Induction of Anaerobic Gene Expression in Rhodobacter Capsulatus Is Not Accompanied by a Local Change in Chromosomal Supercoiling As Measured by a Novel Assay DAVID N

Home , Rhodobacter capsulatus

JOURNAL OF BACTERIOLOGY, Sept. 1989, p. 4836-4843 Vol. 171, No. 9 0021-9193/89/094836-08$02.00/0 Copyright © 1989, American Society for Microbiology Induction of Anaerobic Gene Expression in Rhodobacter capsulatus Is Not Accompanied by a Local Change in Chromosomal Supercoiling as Measured by a Novel Assay DAVID N. COOK, GREGORY A. ARMSTRONG, AND JOHN E. HEARST* Department of Chemistry, University of California, Berkeley, and Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, Berkeley, California 94720 Received 21 March 1989/Accepted 10 June 1989

In the photosynthetic bacterium Rhodobacter capsulatus, the enzyme DNA gyrase has been implicated in the expression of genes for anaerobic metabolic processes such as nitrogen fixation and photosynthesis. To assess the involvement of supercoiling in anaerobic gene expression, we have developed an assay to detect in vivo changes in superhelicity of small regions of the bacterial chromosome. Our method is based on the preferential intercalaction of psoralen into supercoiled versus relaxed DNA, and we have demonstrated the sensitivity of the assay in vivo on chromosomal regions from 2 to 10 kiobases in size. In experiments with inhibitors of gyrase, the reactivity of individual chromosomal fragments to psoralen decreases by a factor of 1.8 compared with DNA from control cultures. We used our assay to determine whether there is a change in superhelicity near the genes coding for essential proteins for photosynthesis upon a shift from respiratory to anaerobic photosynthetic growth. For comparison, we also examined a restriction fragment containing the Jbc operon, which codes for the subunits of cytochrome bcl, a membrane-bound electron transport complex utilized during both aerobic and anaerobic photosynthetic growth. During this shift in growth conditions, the pufand puh mRNAs, coding for structural polypeptides of the photosynthetic apparatus, underwent a six- to eightfold induction, while the amount of mRNA from thejbc locus remained constant. However, we detected no change in the superhelicity of either the genes for photosynthesis or those for the bc, complex during this metabolic transition. Our data thus do not support a model in which stable changes in chromosomal superhelicity regulate anaerobic gene expression. We suggest instead that the requirement for DNA gyrase in the transcription of photosynthesis genes results from the requirement for a swivel near heavily transcribed regions of the chromosome.

A recent model for the general control of gene expression latus is also capable of nitrogen fixation under anaerobic, for aerobic and anaerobic metabolism has postulated a role nitrogen-limiting conditions. Kranz and Haselkorn (17) have for DNA topology in regulating the transition between shown that synthesis of the R. capsulatus nifHDK gene metabolic modes (40). The model proposes that DNA gy- products is inhibited by a 5-h treatment with the gyrase rase, which introduces negative supercoils into the chromo- inhibitor novobiocin, whereas synthesis of most major solu- some, is essential for anaerobic gene expression, while ble proteins appears to be unaffected as judged by 3H-labeled topoisomerase I, which relaxes the chromosome, is required amino acid incorporation and sodium dodecyl sulfate-poly- for expression of genes for aerobic metabolism. This hypoth- acrylamide gel electrophoresis (SDS-PAGE). In a study of esis is based on the isolation of obligate aerobic strains of mRNA accumulation from essential genes for photosynthe- Salmonella typhimurium which were shown to have muta- sis, Zhu and Hearst (43) showed that inhibition of gyrase tions in one of the genes for DNA gyrase (gyrA or gyrB) and results in the loss of detectable mRNA from the puhA locus, of obligate anaerobes with mutations mapping to the gene for which codes for the H subunit of the photosynthetic reaction topoisomerase I (topA) (40). This model of a gyrase-induced center, and from the puf operon, which codes for the switch in metabolic modes has been extended to other pigment-binding reaction center subunits L and M and for facultative anaerobes as well through the study of the effects the light-harvesting I antenna polypeptides. Loss of detect- of gyrase inhibitors on expression of essential genes for able puhA mRNA occurred within 15 min after treatment anaerobic metabolism. In Klebsiella pneumoniae, expres- with gyrase