Redox Regulation of the Genes for Cobinamide Biosynthesis in ...

JOURNAL OF BACTERIOLOGY, Dec. 1989, p. 6734-6739

Vol. 171, No. 12

0021-9193/89/126734-06$02.00/0 Copyright C 1989, American Society for Microbiology

Redox Regulation of the Genes for Cobinamide Biosynthesis in Salmonella typhimurium DAN I. ANDERSSONt AND JOHN R. ROTH* Department ofBiology, University of Utah, Salt Lake City, Utah 84112 Received 28 April 1989/Accepted 5 September 1989

Transcription of the cobinamide biosynthetic genes (the CobI operon) was induced under three different physiological conditions: anaerobiosis (anaerobic respiration or fermentation), aerobic respiration at low oxygen levels, and aerobic respiration with a partial block of the electron transport chain. After a shift to inducing conditions, there was a time lag of approximately 50 min before the onset of CobI induction. Under conditions of anaerobic respiration, the level of CobI transcription was dependent on the nature of both the electron donor (carbon and energy source) and the acceptor. Cells grown with electron acceptors with a lower midpoint potential showed higher CobI expression levels. The highest level of CobI transcription observed was obtained with glycerol as the carbon source and fumarate as the electron acceptor. The high induction seen with glycerol was reduced by mutational blocks in the glycerol catabolic pathway, suggesting that glycerol does not serve as a gratuitous inducer but must be metabolized to stimulate CobI transcription. In the presence of oxygen, CobI operon expression was induced 6- to 20-fold by the following: inhibition of cytochrome o oxidase with cyanide, mutational blockage of ubiquinone biosynthesis, and starvation of mutant cells for heme. We suggest that the CobI operon is induced in response to a reducing environment within the cell and not by the absence of oxygen per se.

distinct regulatory genes. The oxrA gene (39), equivalent to thefnr gene of Escherichia coli (9, 25, 28), encodes a protein that positively controls a number of genes in S. typhimurium, including some terminal reductases (4, 9, 25, 38). The oxrB gene product is involved in anaerobic expression of the oxrA-dependent genes (39). Jamieson and Higgins (18) showed that mutations in the gene for phosphoglucose isomerase (oxrC) prevent anaerobic expression of hydrogenases 1 and 3. It was established earlier (13) that repression of CobI transcription in the presence of oxygen is independent of the oxrA gene. In the accompanying paper (1) we confirm this and show that CobI transcription is also independent of the oxrB and oxrC genes. To elucidate control of the CobI operon in response to oxygen, we examined the regulation of expression and the kinetics of induction of a CobI::lac operon fusion under various growth conditions. Results suggest that oxygen per se does not signal repression.

The B12 biosynthetic genes are organized into three functionally related gene clusters located at 41 min on the chromosome (19, 20); all genes are transcribed counterclockwise and appear to comprise three operons. The CobI operon encodes cobinamide biosynthetic functions, the CoblI operon encodes dimethyl benzimidazole biosynthesis, and the CobIII operon encodes functions required to join cobinamide and dimethyl benzimidazole, forming cobalamin. Most B12 biosynthetic genes appear to be included in these three operons. It was established by Jeter et al. (19) that de novo biosynthesis of vitamin B12 in Salmonella typhimurium occurs only under anaerobic growth conditions. Under aerobic conditions, S. typhimurium can make B12 if provided with cobinamide, suggesting that only synthesis and/or decoration of the corrinoid ring (CobI operon functions) are absent aerobically. Cells can synthesize dimethyl benzimidazole and assemble B12 from cobinamide and dimethyl benzimidazole (CoblI and CoblII operon functions, respectively) under both aerobic and anaerobic conditions. Only four functions in S. typhimurium are known to require cobalamin (8, 14, 33, 34, 40; R. M. Jeter, submitted for publication). Escalante-Semerena and Roth (13) used Lac operon fusions to examine transcriptional regulation of the cobalamin biosynthetic genes. Their studies revealed that transcription of the cobinamide biosynthesis genes (CobI operon) is stimulated by cyclic AMP (cAMP) and that the end product (cobalamin or a derivative) signals repression. Furthermore, CobI transcription is strongly repressed in the presence of oxygen, suggesting the existence of an oxygen-sensing transcriptional regulatory mechanism. Previous studies of several other oxygen-regulated genes in S. typhimurium have revealed evidence for at least three *

