HEMIN BIOSYNTHESIS IN HAEMOPHILUS

DAVID C. WHITE' AND S. GRANICKThe Rockefeller Institute, New York, New York

Received for publication 19 November 1962

ABSTRACT

WHITE, DAVID C. (The Rockefeller Institute,New York, N.Y.) AND S. GRANICK. Heminbiosynthesis in Haemophilus. J. Bacteriol. 85:842-850. 1963.-Hemin-independent Haemophilusspecies have been shown to form hemin bv theclassical hemin biosynthetic pathway. Threedistinct species of Haemophilus [H. influenzae,H. aegyptius, and H. canis (H. haemoglobino-philus)] all lost the enzymatic capacities toconvert 3-aminolevulinic acid to protoporphyrin,which accounts for their dependence on heminfor growth. The strain of H. aegyptus testedcannot form hemin from protoporphyrin, can betransformed with deoxyribonucleic acid (DNA)from H. influenzae, and the resultant progenyhave the enzymatic activity to convert proto-porphyrin to hemin. Attempts to transform thesespecies to hemin independence with DNA fromhemin-independent H. parainfluenzae are unsuc-cessful under conditions where streptomycinresistance is readily transformed.

The hemin biosynthetic pathway is peculiarin that in almost every detail the same enzymaticsteps are used in animals, plants, and microor-ganisms (see review by Granick and Mauzerall,1961). The steps in the formation of protopor-phyrin (PROTO) are the same whether theend product is cytocbrome-hemin, hemoglobin-hemin, or chlorophyll.The first step in this biosynthesis consists of

the formation of 6-aminolevulinic acid (ALA).This amino ketone is formed from either a-keto-glutarate or suceinate and glycine at a branchpoint in the tricarboxylic acid cycle. Two linearALA molecules are condensed to form into thefive-membered pyrrole ring, porphobilinogen(PBG). Four of these PBG molecules thencondense to form uroporphrinogen III (isomerIII), the reduced hexahydroporphyrin with side

l Present address: Department of Biochemistry,University of Kentucky College of Medicine,Lexington

chains of four acetic and four propionic acids.The uroporphrinogen acetic acid side chains arethen successively decarboxylated to copro-porphyrinogen. Two of the propionic acid groupsare oxidatively decarboxylated to form proto-porphrinogen. Protoporphyrinogen is then oxi-dized to PROTO, and iron is chelated to formhemin. The hemin thus formed is modified intothe various cytochrome hemins, used directlyin hemoglobin or catalase, or formed into chloro-phyll.

Naturally occurring hemin-requiring speciesare rare and confined to microorganisms (Las-celles, 1961). The most extensively studied ofthe hemin-requiring microorganisms is Haemo-philus influenzae. The examination of the miss-ino enzymatic steps in this biosynthetic pathwayis the subject of this paper.

Davis (1917) reported that the heat-stablehemoglobin requirement for growth of H.influenzae could be replaced with hemin. Lwoffand Lwoff (1937) showed that this species grownin limiting hemin had both an immediate increasein respiratory activity and a final level of growththat was directly proportional to the amount ofhemin added to the medium. Granick and Gilder(1946) and Gilder and Granick (1947) showedthat H. influenzae could utilize mesohemin,deuterohemin, and hematohemin as well ashemin, but that the vinyl groups had to bepresent on the porphyrin ring for the bacteriato be able to use the porphyrin itself as thegrowth factor. They showed that aerobic andanaerobic growth is dependent on hemin inproteose peptone medium. Brumfitt (1959)used porphyrin intermediates as growth-stimu-lating substances on agar surfaces containinginhibitory peroxides. ALA, PBG, uroporphyrin(URO), or coproporphyrin (COPRO) did notstimulate the growth under these conditions.

MATERIALS AND METHODS

Solutions of hemins and porphyrins wereprepared as described by Granick and Gilder(1946). URO and PBG were synthesized by and

842

HEMIN BIOSYNTHESIS IN HAEMOPHILUS

provided through the generosity of S. F.McDonald. COPRO III was isolated from diph-theria toxin broth supplied by F. H. Clarke ofLederle Laboratories. Coproporphyrinogen wasprepared from COPRO, using methods describedby TMauzerall and Granick (1958) and Sano andGranick (1960). ALA, aminoacetone, and PBGwere assayed by the methods of MIauzerall andGranick (1956). Aminoacetone, PBG, and ALAwere separated chromatographically by themethods of Elliot (1960) and Urata and Granick(unpublished data).

