INTERNATIONALJOURNAL OF SYSTEMATIC BACTERIOLOGY. Jan. 1976, p. 66-73 Vol. 26, No. 1 Copyright 0 1976 International Association of Microbiological Societies Printed in U.S.A.
Nitrate Reductase Activities in Lysogenic and Nonlysogenic Strains of Corynebacterium diphtheriae and Related Species
SHELDON B. ARDEN' AND LANE BARKSDALE Department of Microbiology, New York University Medical Center, New York, New York 10016
Corynebacterium diphtheriae and closely related corynebacteria are gram- positive, pleomorphic, facultatively anaerobic bacteria which have been classi- fied (in part) according to their capacity to reduce or not reduce nitrates to nitrites. An investigation of the presence or absence of nitrate reductase activity in C. diphtheriae, in particular, and in C. beZfanti, C. ulcerans, C. ouis, C. hofmanni, and C. xerosis, in general, indicates that (i) the control of the synthesis of the enzyme is a readily mutable property, (ii) enzyme synthesis occurs under anaerobic conditions, and (iii) maximal activity is associated with the "pellet" or "membrane" fraction. Additional evidence indicates that the gene N-red is not on the phage chromosome and, therefore, is not linked to the gene governing the synthesis of diphtherial toxin (tox) as previously claimed.
The bacterial nitrate reductases, which re- Institute of Pathology, Washington, D.C.); C. bel- duce nitrates to nitrites, serve one of two physio- fanti strains B1, B6, B8, 1170/0re (received from logical functions: (i) that of nitrate assimilation Wenche Blix Gundersen; 12); 106, 107, 109 (received in which nitrate is ultimately reduced to ammo- from N. B. Groman, University of Washington, nium ion and is used as the cell's sole source of Seattle) and 1030 (received from A. Saragea, Bucha- rest, Rumania; 20); C. ulcerans strains 603/50 D.L.C. nitrogen under aerobic and anaerobic condi- (13) and 842/50 D.L.C. (27); C. ovis strains CSIR 6 tions, and (ii) that of nitrate respiration (dissim- (received from H. R. Carne; 7) and 21 (received from ilation), an energy process in which nitrate is A. Saragea; 21); C. hofmanni strains ATCC 10700 utilized in place of oxygen as the terminal elec- and 10701 and E.B. 80 (New York University De- tron acceptor, under anaerobic conditions. The partment of Microbiology); and C. xerosis strains latter process has been referred to as "nitrate or ATCC 373, 7094, 7711 and 9016. anaerobic respiration" (8, 22). Bacteriophages. Phages ptox', PL'Ir (phvs4loS+ 1 The capacity to reduce nitrates to nitrites has and Itox+have been described elsewhere (14). Phage been used as a key property of Corynebacte- 76clovis 21, originating from a gruuis strain, was rium diphtheriae separating it from a cory- received from A. Saragea (21). It is hereafter re- (7), ferred to as Ztox+, nebacterium of ozaena, belfanti (5). It has C. Procedures for the preparation of lysogenic coryne- been reported that corynebacteriophage pros+, bacteria and phage stocks, phage assays, spot test- when used to lysogenize C. belfanti, converts it ing for the detection of bacteriophage, and measure- to nitrate positivity (N-red+)and toxinogenicity ment of phage adsorption were carried out as de- (toxf)(11, 12). This putative Iysogenic co-con- scribed by Holmes and Barksdale (14) and Lampidis version to two positive properties aroused our and Barksdale (16). interest. Here we describe certain properties of Media and general methods. The compositions of corynebacterial nitrate reductase(s) and show the PGT medium, maltose and glucose supplements, that the genetic control of the synthesis of this and tryptose agars, methods of cultivation of bacte- enzyme is not linked to the gene controlling the ria, and the intracutaneous test for toxinogenicity have been described elsewhere (14, 16). Tryptose- synthesis of diphtherial toxin. yeast extract broth contained per liter: tryptose, 10 MATERIALS AND METHODS