inhibitors, a time comparable with the half-life of sion of the nifHDK genes, which code for and nitrogenase the message. In contrast, mRNA levels for the fbc operon nitrogenase reductase, is blocked by drugs which inhibit coding for the cytochrome complex, which is utilized for gyrase (17). In Bradyrhizobium japonicum, expression of bc, both respiration and photosynthesis, are unaffected by gy- enzymes for hydrogen metabolism, an anaerobic process, is rase inhibitor treatment. The rapidity of the decrease in also repressed by drugs which target gyrase (22). For the purple photosynthetic bacterium Rhodobacter puhA and puf mRNA has been interpreted to imply that the observed effects result from gyrase inhibition and capsulatus, recent papers have documented the inhibition of directly are not a to treatment anaerobic gene expression by drugs which inhibit gyrase. secondary response drug (43). Under anaerobic conditions in the light, R. capsulatus An attractive hypothesis is that DNA superhelicity in the develops an extensive photosynthetic membrane system in region of the genes for photosynthesis is altered by DNA which pigment-protein complexes carry out light-driven gyrase, leading to repression or derepression of transcription electron transport to generate metabolic energy. R. capsu- (43). This model might explain why most of the known essential genes for photosynthesis are clustered on a 46- kilobase (kb) section of the chromosome (20), as shown in * Corresponding author. Fig. 1. This section of the chromosome codes for at least 4836 VOL. 171, 1989 CHROMOSOMAL SUPERCOILING AND ANAEROBIC GENE EXPRESSION 4837

puhA ratio of 80:20:2. Photosynthetic cultures were illuminated at 15 W m-2 by a bank of General Electric Lumiline lamps and 4- sparged with a mixture of N2 and CO2 in a ratio of 80:2. Gas bch crt bch crt bch flow rates were approximately 250 cm3 min-1 for each BamHI N K F D L 01 E H G MJ culture. Gas flow rate and composition were controlled with . . . . -f a Matheson Gas Products Multiple Dyna-blender, model i: .y :i}:: i:: i:: i: s -- i-ER--E :iSE::iED:yiS: :: 7:::: T T sw s :::::::::: :: :::::: :::::: ::: o / ::::::::: :: ::::::: :i:: iL:7:X: :i: v # ! v / / ::: ::::::: ::::::::::::::::::::: :::::::::::::::: ::::: :::::::: ::: :::: :::::: ::::::::::::::::::::::::: ::::::: :: > > 8219. Growth rates were monitored at 680 nm on a Bausch & r :::E +E iEEriL.: 7:E :0: :X: :i:::i:|:E: ::: S S ' Lomb Spectronic 21. H H H H Irradiation of cells. Logarithmically growing cultures were quickly cooled in an ethanol-dry ice bath and pelleted for 6 min at 12,000 x g in an SS34 rotor. All subsequent steps were performed in dim light to eliminate undesired trimeth- FIG. 1. Physical-genetic map of the R. capsulatus photosynthe- ylpsoralen (TMP) cross-linking. Cells were suspended in sis gene cluster. BamHl sites are denoted by solid circles, and ice-cold TES buffer (50 mM Tris hydrochloride [pH 8], 10 fragment designations are shown in capital letters. Bacteriochloro- mM EDTA, and 50 mM NaCl) and 0.25 .g of TMP per ml for phyll biosynthesis genes (bch) are indicated by stippled regions, and 10 min on ice. Irradiations were performed on 6-ml samples carotenoid biosynthesis genes (crt) are indicated by crosshatched in a six-well tissue culture plate on ice. UV regions. Directions of transcription for the puf and puh genes are Long-wavelength denoted by the arrows. Note that 35 kb of DNA separates the puf light at a power density of 2 mW cm-2 was provided by a and puh promoter regions even though both operons code for bank of blacklight bulbs (Southern New England Ultraviolet structural proteins of the photosynthetic reaction center. Locations Co.; Xmax = 350 nm). Irradiated samples were pelleted, of M13 probes used in both Southern and RNA dot blotting are suspended in a lysis solution consisting of 0.5% SDS, 10 mM indicated below by bars. For a more detailed map, see reference 41. EDTA, and 23 pLg of proteinase K per ml, and stored at -70°C for later use. DNA isolation. Cells were lysed by three freeze-thaw five structural proteins of the photosynthetic apparatus cycles between a 37°C bath and dry ice. After a 20-min (pufBALM and puhA) as well as all known enzymes for incubation at 37°C, DNA was extracted once with phenol- carotenoid and bacteriochlorophyll biosynthesis (32, 45). chloroform-isoamyl alcohol (25:24:1) and twice with chloro- Nonphotosynthetic functions have not been mapped to this form-isoamyl alcohol (24:1). All steps though the first chlo- section of the genome (45). Since the bacterial chromosome roform extraction were performed in dim