Bacterial fractions were prepared either withalumina, as described by White and Smith (1962),or by shattering at the temperature of liquidnitrogen (Moses, 1955).

Porphyrin synthetic cap)acities of bacteriaand bacterial fractions were measured by incuba-tion in 13 ml of 0.05 M phosphate buffer (pH7.0) in 25-ml Erlenmever flasks shaken in thedark at 37 C. At zero and various times thereafter2-ml samples were withdrawn and added to 2 mlof 0.3 M trichloroacetic acid. After 10 min at roomtemperature, the mixture was centrifuged at8,000 X g for 10 min. Part of the supernatantliquid was used directly to measure PBG, andpart was chromatographed to separate amino-acetone and ALA and then assayed. The porphy-rins were measured after extracting the incuba-tion mixture with 2 N HCl in a dim light for 30min to allow auto-oxidation to the porphyrin(Sano and Granick, 1960). After centrifugation,the absorption maximum was measured spectro-photometrically and calculated as COPRO[ '402 - 470, 1 N HCl].The porphyrins were identified chromato-

graphically by extracting the incubation solution(made to pH 3.5 with acetic acid) with cyclo-hexanone. The organic phase was then washedwith water, concentrated by flash evaporation inthe presence of ethylenediaminetetraacetic acid,and separated by ascending chromatographyusing 2.6 M lutidine and 0.7 M NH40H (10: 7; v/v)as solvent. The porphyrins separate by carboxylgroup number (Nicholas and Rimington, 1951).The isomers of COPRO were separated using thesystem developed by Mauzerall (1960).

Bacterial growth was measured by the tur-bidity with the Klett colorimeter using the bandpass filter (580 to 600 m,u) in 13-mm test tubes.The concentration of intermediates used were:ALA, 3 X 10-s M; PBG, 5 X 10- M, COPRO,2 X 10-5 M, and hemin, 6 X 10-6 M. ALA was

sterilized by filtration and added from a stocksolution in 0.05 M acetate buffer (pH 4.5). PBGwas dissolved in 0.1 M KOH and added to themedium. COPRO, PROTO, and hemin wereadded from stock solutions made up of 50%17oethanol and 0.01 M KOH. The inoculum con-sisted of about 105 late stationary-phase cellsincubated for 4 hr without hemin at 37 C. Growthwas measured after 36 hr at 37 C in 50-mlErlenmeyer flasks containing 10 ml of medium.The pH at the beginning and the end of theexperiment was 7.6. For growth in copropor-phyrinogen, flasks fitted with fritted-glassdispersion discs and T joints were thoroughlydeoxygenated with hot copper scrubbed nitrogenafter inoculation, and the very alkaline solutionof coproporphyrinogen formed during the reduc-tion of COPRO was added rapidly. The buffercapacity of the medium neutralized the addedalkali, and the flasks were incubated in the darkfor 48 hr at 37 C. Doubling time was measuredfrom the linear portion of the growth curve in125-ml side-arm Erlenmeyer flasks incubated at37 C without shaking.

Bacterial strains used in this study were: H.influenzae rd Garf, H. influenzae rd Sano, H.aegyptus 15, and H. parainfluenzae Boss (suppliedby G. Leidy of Columbia University); H. suis3812, H. suis 3090, and H. suis 7356-8 (isolatedby R. E. Shope, Rockefeller Institute); H.parainfluenzae J-66 and H. parainfluenzae K-17(isolated by E. L. Biberstein, University ofCalifornia); H. haemolyticus 10014, H. para-influenzae 9796, and H. piscium 10801 (AmericanType Culture Collection); H. canis 1659 (H.haemoglobinophilus) and H. aphrophilus 5886(National Collection of Type Cultures, London).A proteose peptone base medium described byWhite (1963), in which growth of hemin-requiringHaemophilus is dependent on added hemin, wasused in this study.The transformation techniques and deoxy-

ribonucleate preparations were those describedby Leidy, Jaffee, and Alexander (1962). Differ-ence spectra were measured as reported previ-ously (White, 1962). Protein was measured bythe biuret method (White, 1962).