g; yeast extract, 3 g; and phenol red, 0.005 g. Assays for nitrate reductase activity. (i) Quali- Bacterial strains. The strains of corynebacteria tative determination. Organisms to be screened for used were from this laboratory, except where other- nitrate reductase activity were inoculated (in dupli- wise indicated: Corynebacterium diphtheriae, mitis, cate sets) into PGT liquid media or swabbed onto C7,(-)'""- (hereafter referred to as C7), C~,(P)'""+ 1.5% (wthol) tryptose agar each containing 0.5% (191, C4,(-)tor- (9) and PW8,(P)toSt (16); gravis, (wt/vol) maltose and 0.2% (wt/vol) potassium ni- C8,(-)'0s- (Halifax flat), C8,(4)t0x+(Halifax) (3); in- trate. Tubes and plates were incubated at 37 C for 3 termedius, E.B. 79 (New York University School of to 5 days aerobically (standard incubator atmos- Medicine, The Class Collection of the Department of phere) and anaerobically. Anaerobic conditions Microbiology) and N.S. 1057 (U.S.Armed Forces were obtained by placing the tubes and plates in a Torbal jar (model AJ-2, Torsion Balance Company, Present address: Department of Pathology, Cabrini Clifton, N.J.) and flushing for 5 min with 10% car- Health Care Center, New York, N.Y. 10003. bon dioxide in nitrogen. Three pounds of gas pres- 66 VOL. 26, 1976 CORYNEBACTERIAL NITRATE REDUCTASE 67
sure per square inch were maintained during incuba- expressed as units of enzyme per milligram of pro- tion of the jar. tein (28). The product of the nitrate reductase reaction, ni- Qualitative effect of pH and glucose on nitrate trite, was assayed by the addition to the tubes or reductase activity. Duplicate cultures of C7 grown plates of a solution consisting of equal parts of sul- in complete PGT medium were centrifuged, sus- fanilic acid (8 g/liter of 5 N acetic acid) and of a- pended in 25 ml of PGT plus 1% (wthol) glucose naphthylamine (5 gfliter of 5 N acetic acid) (29). supplement and 0.0005% (wthol) phenol red (final (ii) Quantitative determinations. When the ni- pH 6.8) to an OD of 0.5 to 1.0 in 125-ml Erlenmeyer trate reductase activity of a growing culture was flasks, and incubated in a water bath shaker at being examined, organisms were inoculated into 35 C, 120 rpm (quasi-anaerobiosis). As the glucose complete PGT medium containing 2% (wt/vol) mal- was fermented and the pH was lowered, potassium tose supplement and incubated at 37 C under three hydroxide was added to one flask to neutralize the different conditions: (i) aerobically in a shaking cul- acidity. At l-h intervals, over a 7- to 8-h period, 1.5- ture (300 rpm), (ii) anaerobically by continuous ml samples were removed from each flask and fro- sparging with nitrogen, and (iii)under quasi-anaero- zen. The samples were melted and qualitatively as- bic conditions employing a magnetic stirrer in a sayed for nitrite. In addition, pH values were deter- stoppered bottle. Potassium nitrate at a final concen- mined on each sample with pHydrion papers (Micro tration of 0.8% (wt/vol) was added to each culture at Essential Laboratories, Brooklyn, N.Y .). zero time. At specific intervals 2.5-ml samples were The effect of pH on nitrate reductase activity was removed to small glass tubes and heated in a boiling determined by inoculating C7 to an OD of 1.0 into water bath for 2.5 min to stop enzymatic activity. tryptose-yeast extract broth containing 0.5% The samples were clarified by centrifugation at (wthol) potassium nitrate unbuffered, as well as 1,200 x g for 5 min. buffered with tris(hydroxymethy1)aminomethane When cell-free sonic extracts or their fractions (Tris) maleate to pH 5.5, 6.3, 7.1, and 8.2 in screw- were being assayed for activity, a modification of the cap tubes to one-half volume capacity and incubated assay procedures of Showe and DeMoss (28) and of at 37 C (without shaking). After 1, 2, and 3 days, Ruiz-Herrera et al. (26) was used. Extracts plus samples were taken and qualitatively