SPECIFICITY OF THE VITAMIN B12 REQUIREM\IENT INCERTAIN MARINE BACTERIA'

WILLIAM A. AYERS

Kitchawan Research Laboratory of the Brooklyn Botanic Garden, R.F.D. 1, Ossining, New York

Received for publication April 14, 1960

There is considerable evidence that vitaminB,, and related cobalamins are important factorsin microbial ecology of the sea. Cowey (1956),Burkholder and Burkholder (1956, 1958), Starr(1956), and others have shown that not onlyeyanocobalamin, but other cobalamins as well,are present in sea water and marine sediments.The reviews by Provasoli (1958a, b) list manymarine protozoa and unicellular algae whichrequire vitamin B,2; in some cases, members ofthe pseudovitamin B,2 group (i.e., analogues ofeyanocobalamin containing purines in place ofthe dimethylbenzimidazole moiety) were utilizedin place of the true vitamin. Droop et al. (1959)reported that about 70 per cent of marine proto-phytes grown in pure culture require an exog-enous supplv of vitamin B12. Although "mam-malian" specificity was the most common type,nevertheless, a significant portion of the specieshad other specificity patterns, lending support tothe conclusion that, for microorganisms, ana-logues of vitamin B12 have an ecological impor-tance equal to the vitamin proper.

Production of cobalamins by marine bacteriain pure culture, as well as in marine mud, hasbeen shown by the Burkholders (1956, 1958,1959) and Starr, Jones, and Martinez (1957).Apparently, a substantial part of the Bl2-activesubstances produced under natural conditionsby marine bacteria is in the form of pseudo-vitamin B12 and related forms.Most of the information about the specificity

of the vitamin B12 requirement in bacteria isbased on studies of Escherichia coli strain 113-3and Lactobacillus species used for assay purposes(Kon and Pawelkiewicz, 1960). Other than thespecificity patterns of 19 soil isolates studiedby Ford and Hutner (1957), little information isavailable about the specificity of the vitamin

'This research was supported in part by a grantfrom the National Science Foundation, G-8917,and by contract Nonr-3018(00) with the Office ofNaval Research.

B12 requirement of naturally occurring variantbacteria, especially in the marine environment.Such information should be helpful as an aidto understanding the interrelationship of vitaminproduction and utilization by marine organisms.In addition, salt-tolerant, vitamin B12-requiringmarine bacteria may be useful as agents of assayfor cobalamin compounds in sea water or othermaterials which are hypertonic to conventionalassay organisms.

MATERIALS AND METHODS

Cultures and media. The vitamin B12-requiringbacteria studied were selected strains from acollection of bacterial isolates made during ascreening program for vitamin-requiring andvitanmin-producing bacteria from marine mate-rials (Burkholder, 1959). Isolations were madefrom marine mud and sea water on a mediumconsisting of the following: sea water, 1 liter;trypticase, 4 g; soytone, 2 g; yeast extract, 3 g;vitamin B,2, 1 ,ug; and agar, 15 g.The initial studies of the requirements of the

isolates for several B-vitamins were made in abasal medium containing vitamin-free casaminoacids (Difco) as the nitrogen source along withother supplements. Since methionine was foundto replace the vitamin B12 requirement for manyof the cultures, the medium shown in table 1 wasadopted for vitamin B,2 studies. The amino acidcomposition of this medium is similar to that ofhydrolyzed casein, except for the exclusion ofmethionine. Artificial sea water prepared accord-ing to Lyman and Fleming (1940)2 was used inmedia for nutritional studies to eliminate thepossible presence of growth factors in naturalsea water.

Pad-plate tests. The pad-plate assays weremade in the test medium solidified with 1.5 per

2Composition of artificial sea water used (perkg): NaCl, 23.48 g; Na2SO4, 3.92 g; NaHCO3,0.19 g; KCl, 0.67 g; KBr, 0.10 g; MgCl2, 4.98 g;CaCl2, 1.10 g; SrCI2, 0.02 g; H3BO3, 0.03 g.