RESULTS

Intermediates of hemin biosynthesis as growthfactors. In proteose peptone medium, the finalgrowth achieved and the generation time ofhemin-requiring Haemophilus species were pro-

VOL. 85, 1963 84t3

WHITE AND GRANICK

TABLE 1. Intermediates of hemin biosynthesis asgrowth factors for Haemophilus species

Turbidity (Klett units)

Supplement Hemin requiring Hemin in-to medium dependent

H. influenzae H. aegyptus H. canis H. parain-fluenzae

None 0 0 0 215Hemin 110 115 98 197ALA 0 0 0 210ALA + 112 100 92 190hemin

PBG 0 0 0 220PBG + 105 120 99 198hemin

COPRO 0 0 0 215COPRO + 110 117 100 200hemin

PROTO 105 0 100 170PROTO + 110 110 98 190hemin

portional to the concentration of hemin added tothe medium (unpublished observation). Using thismedium, the various intermediates of heminbiosynthesis can be tested for growth-factoractivity (Table 1). H. parainfluenzae, H. piscium,H. aphrophilus, H. suis, H. parainfluenzae J-66,and H. parainfluenzae K-17 grew in mediumcontaining less than 109 M hemin or 10-8 M

ALA. All these species can form a cytochromesystem as detected by a typical oxygen uptakewith reduced diphosphopyridine nucleotide(DPNH) or by difference spectra (White, 1963)and presumably have the complete heminbiosynthetic system. Three species, H. infiuenzae,H. aegyptus, and H. canis, were unable to grow

in proteose peptone medium unless hemin was

present. H. influenzae and H. canis were able togrow with PROTO in this medium. The inter-mediates ALA, PBG, and COPRO were neitherinhibitory nor stimulatory to growth (Table 1).The fully reduced hexahydroporphyrins ratherthan the porphyrins, themselves, are the actualintermediates in this biosynthetic pathway. Ifthe porphyrins were reduced and added to an

inoculated thoroughly deoxygenated cultureapparatus and incubated in the dark, a repro-

ducible increase in turbidity always resulted(Table 2). This increase consisted of betweenthree and five cell divisions after which growth

TABLE 2. Anaerobic growth of Haemophilus withcoproporphyrinogen

Bacteria

Turbidity (Klett units)

Con-trol

Hemin-requiringspecies

H. influenzae...... 0H. aegyptus.... 0H. canis....... 0

Hemin-independ-ent species

H. parainflu-enzae. 110

stopped. This growth

Control +2 X 10-6 Mcopropor-phyrinogen

131519

120

Control+2 X10-6 Mhemin

353242

108

Control+ 2

X 10-6Mcopropor-phyrin-ogen and2 X 10-6M hemin

755766

was dependent on addedcoproporphrinogen. The porphyrin remainedreduced throughout the growth period as indi-cated by the development of a characteristicpink color upon opening the vessel at the endof the experiment. No new hemin formationcould be detected after growth in coproporphy-rinogen.The hemin-requiring species could store

sufficient hemin to undergo five divisions if grownin a medium where hemin was not growth-limiting. Incubation of a large inoculum inhemin-free growth resulted in an increase in celldensity that stops after 3 to 4 hr. If hemin wasadded there was an immediate resumption ofgrowth.

Bacteria excrete porphyrins into the mediumduring growth (Laseelles, 1961); this is primarilyCOPRO III. The hemin-independent speciesproduce between 10 and 30 ,moles COPRO perliter during a growth cycle, while the hemin-requiring species form no detectable porphyrinduring growth. The porphyrin was extracted byacetic acid-ethylacetate, recovered, and assayedspectrophotometrically in 1 M HCl. No UROcould be detected by extraction with cyclo-hexanone.

Enzymatic activities in hemin biosynthesis. Theinitial step in the formation of hemin is the pro-duction of ALA from succinyl-coenzyme A(CoA), pyridoxal phosphate, and glycine. Theenzyme systems responsible for these reactions

844 J. BACTERIOL.

HEMIN BIOSYNTHESIS IN HAEMOPHILUS

have not thus far been demonstrable in Haeno-philus preparations. The incubation mixturesused successfully by Lascelles (1959, 1960) andKikuchi et al. (1958) for photosynthetic bacterialpreparations and Granick and Urata (1962) formitochondrial preparations were tried withvarious whole- and broken-cell preparations ofhemin-independent Haemophilus species withoutsuccess.Another amino ketone, aminoacetone, appeared

in the medium after growth, at concentrationsof 10 to 30 jiM per liter of medium for both hemin-requiring and hemin-independent species. It canbe formed from threonine as discovered by Elliott(1960) or from acetyl-CoA, pyridoxal phosphate,and glycine by an enzyme with similar activityto the ALA-forming system.