assayed for 0.125 M potassium nitrate and 1.25 x lo-, M methyl nitrite. viologen (SchwardMann) were made up to a final Protein determinations. Protein was precipi- volume of 2.4 ml with 0.05 M K,HPO,-KH,PO, tated overnight at 4 C from cell-free sonic extracts buffer (pH 7.3) in test tubes (1.5 by 15 cm). The tubes or their fractions by the addition of an equal volume were flushed for 30 s with argon, stoppered with of 10% (wthol) trichloroacetic acid. Precipitates plastic caps, and incubated for 5 min at 35 C. The were centrifuged and washed two times in 5-ml por- reaction was initiated by the quick addition of 0.1 ml tions of cold 2.5% (wthol) trichloroacetic acid and of a freshly made Solution of 50 mg of Na,S,O, in 10 redissolved in 0.1 N NaOH. Protein content was ml of 0.01 N NaOH. After 10 min at 35 C the reac- measured by the method of Lowry et al. (18) using tion was terminated by vigorously shaking the mix- bovine serum albumin as a standard. ture until the Na,S,O, was completely oxidized as Growth and disruption of bacteria. Cultures of indicated by the disappearance of the blue color of C. diphtheriue C7 were inoculated into l-liter Erlen- the reduced methyl viologen. Clarification was car- meyer flasks in 800-ml volumes containing PGT me- ried out as already indicated. dium plus 0.5% (wt/vol) maltose and 1% (wthol) Nitrite content was measured by the method of potassium nitrate. To maintain a low oxygen ten- Showe and DeMoss (28). To 2.5 ml of the nitrite- sion, the flasks were incubated as still cultures at containing samples, 0.75 ml of a solution consisting 37 C. After 4 days the cultures were harvested, cen- of two parts of 4% (wthol) sulfanilamide in 25% trifuged (at 4 C) at 9,000 x g for 20 min and washed (vol/vol) HCl and one part of an aqueous solution of two times with 0.05 M K,HPO,-KH,PO, buffer (pH 0.08% (wt/vol) N-l-naphthylethylenediaminedihy- 7.3). The packed, washed cells were resuspended in 9 drochloride was added. After 10 rnin at room temper- to 10 ml of the same buffer. The concentration of ature the absorbancy at 540 nm was read on a Beck- bacteria was always equivalent to at least 3 mg of man model DU spectrophotometer and determina- bacterial N per ml (4). Powdered glass beads (type tions made using a standard curve prepared using 110 5005, 3M Co., St. Paul, Minn.) were added to the potassium nitrite. resuspended cells which were then subjected to sonic Nitrate reductase activity was followed in grow- vibration (Sonifier Cell Disruptor, model W185, ing cultures by periodic sampling. Nitrite determi- Heat Systems-Ultrasonics, Inc., Plainview, Long Is- nations were made on bacteria-free samples as indi- land, N.Y.) in an iced container for two 2-min inter- cated above. To ascertain the nanomoles of nitrite vals, spaced 1 min apart. The material from dis- released per microgram of bacterial nitrogen, opti- rupted cells was clarified by centrifugation at 25,000 cal density (OD) measurements (590 nm, Bausch x g for 30 min. The supernatant was further frac- and Lomb Spectronic 20) were determined at each tionated by centrifugation at 100,000 x g for 240 rnin interval (optical density of 0.1 = 1.32 x lo8 bacteria at 4 C. Sedimented material was suspended in 0.05 per ml = 5.6 pg of bacterial nitrogen per ml [41). M K,HPO,-KH,PO, buffer (pH 7.3) and is referred to In the case of sonic extracts or their fractions (see as the “pellet” or “membrane” fraction; the superna- below), one unit of enzyme activity is defined as the tant fluid is referred to as the soluble fraction. amount of enzyme which will convert 1 pmol of Isolation of nitrate reductase-deficient mu- nitrate to nitrite per min. The specific activity is tants. Under anaerobic conditions the nitrate reduc- 68 ARDEN AND BARKSDALE INT. J. SYST.BACTERIOL. tases (of certain members of the Enterobacteriaceae) RESULTS catalyze the reduction of nitrate to nitrite and of