A synthetic medium, consisting of inorganic salts and any of a number of carbon sources, supported the aerobic growth of Paracoccus halodenitrificans when supplemented with thiamine. The same medium plus a nitrogenous oxide supported anaerobic growth when additionally supplemented with methionine. The observation that vitamin B12 or betaine replaced methionine suggested that P. halodenitrificans had a defect in the cobalamin dependent pathway for methionine biosynthesis, as well as the inability to synthesize betaine when growing anaerobically

Hochstein, L. I.

Tomlinson, G. A.

English

NASA Technical Reports Server

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NASA Technical Memorandum 84411 
ASA-TM-8441 1 19840002656 
The Growth of Paracoccus 
halodenitrificans in a 
Defined Medium 
Lawrence I. Hochstein and Geraldine A. Tomlinson 
October 1983 
National Aeronautics and 
Space Administration 
1 18 
LANGLEY RFsrAR MfR 
L RY NASA 
HI\.. PTON. VIRG N A 
J 
https://ntrs.nasa.gov/search.jsp?R=19840002656 2020-03-21T00:48:21+00:00Z
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NASA Technical Memorandum 84411 
The Growth of Paracoccus 
halodenitrificans in a 
Defined Medium 
Lawrence I. Hochstein, Ames Research Center, Moffett Field, California 
Geraldine A . Tomlinson, Department of Biology, University of Santa Clara, 
Santa Clara,. California 
NJ\S/\ 
National Aeronautics and 
Space Administration 
Ames Research Center 
Moffett Field. California 94035 
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ABSTRACT 
A synthetic medium, consisting of inorganic salts and ~ny of ~ 
number of carbon sources, supported the aerobic growth of Paracoccus 
halodenitrificans when supplemented with thiamine. The same medium 
plus an appropriate nitrogenous oxide supported anaerobic growth 
when additionally supplemented with methionine. The observation that 
vitamin B12 or betaine replaced methionine suggested that 
P. halodenitrificans had a defect in the cobalamin-dependent pathway 
for methionine biosynthesis, as well as the inability to synthesize 
betaine when growing anaerobically. 
1 
INTRODUCTION 
Paracoccushalodenitrificans is a moderately halophilic denitrifying 
bacterium isolated by Robinson and Gibbons from a bacon curing brine 
(Robinson and Gibbons 1952). The organism is thought to have complex 
growth requirements (Doudoroff 1974), and is grown in media containing 
protein hydrolysates (Hochstein and Dalton 1968; Forsyth and Kushner 
1970; Rosso et al. 1973). While attempting to develop a de fined medium 
for the purpose of examining denitrification during steady-state gr owth, 
we observed that a combination of sodium chloride, potassium phospha te, 
magnesium sulfate, sodium lactate, and trace quantities of yeast ext ra ct 
supported aerobic and anaerobic growth (the latter in the presence of 
nitrate). This paper identifies thiamine as the factor tha t supported 
the aerobic growth of P. halodenitrificans. The same medium (plus 
nitrate, nitrite, or nitrous oxide) supported anaer obic growth when 
supplemented with vitamin B12, betaine, or methionine. After completing 
this work, we learned of another thiamine-containing defined medium that 
was suitable for the aerobic growth of P. halodenitrific a ns (Flannery, 
w. L., Bacteriol. Proc. 1955. A25). 
MATERIALS AND METHODS 
Conditions of growth 
P. halodenitrificans (ATCC 13511) was grown aerobically in SOO-ml 
Nephelo flasks (Bellco Glass Co.) containing 50 ml of medium. Aeration 
was achieved by shaking the flasks on a rotary shaker. Anaerobic growth 
was carried out in 500-ml Nephelo flasks containing 200 ml of medium and 
2 
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modified to permit continuous passage of a gas (usually nitrogen) 
through a fritted glass disc. The cells were grown at 30°C and growth 
was determined with a Klett-Summerson colorimeter using a blue No. 42 
filter . When the turbidity exceeded 150 Klett units, the values were 
corrected as described by Sadler et al. (1980). 