MATERIALS AND METHODS Bacterial strains and genetic methods. The genotypes of all bacterial strains used are given in Table 1. All bacterial strains are derivatives of S. typhimurium LT2. A transposition-defective derivative of the specialized transducing phage Mu dl(Ampr lac cts) of Casadaban and Cohen (5) was used. This derivative, Mu dll734Kanr (6), is referred to herein as Mu dJ. One transposition-defective derivative of TnlO, TnJOdell6dell7Tetr (42) was used and is referred to as TnlOdTetr. All transductions were performed with the highfrequency, generalized transducing phage mutant P22 HT105/1 int-201 as previously described (7). Transductants were purified and made phage free by streaking for single colonies on nonselective green indicator medium (7). Culture media, growth conditions and assays. Difco nutrient broth (0.8%) containing 0.5% NaCl was used as a complex medium. The no-carbon E medium of Vogel and Bonner (41) was used as a minimal medium. Solid media

Corresponding author.

t Present address: Department of Microbiology, University of Uppsala, Biomedicum, S-75123 Uppsala, Sweden. 6734

REDOX REGULATION OF cob GENES

VOL. 171, 1989 TABLE 1. Strains of S. typhimurium LT2 used in this study Strain Genotype

TT10852 TT11297 TT14735 TT14736 TT14737 TT14738

TT14739 TT14740 TT14741 TT14742

TT14743

metE205 ara-9 cob-24::Mu dJ metE205 ara-9 cob-24::Mu dJ crp*-771 cya::TnJO cob-24::Mu dJ cob-24::Mu dJ glpK cob-24::Mu dJ glpA cob-24::Mu dJ glpDl09 zhd-1553::TnlO cob-24::Mu dJ glpADJ09 zhd-1553::TnIO cob-24::Mu dJ metE205 zie-1869::TnJOdret cob-24::Mu dJ metE205 zie-1869::TnJOdIet hemA341 cob-24::Mu dJ metE205 ara-9 zbe-1028::TnJO cob-24::Mu dJ metE205 ara-9 zbe-1028::TnJO ubiF2

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contained 15 g of Bacto-Agar (Difco Laboratories) per liter. Carbon sources were present at 0.4% (final concentration) and electron acceptors were present at 20 mM. Auxotrophic supplements were added at concentrations described elsewhere (12). The final concentrations of tetracycline were 20 ,g/ml in complex media and 10 ,ug/ml in minimal medium. To monitor B12 synthesis genetically, strains used carried a metE mutation, which eliminates the B12-independent homocysteine methyltransferase; in such strains, thionene-independent growth depends on B12, a cofactor of the alternative metH methyltransferase (23, 36, 40). Presence of Ado-B12 was monitored by testing growth on ethanolamine; the enzyme ethanolamine lyase requires Ado-B12 (8, 33, 34). In aminolevulinic acid (ALA) starvation experiments, hemA mutant cells were grown overnight in high (100 ng/ml)-ALA medium (nonstarvation) and transferred to low (5 ng/ml)ALA medium (starvation) and then grown for 6 to 10 h to achieve maximum CobI::lac induction. To block electron transport with cyanide, KCN was added to exponentially growing cells to a final concentration of 25 to 100 ,uM, and then cells were grown for 6 to 8 h before being harvested for enzyme assays. Cultures were grown aerobically in 0.5-ml volumes in 10-ml tubes at a speed setting of 8 in a Gyrotory shaker (New Brunswick Scientific Co.). Anaerobic cultures were grown as described earlier (13). The addition of specified amounts of air to sealed anaerobic tubes was achieved by withdrawing with a syringe a given volume of the original gas mix (90% N2-5% H2-5% C02) from the headspace above the culture and injecting the corresponding volume of air. Anaerobic cultures were inoculated by injecting 0.05 to 0.1 ml of aerobic overnight culture into a prewarmed anaerobic culture tube (5 ml). Cells were normally grown overnight to the stationary phase. Extensive control experiments showed that similar f-galactosidase activity was obtained from logarithimically growing cells and cells that had entered the stationary phase after anaerobic growth. Cell density was monitored with a Lambda 4B UV/VIS spectrophotometer (Perkin-Elmer Corp.). f3-Galactosidase activity was assayed as described by Miller (27) by using CHCl3 and sodium dodecyl sulfate to permeabilize the cells. All ,-galactosidase activity values presented are Miller units. RESULTS Expression of the CobI::lac fusion is induced during anaerobic growth. As shown previously (13), transcription of the CobI genes increases in response to anaerobisis. During fermentative growth on glucose, CobI::lac expression increased sixfold compared with growth under aerobic conditions. Anaerobic growth on glycerol-fumarate resulted in a