chlorate to chlorite. This latter product when formed Nitrate reductase activity among species in amounts lethal for the bacteria serves to select of Corynebacterium. Twenty-six strains of nitrate reductase-deficient mutants, because only Coryne bacterium examined under aerobic and those cells lacking this enzyme can survive under anaerobic conditions with regard to nitrate re- anaerobic conditions in the presence of chlorate (23). ductase activities are listed in Table 1. All C. diphtheriae strains C7 and C7(p) were plated seven Cory ne bacteriu d iphtheriae strains out onto tryptose agar containing 0.5% (wt/vol) po- rn tassium nitrate and 0.2 to 0.5 M potassium chlorate tested, mitis, gravis, and intermedius, as well and incubated at 37 C for 5 days under anaerobic as C. hofmanni strains and two strains of C. conditions. The plates were incubated aerobically xerosis, showed strong activity anaerobically for an additional 2 to 3 days to allow the colonies to and weak (to moderate) activity aerobically, increase in size. Isolated colonies were picked and whereas two strains each of C. ulcerans, C. tested for nitrate-reducing activity. ovis, and C. xerosis and eight C. belfanti In addition to this procedure, N-red- mutants of strains were negative under both sets of condi- C7 were obtained by treatment with N-methyl-N- tions. nitroso-N'-nitroguanidine (K and K Laboratories, Corynebacterial nitrate reductase. When Plainview, N.Y.). An overnight culture of C7 was subcultured into complete PGT medium to an OD of C7(-)to~-~-~ed+was grown in complete PGT 0.1 to 0.2 and grown up to approximately 8 x los medium containing 0.8% (wthol) potassium cellslml (OD = 0.6). A 10-ml volume of culture was nitrate and incubated (i) aerobically in a shak- centrifuged, washed once in 10 ml of special Tris- ing culture (300 rpm), (ii) anaerobically by con- maleate buffer (T-M), pH 6.0 (11, resuspended in 10 tinuous sparging with nitrogen, and (iii) em- ml of T-M buffer containing 100 to 300 pg of NTG per ploying a magnetic stirrer in a stoppered bottle ml, and incubated for 15 min, at 35 C, and at 200 under quasi-anaerobic conditions, the greatest rpm. The treated culture was then centrifuged (4 C), amount of nitrate reductase activity was found washed once in cold T-M buffer, and suspended in saline to an OD of 0.5. These cells were serially under conditions of reduced oxygen tension diluted, plated out onto tryptose agar (plus or minus (conditions ii and iii) (Fig. 1). potassium chlorate), and incubated anaerobically as It has been reported that when glucose is above. Isolated colonies were scored for nitrate re- used as a carbon source for Citrobacter sp. ductase activity. Selected mutants were cloned and nitrate reductase activity is repressed (15).Pre- retained for further study. liminary tests with glucose and maltose indi-
- TABLE1. Nitrate reductase activity of various corynebacterial "species"" Nitrate reductase activity Bacterial strains - Aerobicb Anaerobicb
Corynebacterium diphtheriae mitis C7,( -Itoz-, C7,(/3)toz+, PW8,(P)*"5+ Weak to moderate Strong grauis C8,(-)t""-, C8r(4)10z+ Weak to moderate Strong intermedius E.B. 79, NS 1057 Weak to moderate Strong C. ulcerans 603150, 842150 Negative Negative c.ovis 21, CSIR 6 Negative Negative C. belfanti B1, B6, B8, 1170/0re, 106, 107, 109, 1030 Negative Negative C.xerosis 9016, 373 Weak to moderate Strong 7094, 7711 Negative Negative C. hofmanni - 10700, 10701, E.B. 80 Weak to moderate Strong a Qualitative nitrate reductase activity was determined by the intensity of the reddish color formed as a result of the diazo-coupling reagents added. Negative, no color change; weak, pink; moderate, red; strong, dark reddish-brown. Condition of growth. VOL. 26, 1976 CORYNEBACTERIAL NITRATE REDUCTASE 69 tained its N-red- character. N-red- mutants of C7(PYost were still lysogenic and maintained their tox+ status (Fig. 2).