light. Nucleic acids has been shown to be composed of topologically indepen- were precipitated with ethanol and suspended in 0.5 ml of dent domains of torsionally strained DNA (30, 38), the TES. RNase A digestion (30 p.g ml-1) for 15 min at 37°C was photosynthesis cluster might conceivably be contained on a followed by proteinase K digestion (50 pLg ml-') for 30 min at single domain and thus be subject to facile regulation by 55°C. Samples were again extracted with phenol-chloroform- gyrase. Topological isolation of the photosynthesis gene isoamyl alcohol and chloroform-isoamyl alcohol, followed cluster might allow it to be regulated by gyrase indepen- by ethanol precipitation. Before restriction, samples were dently of other regions, such as the Jbc locus. irradiated with 36 kJ of UV-A per m2to cross-link any TMP Heretofore, only an overall change in superhelicity of monoadducts in potentially cross-linkable sites. chromosomes has been amenable to analysis either by Gel electrophoresis and Southern blotting. Gels and DNA ethidium titration (7) or by isotopically labeled psoralen samples were handled essentially by the method of Vos and binding (29). These methods allow one to detect changes in Hannawalt (35). From 4 to 5 pLg of BamHI-restricted ge- bulk chromosomal superhelicity but cannot test whether the nomic DNA was denatured with 0.1 volume of 1 M NaOH at torsional state of several domains changes independently 55°C for 2 min, rapidly neutralized with 0.12 volume of 1 M without altering the average superhelical density of the Tris hydrochloride (pH 4.2), and placed on ice. Samples chromosome. We have therefore developed an assay that were run on Tris-phosphate-agarose gels and blotted to uses psoralen cross-linking to detect changes in the super- nitrocellulose overnight. Southern blots were probed with helicity of small segments of the chromosome. Our results primer-extended DNA made from the single-stranded M13 indicate that there is no stable change in the unrestrained subclones shown in Fig. 1. The BamHI D fragment of the superhelicity of DNA in the photosynthesis gene cluster photosynthesis gene cluster (20, 32) and a 9-kb BamHI during a shift of growth conditions in which genes for fragment hybridizing tojbc were nick translated on plasmids photosynthesis are induced six- to eightfold. Similarly, we containing these inserts. The probe forfbc was constructed observed no change in the superhelicity of DNA coding for from pRSF1 (8) by removing the BamHI fragments which do the constitutively expressed cytochrome bc1 complex. Our not contain the fbc genes. The resulting plasmid was named data thus do not support a model in which gyrase activates pDC100. After hybridization and autoradiography, radioac- gene expression by adjusting local superhelix density under tive bands were cut from the filter, dissolved in 1 ml of ethyl anaerobic conditions. We speculate that the requirement for acetate, and counted in 5 ml of Opti-fluor (Packard Co.) in a DNA gyrase in the transcription of anaerobically induced Packard model 3385 scintillation counter. genes may result from the topological requirement for a RNA isolation and dot blotting. RNA was extracted by a DNA swivel near heavily transcribed regions of the chromo- scaled-down version of the procedure of Zhu and Kaplan some. (44). Equal amounts of purified RNA, about 5 ,ug per time point, were dot blotted onto a Gene Screen membrane (New MATERIALS AND METHODS England Nuclear Corp.) with a Minifold dot blot apparatus (Schleicher & Schuell) by the method of Schloss et al. (28). Bacterial strains and growth conditions. R. capsulatus Probes were made as stated above. In order to account for SB1003 was grown in 150 ml of RCV medium (37) in 250-ml fluctuations in the amount of RNA dotted, we also prepared side-arm flasks at 32°C. Aerobically grown cultures were dots from serial dilutions with about 3 ng of RNA per time sparged in the dark with a mixture of N2, 02, and CO2 in a point. These diluted samples were probed with either labeled 4838 COOK ET AL. J. BACTERIOL. pRC1 (42), which encodes one set of R. capsulatims rRNA genes, or with nick-translated R. capsulatus chromosomal DNA. After hybridization and autoradiography, dots were quantitated by scintillation counting as described above, except that the nylon membranes were not dissolved in ethyl acetate. Data for the puf, puh, and fbc probes were normalized for both rDNA (see Fig. 4B and C) and chromosomal DNA hybridization (data not shown), yielding similar re- B sults. Chemicals. TMP and novobiocin were purchased from Sigma Chemical Co., St. Louis, Mo.