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200 400 600 Time (minutes) FIG. 1. (a) Kinetics of induction of a CobI::lac operon fusion at different initial oxygen concentrations. The values on the ordinate are Miller units of ,3-galactosidase. The initial oxygen concentrations were 0.09% (C1), 0.48% (*), 4.8% (-), 9.5%, (A), and atmospheric oxygen (0). The experimental details are described in Materials and Methods. (b) Cell density (A6w) as a function of time at different oxygen concentrations. The symbols and oxygen concentrations used are as in panel a. 0

170-fold increase in CobI::lac expression, compared with that in cells grown with aerobic respiration of glycerol. All assays are done in the absence of B12 to prevent its repressive effect. Anaerobic respiration at low oxygen levels induces CobI transcription. Several conditions were checked to determine whether a high level of CobI::lac transcription could be achieved under conditions other than strictly anaerobic respiration (Fig. 1). For example, would aerobic respiration to low oxygen levels, in the absence of other electron acceptors, result in high expression levels? A CobI: :lac fusion strain (TT10852) was grown in sealed tubes containing glycerol as the carbon source and various initial concentrations of oxygen as the sole electron acceptor. Induction of the CobI operon occurred after a time lag that depended on the amount of oxygen provided initially (Fig. la). We conclude that the lag reflects the time necessary for the cells to

ANDERSSON AND ROTH

6736 -

J. BACTERIOL.

induction time measured represents the sum of the time

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200 300 400 time (minutes) FIG. 2. Kinetics of CobI: :Iac induction after a shift from aerobic to anaerobic conditions. ,3-Galactosidase levels (left ordinate; El) are plotted as the square root of the Miller unit value at time t after the shift minus the value at time zero immediately before the shift. The right ordinate shows cell density (*) measured as A,6.. Strain TT10852 was grown to the log phase in aerobic glycerol-nitrate medium and purged for 2 min with N2 to remove any oxygen present, and then a 0.2-ml inoculum was transferred to 5 ml of anaerobic prewarmed glycerol-nitrate medium. The tubes were incubated at 37°C with shaking, samples (0.5 ml) were removed at regular time intervals, and ,-galactosidase activity and cell density were determined. 0

1 00

consume oxygen and reduce its concentration to a level that causes induction. After induction of the operon, enzyme levels increased to a final level of approximately 400 U of

P-galactosidase. The rate of increase of P-galactosidase levels and the final level attained were independent of the initial oxygen concentration. The question of whether induction occurs at low oxygen concentrations or only after all oxygen is consumed is difficult to answer directly. However, observation of the growth rate of cells suggests strongly that there is still residual oxygen present at the time of induction. Recall that the sole carbon source, glycerol, can only be utilized by S. typhimurium in the presence of an electron acceptor. Since oxygen was the only electron acceptor provided and since cell growth continued through the induction period, we conclude that some oxygen remained throughout the induction period. These results suggest that high expression of the CobI::lac operon does not require an alternative electron acceptor but can occur when the oxygen level is low. From this type of experiment, we cannot assess the absolute oxygen concentration at which induction occurs, but clearly it must be lower than the lowest initial oxygen concentration given (0.09%). Time course of CobI::lac transcription after oxygen deprivation. To determine the time required for induction of CobI::lac transcription, strain TT10852 (cob-24::Mu dJ) was grown aerobically in glycerol-nitrate medium and then shifted to completely anaerobic conditions (Fig. 2). Induction of ,-galactosidase started approximately 50 min after the shift in growth conditions. In this experiment the square root of the enzyme activity was plotted versus time after the shift; this value showed an initial linear dependence on time (as it does for other operons) and allowed us to extrapolate to estimate the time delay before the onset of induction. The