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TABLE 1Ingredients in vitamin B12 testing medium per

liter of artificial sea water; pH 6.8

Ingredient Amt Ingredient Amt

g g

DL-Alanine ...... 0.275 DL-Threonine 0.190L-Argine ....... 0.215 L-Tyrosine ..... 0.335

DL-Trypto-L-Aspartic acid.. 0.305 phan ........ 0.060L-Cystine ....... 0.175 DL-Valine .... 0.325L-Glutamic acid. 1.165 Glucose 2.0Glycine ......... 0.025 Na-succinate . 2.0L-Histidine...... 0.105 K2HPO4....... 0.025DL-Isoleucine .... 0.315 KH2PO4....... 0.025

Fe(NH4) 2 -

(SO4)2....... 0.007DL-Leucine..... 0.485 pAgL-Lysine.... 00.380 Biotin.1.0DL-Phenyl- Thiamine ...... 500

alanine ...... 0.250 NicotinicL-Proline ..... 0.375 acid ......... 500

Ca-panto-DL-Serine ....... 0.385 thenate. 100

cent agar. Ten ml of sterile, melted medium were

inoculated with 0.5 ml of a light suspension ofeach organism prepared by suspending a smallamount of growth from a 48-hr slant in sterilesea water, then dispensed into a sterile 10 cm

petri dish. Filter paper disks, 12 mm in diameter(S & S 740 E) were dipped into each of thesterile solutions containing 0.5 ,ug per ml of. theB12-vitamins and placed onto the seeded agar.

The dishes were then incubated for 48 to 72 hrat 28 C and examined for growth suirroundingthe pads. Tests for the growth-promoting activityof methionine and certain nucleotides were

carried out similarly with solutions of 0.3 mg

and 0.4 mg per ml, respectively.Tube tests. Tube tests were conducted in 18-

by 150-mm culture tubes. To duplicate tubescontaining 4.5 ml of the test medium was added0.5 ml of a series of vitamin B12 standards togive final concentrations ranging from 1 to 0.01mAg per ml. Solutions of vitamin B12 analogueswere likewise added to tubes at levels of 0.05 mpgand 1 mAg per ml for all analogues, and at 200and 400 mAg per ml for the less active compounds.Analogues were autoclaved in the medium at121 C for 15 min.Each culture tube was inoculated with one

drop of a light suspension of each bacteriumprepared by suspending a small amount of growthfrom a 48-hr agar slant in sterile artificial seawater. Tubes were placed in an inclined positionon a rotary shaker and incubated at 30 C for 48to 72 hr. The growth of the cultures was measuredturbidimetrically with a Bausch and Lombcolorimeter at 655 m,.

Analogues and nucleotides. The vitamin B12analogues studied, indicated in the text andtables, were kindly furnished by Dr. L. Provasoliof Haskins Laboratories, New York, N. Y. Thecompounds were furnished in stock solutions of20 ,ug per ml each and were kept at 4 C undertoluole. Standard vitamin B12 solutions wereprepared from the crystalline product of theNutritional Biochemicals Corporation. Thenucleotides tested were obtained from theCalifornia Corporation for Biochemical Research.

TABLE 2Classification and vitamin requirements of bacteria

under investigation

Probable StanN.Sea WaterGenera Stmin No. Require- Vitamins Requiredt

ments

Achromo- 392$, 477t R Nonebacter 551$, 571t R Thiamine

N29$, N43t R Biotin, thi-amine, nia-cin, panto-thenic acid

Brevibac- 63, 483, NR Biotin, thi-terium 746, 865 amine

375 R ThiamineMicrococ- 976, 1136 R Biotin, thi-

cus amineM35 R Niacin, thi-

amineN2$ NR Biotin, thi-

amine, panto-thenic acid

Pseudo- 429t R Nonemonas 4591, 646t R Thiamine

Vibrio 415, 430 R ThiamineFlavobac- 5271 NR None

terium

* R, requirement for sea water; NR, growth inmedia made with either sea water or distilledwater.

t Vitamins required in addition to vitamin B12.t These strains required vitamin B12; others

were stimulated by the vitamin.