RESULTS 15:-) Assay for local changes in chromosomal superhelicity. DNA intercalators unwind the double helix and therefore bind .3 more readily to negatively supercoiled than to relaxed DNA. The psoralens are a class of intercalators which are capable -04*- of reacting photochemically with DNA (for a review, see reference 3). Upon irradiation with long-wavelength UV light (UV-A, 320 to 380 nm), psoralens form cyclobutane L J 'DCD | monoadducts and interstrand cross-links with pyrimidine bases of the DNA. The rate of formation of these adducts FIG. 2. Schematic illustration of the TMP assay. (A) DNA is has been shown to be a function of DNA superhelicity both irradiated in vivo to a level of less than 1 cross-linkable TMP adduct in vitro and in vivo (14, 29). In experiments with Escherichia per kb. After isolation, samples are reirradiated in vitro to ensure coli, the rate of addition to chromosomal DNA in control the maximum yield of psoralen cross-links. (B) Following endonu- cells is nearly twice that of cells either treated with the clease restriction, DNA is denatured briefly with alkali. (C) Upon gyrase inhibitor novobiocin or irradiated by a neutralization, TMP-cross-linked DNA reanneals rapidly, whereas 'Co y-emit- un-cross-linked DNA is irreversibly denatured. (D) Samples are ting source (29). These experiments utilized a radiolabeled electrophoresed in an agarose gel, blotted to nitrocellulose, and derivative, [4,5',8-3H]TMP, to determine incorporation into probed for the specific sequence of interest. The level of cross- total DNA and therefore could not determine levels of linking for any fragment in the genome can be determined in this photoaddition to individual sequences. manner. XL, Cross-linked DNA; SS, single-stranded DNA. We have extended the usefulness of psoralens for detecting supercoiling in vivo by exploiting the ability of psoralen- cross-linked DNA to reanneal rapidly after denaturation were run on native agarose gels and blotted, and the frag- (15). Our method allows us to determine the rate of TMP ments of interest were visualized by hybridization. cross-linking to any specific DNA restriction fragment in the These blots characteristically contained two bands in each genome (Fig. 2). After in vivo cross-linking at low levels lane: a lower band which ran at the position of the single- (less than 1 TMP cross-link per kb), genomic DNA is purified stranded DNA, and an upper cross-linked band which mi- and denatured. Under these alkaline conditions, cross-linked grated at the position of double-stranded DNA. Samples strands melt but are held in register by TMP so that they incubated with TMP but not irradiated showed no detectable reanneal rapidly upon a return to neutral pH. The cross-link cross-linked band (Fig. 3A). Longer exposure to light re- provides a nucleation site for helix formation, whereas sulted in a higher percentage of cross-linked DNA. Most unmodified or monoadducted DNA is irreversibly denatured importantly, the rate of appearance of cross-linking was in our procedure. Electrophoresis under native conditions notably slower when cells were treated with novobiocin, separates cross-linked from un-cross-linked DNA, and the indicating that less TMP was intercalated in the relaxed amount of cross-linking of any particular restriction frag- chromosome. ment in a genomic digest can be quantified by Southern Blots can be quantitated by determining the fraction of blotting (35). counts at the single-stranded position for each time point. Figure 3 shows the results of novobiocin-induced relax- When plotted on a semilog scale, these data yielded a linear ation of the chromosome as measured in the TMP assay. R. relationship (Fig. 3B). This behavior would be expected for capsulatus SB1003 was grown aerobically in the dark as a Poisson-type process, in which a single psoralen cross-link described in Materials and Methods. A portion of the culture in a DNA fragment is sufficient to cause the DNA to run as was quickly cooled, pelleted, and suspended in an ice-cold a double strand (35). The ratio of cross-linking rates between buffer containing TMP. Cells were incubated in the dark on control and novobiocin-relaxed DNA for restriction frag- ice to allow the TMP to equilibrate and subsequently irradi- ments in this and other experiments was 1.8 (Fig. 3B and ated while on ice. The remainder of the culture was treated data not shown). We examined a number of BamHI restric- with novobiocin, harvested, and irradiated as described tion fragments from the photosynthesis gene cluster, includ- above. After isolation of genomic DNA and removal of free ing BamHI-C, -D, -F, -G, -H, -J, and -K (Fig. 1). These TMP by organic extraction, samples were reirradiated with restriction fragments contain genes coding for