Medium 
The composition of the defined medium is given in Table 1. When 
nitrite was used, it was autoclaved separately. Concentrated stock 
solutions of thiamine hydrochloride, ferro us sulfate (both made up in 
10- 3 N HCl) and methionine, dissolved in distilled water, were steril-
ized by filtration, using a Nalgene 20-~m Type S filter unit. Appro-
priate aliquots of these solutions were added immediately before start-
ing a growth experiment . 
Thiamine was purchased from CalBioChem. Corp. Betaine, vitamin 
B12 , and methionine were obtained from the Sigma Chemical Company. The 
yeast extract was obtained from Difco. 
RESULTS 
As shown in Table 2, no aerobic growth was observed in the basa l 
salts- lactate medium in the absence of yeast extract. Neither ashed 
yeast extract nor a mixture of amino acids replaced yeast extract. Of 
the various vitamins reported present in yeast extract (Culture Media 
Ingredients, Form No. CMT-l, Baltimore Biological Laboratories), only 
thiamine was effective. When thiamine was present in excess, the final 
turbidity observed in the defined medium was considerably lower than 
3 
that observed in the presence of 0.01% yeast extract (Table 2) . The 
addition of the other vitamins present in yeast extract, alone or in 
combination, did not increase the cell yield. Of various trace metal s 
tested, only ferrous sulfate increased the cell yield, approaching 
those values observed in the presence of yeast extract (Table 2). Nitrate 
did not replace ammonium as the sale source of nitrogen, indicating that 
P. halodenitrificans lacked an assimilatory nitrate reductase. The 
generation time for aerobic growth in the defined medium was 2.5 h; 
it was 1.5 h when P. halodenitrificans was grown in complex medium 
(Sadler et al. 1980). Stock cultures have been mainta ined on agar slants 
of this defined medium for over a year. 
The basal salts-lactate medium, contnining thiAmine <lnd fer rOllS 
sulfate, did not support anaerobic growth in the abs ence of yeast extract. 
Of the various amino acids tested, only methionine replaced yeast extr ac t 
(Table 2) . As in the case of aerobic growth, the addition of ferrous 
sulfate also increased the cell yield. The defined medium designed for 
anaerobic growth supported a much higher population of cells than did 
the yeast extract-containing basal salts-lactate solution. The genera-
tion time during anaerobic growth was 4 h when the cells were grown in 
the presence of nitrate, and 7 h in the presence of nitrite. 
P. halodenitrificans also grew anaerobically in the presence of nitrous 
oxide with a generation time of 21 h (Hochstein, Kritikos, and Betlach , 
unpublished data). No growth was observed when the concentration of 
nitrite was greater than 29 mH. Although P. halodenitrificans grows over 
a wide range of salt concentrations (0.4 to 3.0 M) in complex medium 
(Robinson and Gibbons 1952), the r a nge of NaCl concentrations permitting 
4 
either aerobic or anaerobic growth in synthetic medium was more limited 
(1 M to 2 M). 
The requirement for methionine was not satisfied by aspartate, 
isoleucine, threonine, homocysteine, homoserine, dimethylglycine (alone 
or in combination with homoserine or homocysteine), sarcosine, serine, 
glycine, formate or formaldehyde. All of these are potential intermedi-
ates in the biosynthesis of methionine. Betaine or vitamin B12 
replaced methionine (Table 2). The growth rates in the presence of 
betaine or vitamin B12 were the same as those observed with methionine 
(data not shown). In terms of cell yield, betaine and methionine wer e 
equally effective on a molar basis, and vitamin B12 was about 300 tim es 
more effective than either . At times, choline substituted for methionine, 
but the results were inconsistent. 
Various carbohydrates were used as carbon sources for a e r obic and 
anaerobic growth (Table 3) . Several interesting patt e rns we re obse rved . 
All of the hexoses tested, with the exception of mannose, were used . 
Mannitol was used aerobically and anaerobically, whereas sorbitol wns 
not metabolized. P. halodenitrificans also used a variety of dissa cha rid es, 
including sucrose, lactose, and maltose. Growth in the presenc e of 
carbohydrates resulted in an acidification of the gr owth medium during 
aerobic growth and, with the exception of fructose, ana erobic g rowth. 