If cell-free preparations of four hemin-inde-pendent Haemophilus species were incubatedwith ALA, the intermediates to COPRO couldbe demonstrated. PBG and the porghyrinogenswith eight, seven, six, five, and four free carboxylgroups could be detected. The ALA could beaccounted for within 10% as PBG, porphyrin, orALA (Table 3), using cell-free preparationsmade with alumina at concentrations between100 and 200 mg of bacterial protein. If theporphyrinogens were oxidized in dim light andair and extracted, lutidine-ammonia chroma-tography separated the intermediates by thenumber of free carboxyl groups on the porphyrin

TABLE 3. Formation of porphyrin intermediatesfrom ALA by hemin-independent Haemophilus

cell-free preparations after incubation*

H. H. hae- H. para-Determination aphro- moly- influ- H. suis

philis ticus enzae

ALA added.984 354 1,080 610PBG formed 41 29 64 14Porphyrin formed 43 33 73 22ALA recovered 690 17 520 357ALA accounted for.. 1,116 339 1,230 561

(113%) (96%) (113%) (93%)ALA equivalentsALA unutilized

(%) ............ 70 5 48 59PBG recovered

(%).; 8 16 11 5Porphyrin formed

(%).35 75 54 29

* Incubation was for 6 hr. Results expressedas pmoles per 100 mg of bacterial protein.

Freecarboxylgroups

0 --

I.

Proto2

3 eCopro

4 ,

5

67

uro

Porphyrin Chromatography2,6 -Lutidine, NH3, H20

9

S

0A 000

40

Partially H. Partiallyhydrolyzed Aaemo- hydrolizediUro Copro 'iticus Pr'otoocta- tetra- i+-cal di-methylmethyl methyl'sterester ester

.H . H.aphrophiius parasnfluenzae suis

+dal +6a1 +dal

FIG. 1. Porphyrin chromatogram of intermediatesformed from 5-aminolevulinic acid by Haemophilusspecies. Porphyrins were identified by their redfluorescence.

ring (Fig. 1). Partially hydrolyzed porphyrinmethyl esters can be used as markers.The enzymes responsible for coproporphyrino-

gen formation were found in the supernatantafter centrifugation at 100,000 X g for 1 hr.Repeating the experiment above with the super-natant fraction of this centrifugation gave resultsnearly identical to those of Table 3.The physiologically active isomer of COPRO

is isomer III. The proportions of isomers formedwith the cell-free preparations can be determinedby careful descending chromatography (Fig. 2).The presence of both isomers I and III is evidencethat Haemophilus forms uroporphyrinogen fromPBG by the classical two-enzyme process (Bogo-rad, 1958a, b). Prolonged storage of preparationsat -20 C increased the proportion of isomer I.

Using these methods, results with similarpreparations of the hemin-requiring species areillustrated in Tables 4 and 5. Essentially noPBG or porphyrin was formed from ALA, andALA could be recovered almost quantitatively.After incubation with PBG, about 10% of theporphyrin formed by a preparation of hemin-independent H. parainfluenzae was detected.

VOL. 85, 1963 845

846 WHITE AND GRANICK

Descending Chrtomatography ofCoproporphyvin Isomers

2, 6 - Lutidine, NH3, H20

Coproporphyr'inIsomers

I - . *0 0

JII +I1V 0 le t (a

II p

lisuis Hparaa

influen.

J. BACTERIOL.

Cytochromo FeHs

RI -CH2\ plOH A2s\\\ 0 CH3C'

Hwmo aa FCytodwom a2

CH3m^Coo2H coo

Hf HIcaemo- aphr'o-lyticaus C philu.s

Coproporphyrin- staindard

5olvent

38cm, 250C

FIG. 2. Coproporphyrin isomer chromatogramseparated by descending chromatography in thesystem described in Materials and Methods.