carotenoid UV-A to cross-link any psoralen monoadducts in potentially and bacteriochlorophyll biosynthetic enzymes and for the cross-linkable sites. Since our assay is specific for cross- structural polypeptides which bind these pigments and carry links, this in vitro irradiation step substantially increases the out light harvesting and primary photochemistry in the sensitivity and improves the reproducibility of the assay. photosynthetic membrane (32, 45). These fragments ranged The DNA samples were then restricted and denatured im- in size between 2 and 10 kb, and each was sensitive to the mediately prior to gel electrophoresis. Neutralized samples cross-linking assay in the presence of novobiocin. Larger VOL. 171, 1989 CHROMOSOMAL SUPERCOILING AND ANAEROBIC GENE EXPRESSION 4839

A (-) NOVO (+) NOVO A 0 .5 1 2 4 8 0 .5 1 2 4 8

1.0 _ XL me4.iWU4 me~

.8 0

Ss -t e

.2 B

-2 0 2 4 6

z Time after shift (hours) U')(I) B

0 C4) Zs Shift * (L3 E wi 056 -2, i*;h i f t *~~~~0. 0 0 2 4 6 a 0 4 Time of irradiation (min) 'a) -J I I~~~~~~1 FIG. 3. (A) Southern blot of DNA irradiated in vivo for various 2 amounts of time, digested with BamHI, and probed for the BamHI- 4~ K fragment of the photosynthesis gene cluster (32, 45) without (-) and with (+) novobiocin (NOVO) treatment. Cells were grown aerobically to early log phase, and half the culture was harvested for cr- an irradiation time course at 2 mW of UV-A per cm2 for the number -40 0 40 80 l 20 of minutes shown above each lane. The remaining cells were treated with 100 mg of novobiocin per ml for 15 min and then irradiated. (B) Time after shift (min) Quantitation of results from the blot in panel A and a like blot for the BamHI F fragment. Symbols: *, control culture; +, novobiocin- treated culture. SS, Single stranded; XL, cross-linked. C fragments cross-linked at a faster rate, since they repre- -20 -1 0 0 20 30 45 60 90 1 20 sented a larger target for cross-linking (Fig. 3B). mRNA accumulation during the shift from aerobic to an- puhA aerobic photosynthetic growth. TMP is a hydrophobic com- pound (aqueous solubility of 0.6 ,ug ml-') and, when added to an anaerobic photosynthetic cell culture, would preferen- tially reside in the extensive photosynthetic membrane sys- pufLN * * tem ofR. capsulatus. This fact makes a direct comparison of chromosomal superhelicity between aerobic and photosynthetic cultures a difficult task. We chose instead to perform fbc the assay during a shift from aerobic to anaerobic conditions to alleviate any potential problems caused by sequestering of FIG. 4. (A) Growth curves for two different cultures upon a shift TMP in the mnembrane of steady-state photosynthetic cells. from aerobic to anaerobic photosynthetic growth conditions. (B) Cultures shifted from 20 to 0% oxygen exhibited little or no mRNA accumulation time course for a shifted cell culture for pufL growth for the first hour and thereafter resumed growth at a and pufM (*), puhA (El), andfbcF,fbcB, and ]bcC (K). Data shown slightly faster rate than under aerobic conditions (Fig. 4A). were normalized to rRNA, but similar results were obtained when This result is in agreement with the work of Gray (12), who normalized to total RNA (see Materials and Methods). (C) Autora- of the from showed that, in an identical shift from aerobic-dark to diograms samples panel B. anaerobic-light conditions, Rhodobacter sphaeroides under- 4840 COOK ET AL. J. BACTERIOL. goes a 90-min lag phase. During this period there are no significant increases in cell number or cell mass or in total cellular RNA, DNA, or protein content (12). -0 In the first 2 h after the shift, transcription of the structural 0L) BamHI-K genes for photosynthetic reaction centers (coded for by pufLM and puhA) and the light-harvesting I antennae (coded ci for by pufAB) was induced approximately six- to eightfold (Fig. 4B and data not shown). A time course of mRNA ci) W accumulation demonstrated that these genes were induced shortly after a rigorous aerobic to anaerobic shift and that 0 mRNA accumulated to a maximum approximately 90 min C.) \BamHI-F after the shift, about the same time at which cell growth resumed (Fig. 4A). In comparison, mRNA for the cy- U- bcl tochrome bc, complex (coded for by the 11c operon) did not change significantly from preshift levels (Fig. 4B). This I 2~~% complex is utilized for respiratory as well as photosynthetic 0 2 4 6 8 growth (4) and is therefore already present in the cellular membrane. Time of Irradiation The amount of total RNA used in dot blots for the (min) environmental shift time course experiment described above FIG. 5. Comparison of cross-linking rates of aerobic (*) and was normalized to give equal loadings by hybridization with shifted anaerobic (+) cells. The irradiation of shifted cells was an