When a c id was produced, the final pH was 6 . 5 . A cons ide r a ble amount of 
acid must have been produced during anaerobic growth, since the redu c-
tion of 1% ni~rate to volatile products as a consequence of denitri f i-
cation would have resulted in a ma rked alkaliniza tion or the medium. 
The production of acid was unexpected, since the P. ha lodenitrificans 
is described as not producing acid from glucose during ae robic gr owth 
5 
(Duodoroff 1974). However, since those results wer e probably obtained 
from growth ca rried out in complex medium, acid production could ha ve 
been masked by deamination. In addition to carbohydrates, a numb e r of 
o ther compounds served as carbon sources. Of the amino acids tested 
(and listed in Table 3), aspartate was the only one tha t did not serve 
as a source of carbon. 
DISCUSSION 
The observation that P. halodenitrificans grew in a simple min e r al -
salts medium containing thiamine and a suitable carbon s ource confirmed 
the earlier observation made by Flannery. However, the growth r eq uire-
ments were far less complex than suggested by his medium formulation, 
which consisted of 18 amino acids, 10 vitamins, various salts, as well 
as adenine, guanine, and uracil. The requir ement for thinmine was 
rema rkable. When cells were grown aerobically in de fi ned medium con-
taining 5 pg thiamine ml- 1 , populations of 6 x 10 8 colony formin g unit s 
ml- 1 were obtained. When thiamine was present in exc ess, the numbe rs 
were of the order of 10 10 colony forming units ml- 1 • These popul a tions 
were similar to those observed in the complex medium (Sadler, Kritikos, 
a nd Hochstein, unpublished data). 
The def ined medium did not support anaerobic gr owth in the absence 
of methionine . Methionine is synthesized from aspartate in a re ac tion 
sequence wh ich terminates in the methylation of homocys t eine . In cer-
tain ins tances, s erine is the precursor of this me thyl group , wh ich is 
fo r med in a series of reac tions whereby the hydroxymethyl carbon of 
serine is transferred to tetrahydrofolate resulting , ult imnte ly, in 
6 
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methyl tetrahydrofolate (Taylor and Weissbach 1973). This methyl grou p 
is transferred to homocysteine by a set of reactions, some of which require 
a cobalamin coenzyme (Woods et al. 1965). Betaine may also serve as a 
methyl donor in bacterial systems, presumably by the betaine-homocysteine 
methyl transferase reaction (White et al. 1973). 
The ability of betaine but not homocysteine to replace methionine 
suggests a defect in the synthesis of methyl groups. However, since 
vitamin B12 replaced methionine, anaerobically growing cells must have 
been able to make and transfer methyl groups. The alternative conclusion 
is that the methionine requirement reflected a defect in the synthesis 
of the cobalamin coenzyme. Since betaine replaced methionine, a nae robi c 
cells must also have been defective in the synthesis of bet a ine, at l eas t 
nt rates appropriate to cellular needs. This would be cons ist ent with 
our observation that choline, from which betaine can be de rived by oxi-
da tion via betaine aldehyde (Kensler and Langermann 1954), did no t 
substitute effectively for methionine. Furthermore, choline , which 
served as the sole source of carbon for aerobic growth, was ineffective fo r 
anaerobic growth (Table 3). 
The nutritional requirements exhibited by P. ha lodenitrifica ns 
indicate that the organism potentially has two pathways f or synthes i z ing 
methionine: one involves betaine and, if functional, is so only during 
a erobic growth; the other is the cobalamin-dependent pathway. Whe ther 
this pathway operates during aerobic growth is uncertain. If 
P. halodenitrificans synthesized betaine from choline, the obse rva tion 
that choline at times replaced methionine could have r eflected the 
presence of sufficient oxygen in the growth medium at the initia tion 
7 
of growth so that choline was oxidized to growth-supporting levels of 
betaine. 
Little is known about the synthesis of betaine in bac t eria. 