TABLE 4. Formation of porphyrins front ALA byhemin-requiring Haemophilus cell-free

preparations after incubation*

Determination enzae H. aegypltus H. canis

ALA added ........ 109 4,740 392ALA recovered..... 107 4,600 395

(97%) (99% ) (101%0)PBG formed ....... <0.5 <0.5 <0.5Porphyrin formed.. <0.001 <0.001 <0. 001

* Incubation was for 6 hr. Results expressedas jAmoles per 100 mg of bacterial protein.

TABLE 5. Formation of porphyrin intermediatesfrom porphobilinogen (PBG) by hemin-requiring

Haemophilus cell-free preparations afterincubation*

Determnation H. in- H. H. para-Determination fluenzae aegyptus H. canis influ-enzae

PBG added ........ 37 153 123 121PBG recovered in

6 hr............. 25 115 108 70Porphyrin formed.. 0.5 5.1 2.0 16.5PBG accounted for. 27 135 116 136

(73%) (88%) (95%) (112%)

* Incubation was for 6 hr. Results expressedin pmoles per 100 mg of bacterial protein.

FIG. 3. Hemin biosynthesis in the hemin-requiringHaemophilus. 7'he structures of the cytochromehemins formed by Haemophilus are given: hemin c(Paul, 1951), hemin a, (Lemberg, 1961), and hemina2 (Barrett, 1956).

This porphyrin, formed during incubation withPBG in the hemin-requiring cell preparations,was URO (by chromatography) and was formedby the nonenzymatic condensation of PBG.Boiling the H. influenzae preparation had noeffect on the amount of URO formed on incuba-tion with PBG. The hemin-requiring species wereunable to convert the uroporphyrinogen formednonenzymatically (about 10 m,umoles) tocoproporphyrinogen.The activity of the enzyme which oxidatively

decarboxylates two propionic acid side chainsof coproporphyrinogen III to protoporphyrinogenIX could not be demonstrated, despite repeatedattempts in either hemin-independent or hemin-requiring species. The enzyme that inserts ironinto PROTO to convert it to heme was presentin all Haemophilus, except for the one strain ofH. aegyptus tested. All the Haemophilus specieswere able to convert hemin into the heminprosthetic groups of the cytochromes (Fig. 3).These cytochromes were demonstrated in H. canisgrown with PROTO in the difference spectra ofFig. 4. Here, bacteria whose electron-transportcytochrome system was reduced by DPNH werecompared with a similar suspension of bacteriawhose cytochrome system was oxidized.

Transformation of the hemin requirement. TheHaemophilus group is characterized by thecapacity of transformation of recipient popula-tions with deoxyribonucleate isolated from otherspecies (Leidy, Hahn, and Alexander, 1956,

HEMIN BIOSYNTHESIS IN HAEMOPHILUS

1959; Schaeffer, 1956, 1958). Streptomycinresistance was readily transformed betweenspecies, but neither group was able to transformhemin independence. The hemin requirement isparticularly interesting as it has never beenshown to revert.The finding that H. aegyptus could not grow on

PROTO but required hemin suggested theabsence of the enzyme that catalyzes the insertionof iron into the porphyrin ring. Using deoxy-ribonucleate isolated from H. influenzae (strepto-mycin-resistant, able to grow on protoporphyrin),transformation to the ability to grow on proto-porphyrin occurred readily (Table 6). There was

no evidence of linkage to streptomycin, and this

40.04 _

0

+0.03_

E + 0.02

U

t 0.0)

la

o

< -0.02

DPNH reduced vs. oxidized---- DPNH-CO vs. DPNH reduced--Na25204 reduced vs. oxidized

o-CC

cl (553)

P.

o-CO--\(537) (567) a- .

(559) (635)

400 440 480 520 560 600 640 680

Wavelength (m,it)

FIG. 4. Difference spectra of Haemophilus canisgrown in proteose peptone with protoporphyrin(0.5 ,ug/ml). Bacteria suspended in 0.05M phosphatebuffer (pH 7.0) with 20% (v/v) glycerin.