R. capsulatus rRNA probe, pRC1 (42). Normalization of performed 45 min into the adaptation period to photosynthetic our data by hybridization with nick-translated genomic DNA growth. The BamHI K and F fragments are part of the photosyn- also gave essentially the same results (unpublished data). thetic gene cluster and are 2 and 4 kb in size, respectively. The bc, is a 9-kb restriction all three genes from This was expected, since rRNA accounts for the vast probe fragment containing the Jbc operon. majority of total cellular RNA. Chemoheterotrophic R. capsulatus cultures shifted to low oxygen tension have been claimed to increase their rRNA content sevenfold during the DISCUSSION first 140 min after the shift (16). We have not observed a measurable increase in either total RNA or rRNA content in We have described a novel method for detecting changes R. capsulatus cells during the time course of our environ- in the superhelicity of specific sequences in the bacterial mental shift experiments (unpublished data). In fact, if such chromosome. The ability to detect changes in superhelicity an increase in rRNA were actually occurring, one would in local regions of the chromosome allows direct appraisal of have to argue that thefbc mRNA must also be coordinately some long-standing questions about the relationship between induced with the rRNA genes in order to account for the DNA supercoiling, chromosome structure, and transcrip- apparently constitutive expression pattern observed (Fig. 4B tional activation of genes. The bacterial chromosome has and C). been shown, both in vivo and in vitro, to be composed of Chromosomal supercoiling during the induction of genes for topologically independent domains of torsionally strained photosynthetic metabolism. We used our assay to test DNA (30, 38). Complete relaxation of E. coli chromosomal whether stable changes in superhelicity accompany tran- supercoils by DNase I in vitro (38) or by 'Cofy-irradiation in scription of genes for photosynthesis in the switch from vivo (30) indicates that there are approximately 40 of these aerobic to anaerobic metabolism. R. capsulatus was grown domains. The number of domains correlates well with the aerobically in the dark to mid-log phase. A portion of the number of chromosomal cleavages induced by treatment of culture was harvested and irradiated with UV-A and TMP, E. coli with the gyrase inhibitor oxolinic acid (31). It is while the remainder was shifted to anaerobic conditions in intriguing to postulate that the superhelicity of some do- the light. Forty-five minutes into the shift, when the rate of mains might be maintained at a different level from that of mRNA accumulation for photosynthesis gene was maximal other domains (13, 30). Differential superhelicity among (Fig. 4B), the procedure was repeated on the induced cells. domains might be important in regulating transcription, since There was no detectable change in superhelicity for the the utilization of a variety of promoters by RNA polymerase DNA of the photosynthesis gene cluster (Fig. 5). The BamHI both in vivo and in vitro has been shown to be a function of F fragment shown in Fig. 5 contained puhA, which codes for the superhelical density of the DNA template (reviewed in the H subunit of the photochemical reaction center, while reference 6). BamHI-K was immediately downstream of puhA (Fig. 1). Previously, studies involving superhelicity have been per- We also probed BamHI-J, -G, and -D to confirm this result formed either on total chromosomal DNA (7, 29) or on for other regions of the photosynthesis gene cluster. Simi- plasmids (for example, see reference 19). The former work larly, there was no detectable change in superhelicity of a provided important information about whole-chromosome 9-kb restriction fragment containing the fbc operon encoding structure but did not address questions concerning specific the bc1 complex. We repeated this experiment three times regions of the chromosome. The latter studies used the and examined six different BamHI restriction fragments exquisite sensitivity of gel systems which can resolve indi- from the R. capsulatus chromosome. The deviation in cross- vidual topoisomers and provide exact information on the linking rates between aerobic and shifted anaerobic cultures linking number of plasmids. However, studies of bacterial averaged 5 3%. These results demonstrate that neither strains with mutations in one of the topoisomerase genes highly regulated genes such as pufLM and puhA nor consti- demonstrate that the same mutation can have different tutive genes such as the fbc operon undergo a change in effects on gene expression depending on whether the gene of superhelicity after a shift from aerobic to anaerobic photo- interest is carried on a plasmid or on the