _01^ (19?0). Thin layer chroraatography showed that the mobility of the pjCTnent (Rf Q.'O) is identical to that of i^-bacterioruberin. Both of these findings suggest that V the, - halophilio/ ■ . ■ bac|eriumj. isolated■ from the pond at Soda Springs, Mojave Desert, belongs to the genus Halobacterium.

The possible role of the red pigment has not been invetigated in this study. Kelly et aj. (1970) suggested, that 26

the C:^Q hacteribruherin pigments arise from a linear carotene by addition of two units (Pig. 5)«

Mevalonate-^ •^Cj^Qparotenes-^•®»G^Qbacterioruberin 2G^units I

Retinal

Pig* 5s Biosynthetic pathways of Ci-w-bacterioruberin and retinal. oT

The retinal is very similar in its properties to pigments found in the retina of humans. Additionally, the finding that the jiigrnent may be involved in photosynthetic activity of the. orga.nism (/tolken and Nakagawa 1973» Oesterhelt and ,

StoeGkenius 1971), a rare case of aerobic photoautotrophic metabolism in a bacterium, certainly warrants attention and further study of this interesting baoterium.

In additi n to morphological, physiological, biochemical and o:||;her similarities, DNA base composition may be used as a tool in bacterial taxonomy. Investigations on molar percent guanine plus cytosine of purified DNA have shown that strains closely resembling each other in morphological and biochemical properties have nearly identical percent G/C ratio and genera with similar properties have nearly the sa|rae range of percent GG (Marraur and Doty 1962, Schildkraut i;et al I962). 2?

It has been poihted out also that two strains differing: by or more in Ithe G/C ratio most likely do not have any DNA molecules in common and hence are not phylogenetically closely-relatedi(Sueoka 1961). ■ i ■ ■ ' ■ ■ ■ ■ V\fhen DNA is heated in solution there will , be a sharp increase in its extinction coefficient at the teftperaturedouble Stranded that|istructur8 the transition to the occurs denatured from Single-stranded the nature state. The temilerature at the midpoint of the absorbance rise is. the doni'turatlo.n temperature>■ Tmi .and this is. , linearly relatedli to the DNA base composition (Marmur and Doty 1959, 1962)1 In this Study, DNA characteristics reported in

Nig.5 are typical of other such reports (e.g. Joshi et al 1963). The molar percent guanine plus cytosine isolated from this halophilic strain is 59?^ and 68^ respectively for the two DNA components. This is identical to the value obtained for Haliibaoterium aalinarium (Hill 1966, Joshi ■ I' ' ' ' ' ' ' ' ' ' " ' ^ ^ al 1963). Addjitionally, the observations that the bacterium does not liquify gelatin, produce indole, >reduce nitrate or evolve nitrogen indicates that the halophilic bacterium isolated and now in culture at Oalifornia State College, San Bernidino,I is very. . similar■ ■ to" strains■ of Halobacterium sallnarium. 28

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Buchanan, R.E. 1918. Life phases in a bacterial culture. J., Infect. |is. 23:109-125. ;

Difco. 1953« Mariual of Dehydrated Gulture Media and Reagents for Microbiclilogical and Clinical laboratory procedures. 9"^" Ed. Diflio Laboratories Inc., Detroit, Michigan.

Dussault,, H.P. 19|55» An improved technique for staining ' . red halophiiic bacteria. J. Bact, 70: 811—485. Eimhjellen, K. 1965• Isolation of extremely halophilic bacteria. 2e:htralbl. Pur Bakteriol. Parasiten. Infekt. Krankheiten end Hyg., 1 Abt. Supp. 1:127-13?« Gibbons, lf.E. 1969|i Isolation, grov/th and requirements of halophilic balpteria. In Norris, J.R. and D.W,Ribbons (Eds) Methods in Milprobiology. 3B:l69-l83. Acad. Press, New York.

Gibbonf■nn., N.E. 19.?4£ Family.Halobaoteriaceae. In Buchanan, R.E. and N.E., Gibbdns (Eds.), Bei-gey's Mlanual oT~Deterniinative Bacteriology, 3th Williams and Wilkins Co., Baltimore.

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Gochnauer, M.B. anco D.S. Kushner, 1969. Growth and: nutrition of extremely halophilic bacteria. Can. J. Microbiol. 15:1157-1165.I Gbchnauer, M.B., S.C. Kushwaha, M.Kates and D.J. Kushner. 1972. Nutritipnal control of pigment and isoprenoid compound forma|;ion in extremely halophilic bacteria. Arch. Microbiol.. 84:339-349. 1] ' ■ ■ ■ ■ -29 ■i • • . ■ . . . ■ • . r ■ ■

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