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R'ESULTS

Characteristics of the isolates. The majority ofisolates studied could be classified as marine bac-teria as defined by ZoBell and Upham (1944), i.e.,bacteria requiring a medium containing sea wateras diluient on initial isolation. A few strains wereable to develop in media made with distilledwater; however, growth was generally l.ss vigor-ous than in media containing either natural orartificial sea water.The isolates were studied by standard bacterio-

logical procedures in media made with sea waterand tentatively placed in various genera. Table 2

lists the problable genera of the strains along withtheir requirement for sea water and certain B-group vitamins. As indicated, all but four of thestrains required vitamins in addition to vitaminB12. For most of the strains listed in the generaBrevibacterium, Jlicrococcus, and Vibrio, vitaminB12 was stimulatory rather than being absolutelyrequired, since delayed growth occurred in theabsence of furnished vitamin B12. In determiningthe nonessentialitv of vitamin B12 with thesestrains, the possibility of carryover of the vitaminin the inoculum was obviated by making a mini-mum of 4 consecutive transfers in media lacking

Figure 1. Response of strains 459 (top) and 865 (bottom) to analogues of vitamin B12 by the pad-platetest. Reading clockwise from top of each plate, left plate: B12, factor I, factor B, factor A, benzimidazoleanalogue; right plate: pseudovitamin B12, 2-methylmercaptoadenine analogue, 5-methyl benzimidazoleanalogue, cobalamin phosphoribose, control. The plates were incubated at 28 C for 72 hr.

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the vitamin. The strains marked with a doubledagger in table 2 required vitamin B12 for growth;the remainder were stimulated by the vitamin.

Vitamin B12 specificity patterns. Figure 1 illus-trates the response of two strains to vitamin B12and analoguies of the vitamin by the pad-platetest. The diameters of growth zones with allstrains varied only slightly with each analogueand were of little use for estimating potency.However, the opacity and sharpness of the zonesfrequently varied, thereby indicating that thevarious analogues differed in activity.The results of the pad-plate tests are sum-

marized in table 3. Six patterns of specificitywere indicated by the presence or absence ofgrowth zones surrounding the pads containingthe various analogues: a) the benzimidazole ana-logues (B12, 5-hydroxybenzimidazole, 5-methyl-benzimidazole, and benzimidazole) were activefor all strains; b) 2-methylmercaptoadenine andc) 2-methyladenine supported the growth of moststrains; d) pseudovitamin B12 and e) factor Bwere active only for cultures N2, 429, 477, and

TABLE 3Response of marine bacteria to vitamin B12 and

analogues by the pad plate test*

Analoguet

Strain No.B12 _ BA S PS 9 FB

459 +1136, M35 + + + + - _ - i -N29 ++++-- A-

392, 551, 571, + + + + + + - + -430, 63, 375,415, 483, 527,746, 865, 976,N43

N2, 429, 477, 646 + + + + + + + + +

* Growth (+); slight growth (i); or no growth(-) surrounding pads containing analogues.Incubation period, 72 hr.

t The analogues contained the following sub-stituted groups for the 5,6-dimethylbenzimidazolemoiety of cyanocobalamin: 5-HB, 5-hydroxy-benzimidazole (factor I); 5-MB, 5-methylben-zimidazole; BA, the benzimidazole analogue;2-MMA, 2 methylmercaptoadenine; 2-MA,2-methyladenine (factor A); PS, pseudovitaminB12 (adenine analogue); CPR, cobalamin phos-phoribose (no nucleotide base); FB, factor B(no nucleotide).

646, which responded also to all other compounds;f) cobalamin phosphoribose showed slight activityfor many of the strains. It should be noted thatsince a single concentration of the analogues wasused in these tests, the possibility of a very loworder of activity by some of the compounds wasnot excluded.The tube assays of the various analogues per-

mitted a more discriminating estimate of potencyof the compounds for most organisms. Table 4lists the relative activity of the compounds ascompared to eyanocobalamin. Values for in-dividual compounds frequently varied from onedetermination to the next; consequently, thenumerical values cited in table 4 must be regardedas only approximate. However, the general orderof activity of the analogues was reproducible andgenerally correlated with the plate tests. Severalstrains which responded faintly in the plate testsgrew so poorly in the test medium that a quanti-tative estimate of activity with these strainscould not be obtained with the tube assays.The response of the vitamin B12 requiring

mutant, Escherichia coli strain 113-3, to the com-pounds under study was tested under the sameconditions with the medium of Davis andMingioli (1950) and included in table 4 for com-parison with the marine bacteria.The data indicate that all of the benzimidazole

analogues were much more potent than thepseudovitamins or the incomplete vitamin. In allcases, the 5-methylbenzimidazole analogue wasmost active; frequently, it displayed activityequal to that of cyanocobalamin. The 5-hydroxy-benzimidazole analogue showed more variation inactivity with the various organisms. The ana-logues lacking the benzimidazole moiety gave alow order of activity for those bacteria which re-sponded to them by the pad-plate test.