TABLE2. Effect ofglucose and maltose on pH and on yields (qualitative)of nitrate reductasea Sugar Periodic Nitrate re-
ment tivity (at 8 hT
Glucose
C7 cells were inoculated to an OD of 1.0 into PGT medium containing 0.5% (wt/vol) KNO, supple- mented with 1.0% (wt/vol) sugar and incubated un- der quasi-anaerobic conditions in a 37 C water bath Time (hl at 120 rpm. One glucose-containing culture was pe- FIG. 1. Reduction of nitrate by C7,v(-)los-$--red+ riodically neutralized with KOH. The pH of each grown at 37 C under three different conditions of culture was determined at 0 and 8 h. Nitrate reduc- incubation: (i)aerobically in a shaking culture (300 tase activity was qualitatively determined at 8 h rpm), 0;(ii) anaerobically by continuous sparging and scored as in Table 1. with nitrogen, B; and (iii) under quasi-anaerobic conditions employing a magnetic stirrer in a stop- TABLE3. Effect of pH on yields (qualitative)of pered bottle, *. nitrate red uctasea - I I I Tryptose-yeast ex- Nitrate reduc- cated that much less nitrate reductase activity (at 2 days) was associated with glucose-grown cells. How- ever, the final pH of the medium of the glucose- (Unbuffered) 6.8 6.4-6.8 Strong gown cells was 5.5 or lower. C7 grown in PGT + Tris-maleate 5.5 5.5 Negligible medium containing 0.5% (wthol) potassium ni- + Tris-maleate 6.3 6.3 Moderate to strong trate and 1% (wthol) glucose (under quasi-an- + Tris-maleate 7.1 7.1 Strong aerobic conditions) and continually neutralized + Tris-maleate 8.2 8.2 Strong with potassium hydroxide showed nitrate reduc- tase activity qualitatively similar to that pro- C7 cells were inoculated to an OD of 1.0 into duced by maltose-grown cells (Tables 2 and 3). tryptose-yeast extract broths containing 0.5% In the unneutralized glucose-containing cul- (wt/vol) KNO,, unbuffered, and buffered with Tris- maleate (0.05 M):NaOH to pH 5.5, 6.3, 7.1, and 8.2 ture, the pH fell to 5.5 and there was negligible and incubated as still cultures at 37 C. The pH of activity. each culture was determined at 0 and 2 days. Ni- Maximal activity in the tryptose-yeast ex- trate reductase activity was qualitatively deter- tract-nitrate broth was observed with the un- mined at 2 days and scored as in Table 1. buffered culture as well as the cultures buffered at pH 7.1 and 8.2; a slightly decreased activity TABLE4. Localization of nitrate reductase activity in was observed at pH 6.3; and negligible activity fractions of a cell-free sonic extract of was observed at pH 5.5. Corynebacterium diphtheriae strain C7 When the specific nitrate reductase activity Determination Sp acta of a whole-cell sonic extract of strain (x 10-3) C7(-)tos-N-red+ and that of the soluble (superna- tant) and “pellet” or “membrane” fractions were Extract from sonically disrupted C. compared, about 25% of the original activity diphtheriae C7 . . , . . . . , . . . , . . , , . 0.746 remained in the soluble fraction (see Table 4). Soluble (supernatant) fraction . . . . 0.182 Thus, the major nitrate reductase activity “Pellet” or “membrane” fraction . . . 1.348 appears to be located in the “pellet” or “mem- a Specific activity is defined as enzyme units per brane” fraction of C. diphtheriae C7. milligram of protein. One enzyme unit is defined Lysogenization of N-red- corynebacteria. as the amount of enzyme which will convert 1 When the nitrate reductase-negative mutant pmol of nitrate to nitrite per min. Electron micro- C 7 ( - )tor-.V- red- was lysogenized with fitor+(Itoz+ graphic monitorings of the soluble fraction and the or Ztos+),it became a toxin producer but re- “pellet” fraction comprise Fig. 4. 70 ARDEN AND BARKSDALE INT. J. SYST.BACTERIOL. Two other tox-, N-red- corynebacteria, C. Adsorption to, but not lysis of, C. belfanti ulcerans 603 and C. belfanti 1030 when lysogen- strains by phage PLozT.Although seven C. bel-