chromosome. For synthetic growth. instance, mutations in the topA gene of S. typhimurium can VOL. 171, 1989 CHROMOSOMAL SUPERCOILING AND ANAEROBIC GENE EXPRESSION 4841 suppress the leu-500 promoter mutation when the leu-SOO in the regulation of anaerobic gene expression. They mea- promoter is found on the chromosome but not when it is sured the novobiocin sensitivity of several transcriptional cloned into a plasmid (27). Similarly, topA missense muta- lacZ fusions to oxygen-regulated genes in the chromosome tions in S. typhimurium increase both plasmid superhelicity of S. typhimurium. While one aerobically expressed gene and expression from the proU locus, which is responsible for fusion (tonB-lacZ) is stimulated by gyrase inhibition, most of transport of glycine betaine in response to osmotic stress these oxygen-regulated fusions are unaffected by inhibition (13). In contrast, topA deletions, which also increase plas- of DNA gyrase. Furthermore, at least one anaerobically mid superhelicity, fail to induce expression of chromoso- expressed gene fusion is stimulated by gyrase inhibition (5). mally encoded proU (13). One can argue instead, based on evidence in the literature, Furthermore, observed changes in plasmid superhelicity that the bacterial cell has evolved a homeostatic mechanism are not always equivalent to simultaneous alterations in for maintaining relatively constant chromosomal superhelic- chromosomal supercoiling. Gyrase inhibition in E. coli re- ity (25). Expression of topoisomerase genes can respond to laxes the chromosome within 30 min (7), but the same changes in DNA superhelicity and thereby increase or treatment causes pBR322 to become positively supercoiled decrease the level of topoisomerase proteins. Transcription (19, 39). In a AtopA gyrB double mutant of E. coli, the of gyrA and gyrB is stimulated by relaxed DNA (21), chromosome is less negatively supercoiled than in the wild whereas transcription of topA is activated by increased type, but pBR322 from the same strain is significantly more negative superhelicity of the template (34). This regulation is underwound than plasmids isolated from the wild type, with exactly that which would be required in the homeostasis negative superhelicity exceeding that which can be produced model (21). In addition, the DNA-binding affinities ofthe two in vitro by purified gyrase (24, 26). These plasmid anomalies topoisomerases are dependent on the superhelicity of the have recently been explained as transcriptionally induced DNA substrate, with gyrase binding best to relaxed DNA effects caused by the inactivation of topoisomerase proteins molecules (10) while topoisomerase I prefers negatively (18, 33, 39). For some purposes, such as the study of supercoiled DNA (36). This biochemical evidence indicates topological questions involving DNA replication or chromo- that the topoisomerases might be titrated over the chromosomal domain structure, the TMP assay described here some so as to minimize local differences in DNA superhe- provides a useful alternative to studies on plasmids. licity. We can estimate the maximum possible change in chro- Our data for R. capsulatus argue that the requirement for mosomal supercoiling which might escape detection by our DNA gyrase in the transcription of photosynthesis genes (43) assay. Relaxation of the chromosome by novobiocin in R. is not related to a gyrase-induced change in chromosomal capsulatus is complete in about 20 min, as measured by the DNA superhelicity which activates transcription. The loss of TMP assay, and changes in superhelicity can be consistently mRNAs for photosynthesis after treatment with gyrase detected at much shorter times when the change in TMP inhibitors could be an indirect result of inhibitor treatment. reactivity is small (data not shown). We can reliably detect a Gyrase is clearly an essential enzyme for DNA metabolism, change in the rate of cross-linking which is 15% of the and loss of gyrase activity might have numerous and com- maximal change caused by novobiocin (Fig. 3). This limit is plex effects on the cell which do not directly reflect its greater than the 5% random variation observed between immediate role in bacterial physiology. On the other hand, aerobic and anaerobic cultures in Fig. 5. Sinden et al. (29) data are accumulating that gyrase may play a direct role in have shown that the rate of TMP photoreaction is linearly the transcription of strongly expressed genes and so may be proportional to the unrestrained superhelical density over a essential to the expression of the puf and puh operons (see range comparable to that found in vivo. Thus, a 15% below). threshhold in TMP cross-linking rates would represent a We emphasize that our experiments are designed to detect maximal undetectable change of