Vitamin B12-methionine relationship. It isknown that vitamin B12 functions in methylgroup synthesis, since methionine replaces thevitamin B12 requirement of E. coli mutants andcertain other bacteria, and spares the vitamin re-quirement in animals (Arnstein, 1960). Themarine bacteria were screened for their ability toreplace the vitamin B12 requirement with methio-nine by the pad-plate technique, followed bytube tests with certain strains. The majority ofcultures gave a maximal response to methionine,indicating a complete replacement of the vitamin.Only three strains could be clearly shown to re-

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TABLE 4Relative activities of vitamin B12 analogues (B12 = 100)*

AnaloguetStrain No.

5-HB 5-MB BA 2-MMA 2-MA PS CPR FB

63 13 100 34 0.09 0.08 Ot 0.08 0.02375 8 100 17 0.17 0.24 0 0.03 0429 64 100 55 50 54 10 0.24 0.42459 9 26 9 0 0 0 0.06 0.01483 44 100 41 0.5 0.19 0 0 0527 67 88 28 3.0 0.6 0 0.15 0551 58 98 60 1.4 1.2 0 0.02 0.01571 74 86 70 3.3 3.3 0.002 0.02 0.01646 62 98 34 31 13 2.7 0.07 0.12746 35 100 50 17.3 8.2 0.02 2.5 0.01865 16 55 36 5.6 9.5 0 0.9 0.02976 5.5 50 11 0.03 0.04 0 0.19 01136 <5 60 10 0 0.04 0 0.19 0Escherichia coli strain 113-3 70 88 64 40 30 2.2 - 1.8

* The optical density of the cultures grown in the presence of suboptimal concentrations of thevarious analogues was compared to that of vitamin B12-grown cultures. The numerical values representrelative activities of the analogues with vitamin B12 rated arbitrarily at 100. Incubation time 48 hrexcept for strains 459, 527, and 551 which were read at 72 hr. E. coli strain 113-3 was incubated for 24 hr.

t See footnote, table 3.t Zero = no activity shown at the highest concentration used (0.4 ,ug/ml).

quire cobalamins for maximal growth, althoughseveral grew so poorly that this property couldnot be ascertained.

Table 5 shows that strains N2 and 459 grew inmedia supplemented with vitamin B12, but failedto develop in several levels of methionine duringthe 3-day incubation period; in comparison,strain 527 grew in both media. Upon continuedincubation for 3 days longer, culture 459 (but notN2) developed growth in tubes containingmethionine, possibly as a result of adaptation ofthe culture or selection of variants able to utilizemethionine.

Repeated experiments with strain 527 in-dicated that this organism grew most rapidly inmedia containing vitamin B12. As shown in table5, the organism responded to methionine to a

lesser extent than to vitamin B12, indicating a

B12-sparing action by the amino acid. It can alsobe noted in table 5 that the highest level of me-

thionine was slightly inhibitory to this culture;the growth response of many of the marine bac-teria to methionine was similar in this respect.

Vitamin B12 antagonism. With many of thesoil bacteria studied by Ford and Hutner (1957)vitamin B12 was antagonized by pseudovitamin

TABLE 5Growth of several strains in methionine

or vitamin B12*

Strain No.Addition Amt

N2 459 527

ig/linlControl 0 0 0 0Methionine 1 0

10 0 0 0.2050 0 0 0.65100 0 0 0.72200 0 0 0.50

Vitamin Bl2 1 0.49 0.27 1.30

* Growth expressed in optical density units ofcultures incubated for 72 hr at 28 C.

B12 when the latter was present in relatively highconcentrations. A similar effect on several of themarine bacteria under study was indicated by thepad-plate tests where the zones of exhibition weresometimes distorted or impeded by adjacent padscontaining pseudovitamin B12. A further study ofthe pseudovitamin-vitamin B12 antagonism wasmade in tube tests with media containing 0.1m,ug cvanocobalamin per ml. Pseudovitamin B12

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was added to the series of tubes in gradedamounts; the medium was sterilized, then inocu-lated.