ized with ptos+gained the capacity to synthesize funti strains were capable of removing /3"or' diphtherial toxin but remained N-red-. C. ouis phage particles in a standard adsorption experi- (N-red-), strain 21, was not sensitive to phage ment with an efficiency sometimes equalling but could be lysogenized with phage Zt"+. that of the C7 reference strain, they were incap- Strain 21(Z)tox+ was still N-red-. Thus, al- able of supporting the growth of the three tax+ though lysogenicity was followed in each of corynebacteriophages p, prir and 1, either in these four cases by conversion to toxinogenic- soft agar overlays on plates or during continu- ity, there was no change in the inability to ous subculture in the presence of excess phage reduce nitrates to nitrites (Fig. 3). (Table 5). DISCUSSION Mutation ~ c7,( -)t~s-~+red- ____- Our initial studies concerning the presence of the anaerobic nitrate reductase were in relation C7,( p)tox+hr-?d Mutation C7s(P)tox+Nred- to the proposed linkage of the gene controlling FIG. 2. Established bacterial genomes which indicate its synthesis to the corynebacteriophage gene, that tox+ and N-red+are separate and independent. tox, which governs the synthesis of diphtherial
FIG. 3. Expression of tox+ in relation to N-red+ in four corynebacterial "'species." TABLE5. Behavior of C.belfanti, C.diphtheriae, and C.ulcerans in the presence of various concentrations of coryne bacteriophages Lysis by drops (0.05 ml) of phages p, Formation of Strain Pr,1 plaques by p, Efficiency of ad- pYrr, 1 (replicates sorption of fit"+ 5 x 109 PFU/ml 1 x 106 PFU/ml in)
C. belfanti Ir Strain B-1 0.9 B-6 1.0 B-8 0.9 1170/0re 0.8 106 1.0 107 1.0 109 1.0 C. diphtheriae Strain C7,( -)fox-c + + + 1.0 C4,( -)fox- + + + 1.1
C. ulcerans Strain 603 + + + 0.6
a PFU, Plaque-forming units; +, lysis; -, no lysis. * Strains of C. belfanti undergo lysis from without (first two columns), but do not support phage replication (column 3). The enzymatic, nonproductive lysis which is observed results from the action of murolytic enzymes in the phage stocks. Standard reference strain, C7,(-)f0s-, which adsorbs pfox' phage with an efficiency of 1.0. FIG. 4. Electron micrographs (prepared by K. S. Kim) ofa bacteria-free sonic extract (‘$ellet” and soluble fractions) of Corynebacterium diphtheriae C7,( -)ios-T-rpf~ negatively stained with 2% (wtlvol) ammonium molybdate. (A) 100,000 x g ‘$ellet” fraction consisting mostly of small membrane fragments associated with major nitrate reductase activity. (B)100,000 x g soluble (supernatant) fraction consisting mostly of ribosome- like particles (215 nm in diameter). There are no bacterial cell membrane structures. See text concerning residual activity associated with this soluble fraction. 71 72 ARDEN AND BARKSDALE INT. J. SYST.BACTERIOL. toxin. If this linkage existed, one would expect erties can often be successfully exploited be- that phage ptox+would co-convert a nontoxino- cause mutations in them or affecting their genic (tor), nitrate reductase-deficient (N- expression are selected against in the natural red-) corynebacterium to the ability to synthe- environment. An example in kind is the useful- size both diphtherial toxin and nitrate reduc- ness of lac- in the characterization of Shigella tase. Two independent investigations employ- dysenteriae and Shigella sonnei . Both orga- ing a number of strains of the N-red- Corynebac- nisms carry the 2 gene controlling the synthe- terium belfanti, plus phage @Ox+, purportedly sis of P-galactosidase but lack entirely the E. achieved this co-conversion in one of two ways: coli permease (17, 25). In the laboratory, mu- (i) by infecting strains with high multiplicities tants which are lac+ are easily obtained. Yet of p or (ii) following continuous subculture of these mutants are often susceptible to such C. belfanti in the presence of this same phage counter-selecting agents as bacteriophages and (17, 19; personal communication to L. B. from seem never to be isolated from the wild. In the N. B. Groman, 1964). The N-red- strains C. case of C. diphtheriae in the wild there may be ulcerans 603, C. belfanti 1030, and a C. diphthe- a special advantage to being able to -turn on riae C7 mutant are tox- and sensitive to phage nitrate respiration with the result that selec-