unrestrained superhelicity stable changes in chromosomal superhelicity due to changes in vivo of 15% during a shift from aerobic to anaerobic in gyrase activity. In control experiments, we can easily growth. detect the relaxation of the chromosome after gyrase inhibi- Bliska and Cozzarelli (1) have recently demonstrated, tion (Fig. 2 and data not shown). These experiments, in using a recombinational assay, that 60% of the supercoils in which irradiations are carried out on ice, might not be vivo are probably restrained by interactions with proteins sensitive to transient changes in superhelicity induced by and other cellular components. They estimate that the unre- transcription since RNA polymerase is itself inactive under strained superhelical density in vivo is about -0.02. This these conditions. number agrees with earlier estimates that about half of the in We favor an alternative hypothesis to that advanced by vivo supercoils are restrained (23). Since intercalators are Yamamoto and Droffner (40) for the observed dependence sensitive to unrestrained supercoils but not to DNA wrapped on gyrase in the expression of genes for anaerobic metabo- by protein, a 15% threshold in detection of chromosomal lism in R. capsulatus (17, 43). Redirection of the cellular supercoiling translates to a maximum undetectable differ- metabolism to photosynthesis or nitrogen fixation involves a ence in unrestrained superhelical density of 0.003. substantial commitment by cells to the production of new Yamomoto and Droffner (40) suggest that the transition enzyme systems. This induction, in turn, requires a high from an aerobic to an anaerobic environment results in gross level of transcription of the genes for the structural proteins changes in chromosomal superhelicity, which then activate involved. Recent evidence indicates that on highly ex- expression of a large number of genes. This model has pressed regions of topologically anchored DNA, RNA poly- received wide attention in the scientific literature, since a merase divides the template into domains and introduces true physiological role for changes in chromosomal super- positive and negative superturns by rotating the DNA coiling as a mechanism to regulate gene expression has not through the transcription complex (2, 9, 11, 18, 33, 39). The yet been conclusively demonstrated (for a discussion of requirement for gyrase under these conditions could simply supercoiling as a response to environmental stress, see reflect the need to maintain the chromosome at its steady- reference 13). In a recent paper, Dorman et al. (5) presented state superhelicity by restoring negative supercoils removed genetic evidence against a global role for DNA supercoiling by the process of transcription. Thus, instead of activating 4842 COOK ET AL. J. BACTERIOL. transcription by altering DNA topology, gyrase could be and ribosomal RNA of Rhodopseudomonas capsulata. FEBS required, in certain circumstances, as a consequence of Lett. 177:61-65. transcription. We are currently performing experiments to 17. Kranz, R. G., and R. Haselkorn. 1986. Anaerobic regulation of test nitrogen-fixation genes in Rhodopseudomonas capsulata. Proc. this model. Natl. Acad. Sci. USA 83:6805-6809. 18. Liu, L. F., and J. C. Wang. 1987. Supercoiling of the DNA ACKNOWLEDGMENTS template during transcription. Proc. Natl. Acad. Sci. USA We thank G. Drews for providing pRC1 and N. Gabellini for 84:7024-7027. providing pRSF1. We also thank D. Burke-Aguero, D. O'Brien, J. 19. Lockshon, D., and D. R. Morris. 1983. Positively supercoiled Gingrich, M. Alberti, J. Kahn, D. Falvey, and P. Spielmann for plasmid DNA is produced by treatment of Escherichia coli with critically reading the manuscript and Melanie Beikman for valuable DNA gyrase inhibitors. Nucleic Acids Res. 11:2999-3017. technical assistance. 20. Marrs, B. 1981. Mobilization of the genes for photosynthesis This material is based upon work supported under a National from Rhodopseudomonas capsulata by a promiscuous plasmid. Institutes of Health training grant to D.N.C. and a National Science J. Bacteriol. 146:1003-1012. Foundation Graduate Fellowship to G.A.A. This work was also 21. Menzel, R., and M. Gellert. 1983. Regulation of the genes for E. supported in part by Public Health Service grant GM 30786 from the coli DNA gyrase: homeostatic control of DNA supercoiling. National Institutes of Health and by the Office of Basic Energy Cell 34:105-113. Sciences, Biological Energy Division, Department of Energy, under 22. Novak, P. D., and R. J. Maier. 1987. Inhibition of hydrogenase contract DE-ACO30-76F00098. synthesis by DNA gyrase inhibitors in Bradyrhizobium japonicum. J. Bacteriol. 169:2708-2712. 23. Pettijohn, D. E., and 0. Pfenninger. 1980. 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