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Nitrate Reductase Activities in Lysogenic and Nonlysogenic Strains of Corynebacterium Diphtheriae and Related Species

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Nitrate Reductase Activities in Lysogenic and Nonlysogenic Strains of Corynebacterium Diphtheriae and Related Species 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).
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