@Ox+. The N-red-, tox- C. ovis, strain 21, is tive pressures strongly favor N-red + strains. sensitive to phage Ztox+.Lysogenic strains pre- This would especially be the case if, as is true pared from each of the foregoing corynebacteria for a number of gram-negative bacteria (2, 24, and corynebacteriophages were all tox + and N- 30, 311, nitrate respiration in corynebacteria red- (Fig. 3). There seems, then, no correlation were cofunctional with certain other essential between the presence of a tox+ prophage and reductases. the capacity to reduce nitrates. Were there such ACKNOWLEDGMENTS a correlation, the converted N-red + strains would be heterogenotes since the nonlysogenic This work was supported by Public Health Service grant AI-01071 from the National Institute of Allergy and Infec- strains independently undergo mutation with tious Diseases. regard to nitrate reductase activity. We are grateful to K. S. Kim and Edward Goldzimer for Through further investigation of the coryne- aid in carrying out these studies and to Geraldine Hodgson bacterial nitrate reductase, in particular that of for help with this manuscript. C. diphtheriae strain C7, we have shown that REPRINT REQUESTS it is membrane associated. It has also been Address reprint requests to : Dr. Lane Barksdale, Dept. observed that its activity is “repressed” when of Microbiology, New York University Medical Center, 550 cells are grown in the presence of glucose. This First Ave., New York, N.Y. 10016. is actually a pH effect since it can be obtained by growing C7 in a medium having a pH of 5.5 LITERATURE CITED or below. Whereas a drop in pH is always associ- 1. Adelberg, E. A., M. Mandel, andG. C. C. Chen. 1965. Optimal conditions for mutagenesis by N-methy1-N‘- ated with glucose-grown cells, continuous neu- nitro-N-nitrosoguanidine in Escherichia coli. Bio- tralization of them with potassium hydroxide chem. Biophys. Res. Commun. 18:788-795. leads to the formation of reasonable amounts of 2. Azoulay, E., J. Puig, and F. Pichinoty. 1967. Alteration enzyme. It appears that the corynebacterial ni- of respiratory particles by mutation in Escherichia coli. Biochem. Biophys. Res. Commun. 27270-274. trate reductase is a dissimilatory one since (i) 3. Barksdale, L. 1959. Symposium on the biology of cells the media being used contain organic nitrogen modified by viruses or antigens. I. Lysogenic conver- sources, and one would expect these to be prefer- sions in bacteria. Bacteriol. Rev. 23:202-212. entially utilized to the inorganic nitrogen 4. Barksdale, W. L., and A. M. Pappenheimer, Jr. 1954. Phage-host relationships in nontoxigenic and toxi- source (potassium nitrate), and (ii) maximal genic diphtheria bacilli. J. Bacteriol. 67920-232. activity is observed under conditions of anaero- 5. Bezjak, V. 1954. Differentiation of Corynebacterium biosis or reduced oxygen tension. diphtheriae of the mitis type found in diphtheria and The capacity to reduce nitrates was early ozaena. I. Biochemical properties. Antonie van Leeu- wenhoek J. Microbiol. Serol. 20:269-272. exploited as a character useful in the taxonomy 6. Buchanan, R. E., and N. E. Gibbons. 1974. Bergey’s of corynebacteria (6) and mycobacteria (32). In manual of determinative bacteriology, 8th ed., p. 601. Escherichia coli the nitrate reductase complex Williams and Wilkins, Co., Baltimore. appears to be controlled by several genetic loci 7. Carne, H. R. 1939. A bacteriological study of 134 strains of Corynebacterium ouis. J. Pathol. Bacteriol. 49:313- (10). Nothing is known about the genetic con- 328. trol of nitrate reductase in Corynebacterium. 8. Egami, F. 1973. A comment to the concept on the role of The mutation from N-red+ to N-red- is not nitrate fermentation and nitrate respiration in an uncommon. Yet, somehow in nature it does not evolutionary pathway of energy metabolism. Z. Allg. Mi krobiol . 13:177-181. occur very frequently or there would be more 9. Freeman, V. J. 1951. Studies on the virulence of bacte- reports of isolates of C. diphtheriae which lack riophage-infecting strains of Corynebacterium dzph- the activity. In bacterial taxonomy, trivial prop- theriae. J. Bacteriol. 61:675-688. VOL. 26, 1976 CORYNEBACTERIAL NITRATE REDUCTASE 73