Figure 2 illustrates the antagonistic effect ofpseudovitamin B12 in strain 527. The lower levelsof the analogue produced a slightly stimulatoryeffect; higher concentrations retarded growth. Asimilar pattern of inhibition by pseudovitaminB12 was observed with strains 415, 459, 483, 571and 1136.Vitamin B12 antagonism was also exhibited by

other members of the pseudovitamin group forthose strains which could not utilize the ana-logues. Growth in the presence of a suboptimallevel of vitamin B12 was inhibited or retarded byrelatively high concentrations of 2-methyladeninecobalamin and 2-methylmercaptoadenine co-balamin with strains 459, 976, and 1136. Theeffect, in general, was similar to the antagonismby the adenine analogue of cobalamin.

Pseudovitamin B12-methionine synergism. Asindicated above, the vitamin B12 requirement ofstrain 527 was partially replaced by methionine.When pseudovitamin B12 was supplied to thegrowth medium along with methionine, a markedstimulation of growth occurred, even thoughpseudovitamin B12 alone did not support growthand was actually inhibitory in the presence of thetrue vitamin.Both the synergistic and antagonistic effect of

pseudovitamin B12 is illustrated by the plateculture shown in figure 3. Where pseudovitaminB12 diffused out from the center pad, a sector ofstrong growth resulted at the juncture of themethionine zone. The vitamin B12 zone demon-strated slight inhibition, whereas the growtharound the pad containing the less active com-pound, cobalamin phosphoribose, was markedlyinhibited by pseudovitamin B12.The results of tube tests with strain 527 con-

ducted with methionine, vitamin B12, and pseudo-vitamin B12, singly and in combination, are shownin table 6. Pseudovitamin-vitamin B12 an-tagonism and methionine-pseudovitamin B12synergism are evident. Methionine also stimu-lated growth with the suboptimal concentrationof vitamin B12 furnished. All three compoundstogether supported the most vigorous growth.With varying concentrations of methionine

and pseudovitamin B12 supplied together, theresults in table 7 were obtained. Maximal growthwas achieved with methionine levels of 50 and100 /ug per ml along with a minimum of 4 mAg perml of pseudovitamin B12. Higher levels of pseudo-vitamin B12 had no increased effect. The presenceof 500 ,ug of methionine per ml was definitelyinhibitory. Pseudovitamin B12 apparently

96 HR INCUBATION.4 I

1-.3

zw0

.2-i0

0 .10

0 0.1 I 10 100PSEUDOVITAMIN B12 mpg/ml

Figure 2. Effect of pseudovitamin B12 on thegrowth of strain 527 in the presence of a sub-optimal concentration of vitamin B12. Mediumcontained 0.1 m,ug vitamin B12 per ml.

Figure S. Plate culture of strain 527 showingantivitamin activity and synergism by pseudovi-tamin B12. Top: B12, 0.5 Mg/Ml; center: pseudo B12,0.5 jg/ml; right: methionine, 0.3 mg/ml; left:cobalamin phosphoribose, 0.5 ,g/ml. Incubation96 hr.

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TABLE 6Growth response of strain 527 to methionine, vitamin

B12, pseudovitamin B1,2 alone, andin combination

Additions* Optical Density

None ........................... 0Methionine ........................ 0.31B12 ........................... 0.50Pseudovitamin B12................. 0Methionine + B12.................. 1.10Methionine + Pseudovitamin B12 0.53B12 + Pseudovitamin B12 .......... 0.16Methionine + B12 + Pseudovitamin

B12 ......................... 1.30

* Levels per ml: DL-methionine, 10ug; vitaminB12, 0.1 mpg; pseudovitamin B12, 40 mpg. Incuba-tion time, 72 hr at 28 C.

TABLE 7Growth of no. 527 in variousmethionine-pseudovitamin

B12 combinations*

Pseudo- DL-Methionine (pg/ml)vitaminB-2(mpg/ml) 0 10 50 100 500

0 0 0.14 0.24 0.25 0.064 0 0.06 0.54 0.50 0.26

20 0 0.06 0.50 0.50 0.2780 0 0.07 0.50 0.48 0.27160 0 0.06 0.47 0.48 0.29

* Growth expressed as optical density of cul-tures after 51 hr incubation at 28 C.

protected the culture to some extent from thisinhibition, but this effect did not increase withconcentration of the pseudovitamin.