P. halodenitrificans may prove useful in studying the biosynthesis of 
betaine as well as the pathway associated with the production of the 
metabolically active form of the cobalmin coenzyme concerned with 
methionine biosynthesis. 
ACKNOWLEDGMENTS 
We wish to acknowledge the technical assistance o f Ka thleen Grant 
Gonzales, Michael Trindle, and Brian Wilson. 
This work was supported, in part, by funds from the NASA/Universit y 
Consortium Agreement NCA-OR 685 902. 
8 
References 
I)()III)()I{UI,'F, M. 1,)7 /1. Cl'llliS l'ilr;.l C()('C W '; I);(vi ~; ill Jl:]vl :;, 1)()lId()r()rr, S(;lIlil'r, 
and Mandel 1969, 384. In Bergey's Manual of Determinative Bacteri-
ology, 8th ed. Edited by R. E. Buchanan and N. E. Gibbons. The 
Williams and Wilkens Co., Baltimore, pp. 438-440. 
FORSYTH, M. P. and D. J. KUSHNER. 1970. Nutrition and distribution of 
salt response ' in populations of moderately halophilic bacteria. 
Canad. J. Microbiol. 1Q: 253-261. 
HOCHSTEIN, L. I. and B. P. DALTON. 1968. Salt speci ficity of a 
reduced nicotinamide adenine dinucleotide oxidase prepared from a 
halophilic bacterium. J. Bacteriol.~: 37-42. 
KENSLER, C. J. and H. LANGERMANN. 1954. Metabolism of choline and 
related compounds by hepatic tissue from several species including 
man. Proc. Soc. Exp. BioI. Med.~: 364-367. 
ROBINSON, J. a nd N. E. GIBBONS. 1952. The effect of salts on th e 
growth of Micrococcus halodenitrificans n. sp. Canad. J. Bot. 
lQ: 147-154. 
ROSSO, J. P., P. FORGET, and F. PICHINOTY. 1973. Les nitrate-
reductases bacteriennes solubilisation, purification et properties 
de l'enzyme a de Micrococcus halodenitrificans. Biochim. Biophys. 
Acta 1ll: 443-455. 
SADLER, M., M. McANINCH, R. ALICO, and L. I. HOCHSTEIN. 1980. The 
intracellular Na+ and K+ composition of the moderately halophilic 
bacterium, Paracoccus halodenit rificans. Canad. J . Microbiol. lQ : 
496-502. 
9 
TAYLOR, R. T. and H. WEISSBACH. 1973. N5-methy1tetrahydrofolate-
homocysteine methyltransferases. In The Enzymes, Vol. 9, 
3rd ed., Edited by P. D. Boyer. Academic Press, New York, 
pp. 121-165. 
WHITE, R. F., L. KAPLAN, and J. BIRNBAUM. 1973. Betaine-homocysteine 
transmethylase in Pseudomonas denitrificans, a vitamin B12 over-
producer. J. Bacteriol. lll: 218-223. 
WOODS, D. D., M. A. FOSTER, and J. R. GUEST. 1965. Cobalamin-dependent 
and -independent methyl transfer in methionine biosynthesis. In 
Transmethylation and Methionine Biosynthesis . Edited by S. K. Shapiro 
and F. Schlenk. University of Chicago Press, Chicago, pp. 138-156. 
10 
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TABLE 1. Composition of the defined 
medium for P. halodenitrificans 
Basal Salts 
NaCl 58.4 g 
8.7 g 
l.Og 
0.1 g 
Sodium lactatea 5.0 g 
Thiamine hydrochloride 0.05 mg 
FeS04·7H20 0.03 mg 
L h " " b -met lonlne 14.0 mg 
KN0 3 (or KN02) b 10 (or 2.5) g 
Water 1 liter 
Note: The pH was adjusted to 7.4. 
aAdded as 8.3 ml of a 60% solution of 
sodium lactate. 
bAdded for anaerobic growth. 