required for cells to consume tiny amounts of residual oxygen, signal conditions to the CobI operon, and then transcribe and translate ,-galactosidase from the CobI::lac hybrid operon. We estimate that the time for the culture to become anaerobic was negligible in this experiment, since the only oxygen present was the traces introduced with the innoculum. Transcription and translation of the hybrid operon (5 to 7 kilobases), assuming average rates, would require only a few minutes. Therefore, most of the 50-min delay observed probably represents the time needed for synthesis or turnover of cellular components that are involved in signalling anaerobic conditions to the CobI promoter. Note that induction did not require cell growth; when P-galactosidase first appeared, 50 min after the shift, cell density had not increased compared with that at the time of the shift (0 min). When induction was practically complete (300 min), the cell density had increased less than 50%. Partial blocks of electron transport result in aerobic induction of CobI transcription. The experiment shown in Fig. 1 demonstrated that induction of CobI transcription can occur during aerobic respiration with low oxygen levels. One result of a decreased oxygen level would be a decrease in the rate of electron transport. This suggested the possibility that the CobI operon might be induced in the presence of high oxygen levels if the electron transport chain were inhibited. To test this idea, partial blocks were introduced into the electron transport chain in three different ways. First, the

aerobic terminal oxidase cytochrome o (2, 17, 29) was blocked with low concentrations of the respiratory poison KCN (25 to 100 ,uM). This concentration of cyanide does not inhibit the anaerobic terminal oxidase cytochrome d (2, 17, 29). Second, a block in ubiquinone biosynthesis was introduced by transducing a ubiF mutation (from strain KR42 [43]) into strain TT10852. Two quinones are present in cells; ubiquinone is synthesized and used aerobically, and menaquinone is synthesized and used preferentially under anaerobic growth conditions (4, 17, 32). Third, cells with a hemA mutation were starved for heme by limiting the supply of ALA, which is required for heme biosynthesis in this mutant. All of these conditions caused increased transcription of a CobI::lac fusion despite heavy aeration of the cultures (Table 2). Although the data presented are for samples taken after 6 to 8 h of growth limitation, these levels reflect the highest level obtained during the course of the culture; no transient changes in expression level were seen. Experiments of this general type were performed earlier by Kuritzkes et al. (24) to test regulation of the E. coli glpAB genes (anaerobic dehydrogenase); in this situation, results similar to those reported here were interpreted as suggesting a role of heme proteins in regulation. Finally, we tested whether uncoupling electron transport and oxidative phosphorylation would permit CobI induction in the presence of oxygen. Cells were grown in glycerol minimal medium aerobically, and the uncoupler 2,4-dinitrophenol was added to the culture. At the lowest 2,4-dinitrophenol concentration used (0.1 mM), the growth rate was not reduced significantly, whereas at the highest 2,4-dinitrophenol concentration (2 mM) cell growth stopped. The cells were incubated for 6 to 8 h after the addition of 2,4dinitrophenol and then assayed for P-galactosidase activity. This respiratory uncoupler had no effect on aerobic CobI transcription at any concentration tested (0.1 to 2 mM). Levels of CobI expression depend on the particular electron acceptor present during growth. If a decreased rate of electron transport can stimulate CobI operon expression, one

TABLE 2. Effect of electron transport inhibition on CobI::lac expression Treatment

Cyanide added' Ubiquinone removedc Heme removedd

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1-Galactosidase activitya Before

Aftera

5 5 5

98 31 28

500

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20 6 6

The values are P-galactosidase activity before and after induction is expressed as Miller units (27) under the indicated conditions. b Strain TT10852 (cob-24::Mu dJ) was grown with vigorous aeration in glycerol minimal medium, and KCN was added. The cells were grown for 6 to 8 h after KCN addition and then assayed for P-galactosidase activity. The concentrations of cyanide used decreased the growth rate approximately 10 to 50%. c Strains TT14742 (cob-24::Mu dJ ubiF+) and TT14743 (cob-24::Mu dJ ubiF) were grown in glycerol minimal medium with vigorous aeration to the midlog growth phase and then assayed for p-galactosidase activity. The presence of the ubiF mutation decreased the growth rate approximately 50%o compared with that of the wild type. d Heme starvation was achieved by inoculating strain TT14741 (cob-24::Mu dJ hemA341), grown overnight in glycerol minimal medium with 100 ng of ALA per ml, into the same medium containing only 5 nm of ALA per ml. The cells were grown for 6 to 8 h aerobically and then assayed for ,-galactosidase activity. Since the cells were starved for ALA, the cell growth rate decreased continuously and cell density increased only two to threefold during this time. Growth had stopped at the time cells were assayed for ,B-galactosidase a

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