In view of the synergism of pseudovitamin B12and methionine obtained with strain 527, theremainder of the bacteria, for which pseudovita-min B12 was inactive, were retested for pseudo-vitamin B12 activity in the presence of methio-nine. One other strain, no. 375, produced a slightstimulation by pseudovitamin B12 in the presenceof methionine, whereas all others failed to giveany additional response.

Effect of nucleotides. All of the cultures weretested with the pad-plate technique for the pos-sible replacement of sparing of the vitamin B12requirement by the following compounds: de-oxyguanosine, deoxyuridine, adenylic acid,

guanylic acid, uridylic acid, adenine, and guanine.None of the bacteria responded to these com-pounds tested singly or in combination.

DISCUSSION

The major difference in specificity of the bac-teria for the various analogues of vitamin B12 wasindicated by their response to members of thepseudovitamin B11 group. Whereas all of the bac-teria demonstrated a distinct preference for thebenzimidazole analogues, many were able tosubstitute factor A for the vitamin and severalutilized all of the analogues, including the nucleo-tide-free, factor B. In contrast, only one of theLochhead soil isolates studied by Ford andHutner (1957) was responsive to factor A, factorB, or pseudovitamin B12.

Comparison of the relative activity of the vari-ous analogues on the marine bacteria that wereable to utilize all or most of the compounds indi-cates a sequence of decreasing preference as fol-lows: 5,6-dimethylbenzimidazole cobalamin (vi-tamin B12) > 5-methvlbenzimidazole cobalamin> 5-hydroxybenzimidazole cobalamin > benzi-midazole cobalamin > 2-methylmercaptoadeninecobalamin > 2-methyladenine cobalamin >adenine cobalamin > cobalamin phosphoribose> factor B. The presence of methyl substitutionin both benzimidazole and adenine analogues ap-parently results in a much greater activity. Withthe pseudovitamins, the addition of the methyl-mercapto group to the adenine moiety furtherenhances activity.The order of activity of the analogues for E.

coli strain 113-3 was similar to that of the marinebacteria. It should be noted, however, that thenumerical values for several analogues, especiallypseudovitamin B12 and factor B for this organism,were considerably lower than other publishedresults. For example, the activity of pseudovita-min B12 has been reported by different workersto be 50 and 18 (Kon and Pawelkiewicz, 1960) ascontrasted with the results here of 2.2. Likewise,factor B had an activity of only 1.8 in the presentstudy, whereas this compound has been variouslyreported to have an activity of 8 to 18 and 20 forE. coli by the tube test. Presumably, differencesin technique, time of incubation, or other factorsmay affect the relative activity of the analogues.These results as well as the data cited by Konand Pawelkiewicz (1960) indicate that the rela-tive activity of the analogues does not remain re-markedly constant.

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Presumably, analogues of vitamin B12 are in-trinsically active for responsive organisms ratherthan being converted to the vitamin proper. Thisis suggested by the finding that certain analoguesfurnished to E. coli or Ochromonas malhamensiscan be recovered largely unchanged. The recentisolation of a pseudovitamin B12-type coenzymeby Barker and associates (1958, 1960a) fromClostridium tetanomorphum, as well as benzi-midazole-containing coenzyme under altered con-ditions of culture (Barker et al., 1960b), supportsthe view that a number of B12-active coenzymesmay be produced by bacteria and have identicalor closely similar functions.The observation that pseudovitamin B12

antagonizes the utilization of vitamin B12 by cer-tain of the cultures parallels the findings of Fordand Hutner (1957) with soil bacteria. Pre-sumably, the inactive pseudovitamin B12 com-petes with true vitamin for active sites within thecell. The study by Ford (1959) indicated that in-active analogues of vitamin B12 apparentlysaturate the mechanism of the cells for bindingthe vitamin and prevent its uptake.Low concentrations of pseudovitamin B12 and

other inactive analogues were found by Ford andHutner (1957) and Ford (1959) to stimulategrowth slightly in the presence of vitamin B12.These observations are similar to the results ob-tained in the present study. Ford (1959) explainedthe stimulation effect as possibly resulting from acompetition between the analogue and vitaminfor binding substances released outside of the cell,thereby permitting a higher level of free vitaminB12 to enter.The observation that pseudovitamin B12 stimu-