transformation was completely inhibited byincubating the recipient population with crystal-line deoxyribonuclease prior to the addition oftransforming deoxyribonucleate. Addition ofFeCl2 (1.4 X 10-5 M) or thioglycolate (1.75 X10-3 M) had no effect on the transformation or

the deoxyribonuclease control. The resultanttransformants were able to grow as rapidly andshowed identical final growth in proteose peptonemedium with limiting amounts of protoporphyrinas the naturally occurring PROTO-utilizingspecies of H. influenzae and H. canis (Fig. 5).Demonstration that the transformed activitywas the enzyme in the transformant, H. aegyptus(p+), was suggested by the disappearance of54.5% of added protoporphyrin incubated withwhole cells as compared with 13% in an identicalpreparation of nontransformed H. aegyptua(p-). The incubation mixture contained:protoporphyrin, 0.02 ,umoles; FeSO4, 0.02MAmoles; thioglycolate, 40 ,umoles; 100 ,UM trisbuffer, and bacterial protein, 40 mg per ml; withthe incubation in the dark for 3 hr at 37 C innitrogen, using the technique of Labbe andHubbard (1960).Attempts to transform the hemin-requiring

species to hemin independence were uniformlyunsuccessful under conditions where transforma-tion to streptomycin resistance readily occurred.If the selection for transformants is done inproteose peptone medium, five or six enzymaticactivities must be functional for hemin-independent growth. Addition of ALA (10-4 M)or coproporphyrinogen (10-6 M) to the trans-

TABLE 6. Interspecific transformations of Haemophilus*

Recipient species Donor deoxyribonucleate Transformants in 108 recipients

Haemophilus aegyp- H. influenzae SMis -+ Smr pSm_p + SMP SmrP+tus(Sms, P-) (Smr, P+) 1 X 105 1.1 X 106 0

H. aegyptus H. parainfluenzae Sms -+ Smr H- -* Sm'H- - SmrH+(Sm', H-) (Smr, H+) 5.6 X 106 0 0

H. influenzae H. parainfluenzae Sms -+ Smr H- -- H+ Sm'H-+ SmrH+(Sm', H-) (Smr, H+) 1.7 X 105 0 0

H. parainfluenzae H. canis Sm' -A Smr DPN- -> DPN+(Sm', DPN-) (Smr, DPN+) 2.7 X 101 0

H. parainfluenzae H. aphrophilus Sm' -+ Smr DPN- - >DPN+(Sm', DPN-) (Smr, DPN+) 1.2 X 104 0

* Markers are: Smr, streptomycin resistance; Sm', streptomycin sensitivity; P+, growth onPROTO; P-, no growth with PROTO; H+, growth with hemin; H-, hemin requiring; DPN+,DPN independent; DPN-, DPN requiring.

VOL. 85, 1963 847

WHITE AND GRANICK

60

40

4

H 20

Op-0 -EO

/-UdPF "Doubling Time"/ z(lg/ml protoporphyvirn-I!-o H.cxanis -75rnin.

H.iinf1uenZae-50min.) --- H a.egyptus (P))-70min...1*- H.aegyptus(P)- 20

0.01 0.02'' 1.0

g9 Protoporphyrin/mIFIG. 5. Variation in final growth level in proteose

peptone medium with protoporphyrin concentration.

forming medium would necessitate the activitiesof four or five enzymes for growth in ALA andone enzyme activity for growth in copropor-

phyrinogen, if the bacteria are permeable to thesesubstrates. Adding these substrates had no

effect on the transformation. For copropor-

phyrinogen, incubation must be anaerobic andin the dark. If sufficient hemin to allow twodivisions was added in the presence of thesesubstrates after exposure to deoxyribonucleate,the minimal growth was the same after trans-formation with deoxyribonucleate from hemin-independent species, transformation with deoxy-ribonucleate from hemin-requiring species, andtransformation with the deoxyribonuclease-treated controls, indicating no transformationto hemin independence (Table 6).

If spheroplasts were made of H. parainfluenzaeby growth in 3 M sucrose and 50 units of penicillinand then lysed, the lysate was capable of trans-forming streptomycin resistance to the strepto-mycin-sensitive recipient H. influenzae. Thereare no deoxyribonuclease activity and no viablecells in this lysate. Presumably, this sphero-plast lysate should represent the least-manipu-lated deoxyribonucleate-ribonucleate preparationavailable. Using this preparation, no transforma-tion of H. influenzae to hemin independenceoccurred.The Haemophilus species have another require-

ment for pyridine nucleotide, which is the same

in all species tested (Shifrine and Biberstein,1960). All these species are able to grow on

nicotinamide riboside but not nicotinic acid or

nicotinamide. Again deoxyribonucleate fromspecies not requiring diphosphopyridine nucleo-

tide (DPN) was not able to transform to DPNindependence under conditions where strepto-mycin resistance went readily (Table 6). Inthese experiments, selection for DPN indepen-dence was made directly, in the presence ofnicotinic acid (10-3 M), or after a generation inDPN and treatment with Neurospora diphos-phopyridine nucleotidase.