10. Glaser, J. H., and J. A. De Moss. 1972. Comparison of cerans, Corynebacterium ovis et Corynebacterium nitrate reductase mutants of Escherichia coli selected diphtheriae. Arch. Roum. Pathol. Exp. Microbiol. by alternative methods. Mol. Gen. Genet. 116:l-10. 27:733-750. 11. Groman, N. B. 1960. Conversion by bacteriophage-a 22. Nason, A. 1962. Symposium on metabolism of inorganic factor in bacterial variation, p. 41-46. In J. D. Daw- compounds. 11. Enzymatic pathways of nitrate, ni- son and L. W. Parks (ed.), Proceedings of the twenty- trite, and hydroxylamine metabolism. Bacteriol. first annual biology colloquium. Microbial genetics. Rev. 26:16-41. Oregon State College, Office of Publications, Corval- 23. Pichinoty, F., J. Puig, M. Chippaux, J. Bigliardi-Rou- lis, Oregon. vier, and J. Gendre. 1969. Recherches sur des mu- 12. Gundersen, W. B., and S. D. Henriksen. 1959. Conver- tants bacteriens ayant perdu les activites cataly- sion in Corynebacterium belfanti by means of a tem- tiques liees a la nitrate reductase A. 11. Comporte- perate bacteriophage originating from a toxigenic ment envers le chlorate et le chlorite. Ann. Inst. strain of Corynebacterium diphtheriae, type mitis. Pasteur Paris 116:409-432. Acta Pathol. Microbiol. Scand. 47:173-181. 24. Piechaud, M.,J. Puig, F. Pichinoty, E. Azoulay, and L. 13. Henriksen, S. D., and R. Grelland. 1952. Toxigenicity, Le Minor. 1967. Mutations affectant la nitrate-reduc- serological reactions and relationships of the diphthe- tase A et d'autres enzymes bacteriennes ria-like corynebacteria. J. Pathol. Bacteriol. 64503- d'oxydoreduction. Etude preliminaire. Ann. Inst. Pas- 511. teur Paris 112:24-37. 14. Holmes, R. K.,and L. Barksdale. 1969. Genetic analy- 25. Rickenberg, H. V. 1960. Occurrence of p-galactosidase sis of to%+ and toz- bacteriophages of Corynebacte- in the genus Shigella. J. Bacteriol. 80:421-422. rium diphtheriae. J. Virol. 3:586-598. 26. Ruiz-Herrera, J., M. K. Showe, and J. A. De Moss. 15. Kapralek, F., J. Dolezal, and I. Drahonovska. 1973. 1969. Nitrate reductase complex of Escherichia coli Effect of glucose on the synthesis of bacterial respira- K12: isolation and characterization of mutants una- tory nitrate reductase and tetrathionate reductase. ble to reduce nitrate. J. Bacteriol. 97:1291-1297. Folia Microbiol. (Prague) 18:l-6. 27. Saxholm, R. 1951. Toxin-producingdiphtheria-like orga- 16. Lampidis, T., and L. Barksdale. 1971. Park Williams nisms isolated from cases of sore throat. J. Pathol. number 8 strain of Corynebacterium diphtheriae. J. Bacteriol. 63:303-311. Bacteriol. 105:77-85. 28. Showe, M. K., and J. A. DeMoss. 1968. Localization 17. Li, K.,L. Barksdale, and L. Garmise. 1961. Phenotypic and regulation of synthesis of nitrate reductase in alterations associated with the bacteriophage carrier Escherichia coli. J. Bacteriol. 95:1305-1313. state of Shigella dysenteriae. J. Gen. Microbiol. 29. Society of American Bacteriologists. 1957. Manual of 234~355-367. microbiological methods, p. 153-154. McGraw-Hill, 18. Lowry, 0.H., N. J. Rosebrough, A. L. Farr, and R. J. New York. Randall. 1951. Protein measurement with Folin 30. Stouthamer, A. H. 1967. Nitrate reduction in Aerobac- phenol reagent. J. Biol. Chem. 193265-275. ter aerogenes. 11. Characterization of mutants blocked 19. Matsuda, M., and L. Barksdale. 1967. System for the in the reduction of nitrate and chlorate. Arch. Mikro- investigation of the bacteriophage-directed synthesis biol. 56:76-80. of diphtherial toxin. J. Bacteriol. 93:722-730. 31. Stouthamer, A. H. 1969. A genetical and biochemical 20. Maximescu, P. 1968. New host strains for the lysogenic study of chlorate resistant mutants of Salmonella Corynebacterium diphtheriae Park Williams no. 8 typhimurium, Antonie van Leeuwenhoek J. Micro- strain. J. Gen. Microbiol. 53:125-133. biol. Serol. 35505-521. 21. Maximescu, P., A. Pop, A. Oprisan, and E. Potorac. 32. Virtanen, S. 1960. A study of nitrate reduction by myco- 1968. Relations biologiques entre Corynebacterium ul- bacteria. Acta Tuberc. Scand. Suppl. 47:l-119.