lates strain 527 in the presence of methionine, butinhibits growth in the presence of active vitaminB12 analogues, is an interesting case. With avariety of organisms there is, clearly, an involve-ment of vitamin B12 in the synthesis of methylgroups and methionine; in many cases, me-thionine completely replaces the vitamin. Withstrain 527, however, methionine only spares thevitamin requirement. It would seem that vitaminB12 serves at least two functions in this bacterium.Since no growth occurred when pseudovitaminwas supplied alone, presumably pseudovitaminB12 is unable to function in methyl group bio-synthesis, yet is capable of carrying on some otherfunction if methionine is supplied. It may be thata source of labile methyl groups is required for

activity of pseudovitamin B12 to be expressed ormethionine may simply permit sufficient growthto allow the secondary function of vitamin B12 tobe carried out. A third possibility is that whenmethionine and pseudovitamin are supplied to-gether, the amino acid supports sufficient growthto allow adaptation of the culture or selection ofvariants able to utilize pseudovitamin B12.

Several of the bacteria have promise as agentsof assay for cobalamins in sea water and marinematerials because of their patterns of specificityand tolerance to salt. According to Provasoli(1958b), the use of two assay organisms-onesensitive only to the benzimidazole analogues andanother responsive to all of the cobalamins-may give the most meaningful information aboutthe vitamin B12 distribution in the marine en-vironment. Organisms of both types are repre-sented in the group studied here. These or similarmarine bacteria may prove of value for the assayof vitamin B12 and its analogues in marine ma-terials.

SUMMARY

The response of 21 strains of vitamin B12-re-quiring marine bacteria to analogues of vitaminB12 was studied. The genera represented included:Achromobacter, Brevibacterium, Pseudomonas,Micrococcus, Vibrio, and Flavobacterium. Sixpatterns of specificity were demonstrated basedlargely on the ability of the bacteria to utilizemembers of the pseudovitamin B12 group or theincomplete vitamin. Cultures varied in specificityfrom that of the "mammalian" type, whichshows a response only to the benzimidazole ana-logues, to that like the B12 mutant, Escherichiacoli strain 113-3, which responds to all of thecobalamins.

Benzimidazole analogues were most active forall bacteria followed by the methylmercapto-and methyladenine analogues. PseudovitaminB12, cobalamin phosphoribose, and factor B wereeither inactive or poorly active for most strains.Antivitamin B12 activity was demonstrated bythe pseudovitamins for certain cultures. All butthree of the bacteria were able to replace com-pletely their requirement for vitamin B12 withmethionine, whereas deoxyribosides were inactivefor all.A Flavobacterium isolate which spared the

vitamin requirement with methionine, respondedto pseudovitamin B12 in the presence of the amino

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acid, but not in its absence. It was suggested thatpseudovitamin B12 is unable to carry oIn themethyl group synthetic function of vitamin B12with this bacterium, but can substitute for thevitamin in an unknown second function.

REFERENCESARNSTEIN, H. R. V. 1960 The metabolic func-

tion of vitamin B12. In Vitamin metabolism.Symposium XI, Proc. Fourth Intern. Congr.Biochem., pp. 286-301.

BARKER, H. A., H. WEISSBACH, AND R. 1). SMYTH1958 A coenzyme containing pseudovitaminB12. Proc. Natl. Acad. Sci. U. S., 44, 1093-1097.

BARKER, H. A., R. D. SMYTH, H. WEISSBACH,A. MUNCH-PETERSON, J. I. TOOHEY, J. N.LADD, B. E. VOLCANI, AND M. WILSON 1960aAssay, purification, and properties of theadenylcobamide coenzyme. J. Biol. Chem.,235, 181-190.

BARKER, H. A., R. D. SMYTH, H. WEISSBACH,J. I. TOOHEY, J. N. LADD, AND B. E. VOLCANI1960b Isolation and properties of crystallinecobamide coenzymes containing benzim-idazole or 5, 6-dimethylbenzimidazole. J.Biol. Chem., 235, 480-488.

BURKHOLDER, P. R. 1959 Vitamin-producingbacteria in the sea. First Intern. Oceanogr.Congr., Preprints, pp. 912-913.

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