DISCUSSION

Of those strains of the Haemophilus speciesstudied, the hemin-independent group is com-prised of: H. parainfluenzae, H. aphrophilus, H.suis, H. piscium, and the animal H. parainfluenzaeJ-66 and K-17. All these species will grow inproteose peptone medium, which contains nodetectable hemin, and during growth form a cyto-chrome system containing four types of hemin.The enzyme that converts ALA to PBG, theenzymes converting PBG to uroporphyrinogenIII, and the enzyme that forms coproporphy-rinogen III from uroporphyrinogen III have beendemonstrated to be present. H. haemolyticus isbelieved to be part of this group, as it has theenzymatic activities found in the others, but asyet has not been grown in simplified medium freeof hemin. For H. haemolyticus to grow, no heminneed be added to Brain Heart Infusion Broth.If small amounts of hemin are required, muchless is needed for growth to comparable densitythan in the three species described below. Theevidence that H. haemolyticus can form ALA isthe finding of coproporphyrinogen and COPROin the medium after growth. The conversion ofcoproporphyrinogen to PROTO has not beendemonstrated.The hemin-requiring species must have hemin

present to grow in proteose peptone medium. Thefinal density of growth is proportional to thehemin concentration up to 10-7 M for the threespecies, H. influenzae, H. aegyptus, and H. canis.All three lost the enzymatic capacity to formPBG from ALA, uroporphyrinogen III fromPBG, coproporphyrinogen III from uropor-phyrinogen III, and could not grow on copro-porphyrinogen III. H. aegyptus cannot formhemin from PROTO, but the others can formthe cvtochrome hemins from PROTO. It hasnot been possible to detect the formation of ALAby these three hemin-requiring species.The advantage of the Haemophilus system is

the availability of transformation with isolateddeoxyribonucleate between species. In the case

848 J. BACTERIOL.

HEMIN BIOSYNTHESIS IN HAEMOPHILUS

of the protoporphyrin-iron inserting enzyme,transformation presumably has shown this heminbiosynthetic enzyme to be inherited like otherenzyme systems. The nontransformability tohemin or deoxyribonucleate independence prob-ably represents the fact that a number of genesmust be transformed in a single bacterium toprovide a clone of hemin- or deoxyribonucleate-independent progeny. In the course of manygenerations in the presence of the requiredfactor, secondary mutations that are not now ofselective disadvantage may have occurred, whichmakes the possibility of a recombination to heminor deoxyribonucleate independence even lesslikely. To test whether the genes for the missingenzymes are present but inactive requires theisolation of one-enzyme mutants. These auxo-trophs have thus far not been isolated.

ACKNOWLEDGMENTS

The authors are indebted to G. Leidy, whogenerously provided us with her latest methodsof transformation and growth and transformablestrains of Haemophilus. We wish to thank R. E.Shope and E. L. Biberstein for cultures, S. F.McDonald for porphobilinogen and uropor-phyrin, F. H. Clarke for diphtheria toxin broth,and D. Mauzerall and G. Urata for many stimu-lating discussions.

LITERATURE CITED

BARRETT, J. 1956. The prosthetic group of cyto-chrome a2. Biochem. J. 64:626-639.

BOGORAD, L. 1958a. The enzymatic synthesis ofporphyrins from porphobilinogen I, uropor-phyrin I. J. Biol. Chem. 233:501-509.

BOGORAD, L. 1958b. The enzymatic synthesis ofporphyrins from prophobilinogen II, uropor-phyrin III. J. Biol. Chem. 233:510-515.

BRUMFITT, W. 1959. Some growth requirements ofHaemophilus pertussis. J. Pathol. Bacteriol.77:95-100.

DAVIS, J. D. 1917. Food accessory factors (vita-mines) in bacterial culture with especialreference to Hemophilus bacillus influenzae.J. Infect. Diseases 21:392-402.

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