11 
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TABLE 2. Growth requirements of P . halodentrificans 
Klett unit s 
Medium 
Aerobica Anaerobic 
Basal salts plus lactate o o 
Basal salts, lactate, plus 
0.01% yeast extract 1200 157 
0 . 0001% yeast extract 145 53 
thiamine 250 3 
thiamine , FeS04 1100 0 
thiamine, methionine nd 280 
thiamine, methionine, FeS04 nd 527 
thiamine, betaine (or vitamin B12), FeS04 nd 525 
(or 564) 
NOTE: The reported changes in Klett units were obta ined by inter-
polating the turbidity increase occurring during the stationary phas e 
through the extrapolated value of the exponential growth phase. The 
initial Klett values were subtracted from the value observed at the 
intersection of the lines. The concentrations of the added ingre-
dients were those in Table 1. Anaerobic growth was carried out in the 
presence of 1% potassium nitrate. Betaine and vitamin B1 2 were added 
to a final concentration of 15 . 5 mg and 0 . 4 mg/liter, respective ly. 
and not done . 
12 
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TABLE 3. Substrates supporting 
growth in the defined medium 
Acetate Glycine a 
S -Alanine Lactate 
Arginine Malate 
Choline a Maltose 
Citrate Mannitol 
Dimethylglycine a Proline 
Ethanola Propanol a 
Fructose Succinate 
Galactose Sucrose 
Glucose Trehalose 
Glycerol Sarcosine a 
NOTE: Each compound was tested 
for its ability to support growth 
in the defined medium at a concen-
tration of 0.5%, with the appro-
priate additions for aerobic or 
anaerobic growth as indicated in 
Table 1. All of the amino acids 
were the L-isomers. The follow-
ing compounds did not support 
either aerobic or anaerobic growth: 
arabinose, arabitol, aspartate, 
betaine, butanol, formate, mannose, 
methanol, methionine, ribitol, 
ribose, or sorbitol. 
aSupported only aerobic growth. 
13 
1. Report No. I 2. Government Accession No. J . Recipient'S Catalog No. 
NASA TM-84411 
4. Title .nd Subtitle 6. Report Date 
THE GROWTH OF PARACOCCUS HALODENITRIFICANS IN A October 1983 
DEFINED MEDIUM 
6 . Performing Or\lillnization Code 
7. Author(sl Lawrence I. Hochstein and Geraldine A. 8. Performing O'goniz.tion Repo<t No. 
Tomlinson (Dept. of Biology, University of Santa A-9487 Clara, Santa Clara, California 95053) 10. Work Unit No. 
9. Performing Or!jln ization Name .nd Address T-4615 
Ames Research Center 11. Contract or Gr.nt No . 
Moffett Field, California 94035 
1 J . Type of Report and Period Covered 
12. Sponsoring Agency Name and Address Technical Memorandum 
National Aeronautics and Space Administration 14. Sponsoring Agency Code 
Washington, D. C. 20546 199-60-62-06- 00 
15. Supplementary Notes 
Point of Contact: L. I. Hochstein, Ames Research Center, MiS 239-10, Moffett 
Field, California 94035 (415) 965-5938 or FTS 448-5938. 
16. Abstract 
A synthetic medium, consisting of inorganic salts and any of a number of 
carbon sources, supported the aerobic growth of Paracoccus halodenitrificans 
when supplemented with thiamine . The same medium plus a nitrogenous oxide 
supported anaerobic growth when additionally supplemented with methionine. 
The observation that vitamin B12 or betaine replaced methionine suggested 
that P . ha10denitrificans had a defec t in the cobalamin-dependent pathway for 
methionine biosynthesis, as well as the inability to synthesize betaine when 
growing anaerobically. 
17. Key Words (Su!/ge5ted by Author(sl) 18. Distribution Statement 
P. ha10denitrificans Unlimited 
Synthetic medium 
Methyl group 
Biosynthesis Subject Ca tegory: 51 
19. Security O."if. (of this reportl 20. Security Classif. (of this J)age) 21. No . of Pages 22. Price . 
Unclassified Unclass ified 16 A02 
"For nit by the Nat ional Technical Information Service, Springf ield, Virgin i. 22161 
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The growth of paracoccus halodenitrificans in a defined medium

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