lithium salts of the "2P-labeled ppGpp and pppGpp were then precipitated with 20 ... ['H]guanosine, or [ 'C]- succinate and measuring the rates of incorporation of.
*JolItNALI
OF BACTERIOLOG;Y, Fe). 1979, p). 956-962 ))1-9 19tj3' 79/62-06956/07.$()2.()()/(
Vol.I i, No. 2
Synthesis of Guanosine Tetra- and Pentaphosphates by the Obligately Anaerobic Bacterium Bacteroides thetaiotaomicron in Response to Molecular Oxygen THOMAS L. GLASS, \W. MICHAEL. HOLMES, PHILLIP) 1B. AINIIi,MN AN!) EDMUNDI) STELL WAG 1)Department of/Microhiology, Medicol (ollcge of Virginia. Virgin ia (CommotwOte'aOth I-Oitucrsitv, Ric/hmond, Virginia 2.3298 Received
ftor p)ublicatiol 2
Set pember
1978
Guanosine 5'-diphosphate :3-diphosphate (ppGpp) an(d gctanosine 5'-triphosphate 3-diphosphate (pppGpp) were detected in foirnic acid extracts of airexposed cultures of Bacteroa'(ls thetaiotaomicron. he irleintification of ppGpp and pppGpp in B. thetaiotaomicron was base(d On the following results: (i) cochrormatographv of P-labeledl hNperphosphor-vlatedl nucleotides in two different two-dimensional solvent systemis with authenitic pI)pGpp) anrlc pppGpp; (ii) incorporation of [ H]guanosine into the putative ppGp p andl ppjpGIpp (iii) alkalinie lability; and (iv) iesistance, to periodate oxidatioi. 1iheie was a imaikecl increase in the concentiation of ppGpp anrd pppGpp) after shift fiomn anaerobic to aerobic coinditions, andi accumulation of both ppG)pp ani(l pppGipp was blocked unrlcet these conditions by plrett'eatmient of the cultuie with rifampin oI tetiacycline. Growth and incorpotation of [ H]guanosine, [ H]thvmidine, [C Isutccinate, and
-[:;S]nmethiorline
L iInto nmac-romIolecules were inhibited immediately uI)pon exposure to air. The accumulation of ppGpp and pppGpp in thetaiotaonmicron upon exposure to air- may a novel signal for synthesis (f these
conipoundls.
replresent
Meiimbers of the genus Bacteroides ate strictly anaerobic, gram-negative, non-sp)oreforming bacilli which constitute a large percentage of the total cultivable fecal microflora of animals including humans (12). Several studies have reported that Bacteroides cl/gatus, Bacteroides thecta lotaonmicron, and Bac teroidles (1i'stasoniis are found in fecal sanmples in the range of 10' viable bacteria per g (wet weight) of humlani feces (7, 12). In view of the ecological importance of these bacteria to humans, there is a paucitv of )0ublished infor-mation regarding the imetabolic and genetic regulatory processes of these organisnls.
Escherichia co/'i has been shown to svnthesize guanosine 5 -diphosphate .3-diphosphate (ppGpp) and guanosine 5'-triphosphate :3-diphosphate (pppGpp) in response to a variety of enrivronmental perturbations r anging from armino acid starvation to nutritional shifts (4, 5) It is generallv believed that these c onmpounds p)lay intimate r oles in the regulation of a number of gene functions ranging from synthesis of stable RNA to regulation of a variety of bacterial operons (4, 5, 19). Although we know a good deal about the imietabolisnm of ppGpp and pppGpp in E. coli, relatively little is known regarding the general 950
occurrence
aniI l)lUtati-ve regulatoryirole in other
procarvotes. However, these comploundls have been reported in several other bacterial sp)ecies
including Salmotol cl/a tvph imwiumt, Ba cills and( Kho(lopsecldontionas spiterou(/c.s (4, 19). In this comml1-lunication we repo-t evidence for the productioi-I of p)pGpp and pppGpp in acn obligately anaerolbic bacterlium in response to nmolecular oxygen. s ubtili.s,
MATERIALS AND METHODS Organism and culture conditions. B. thfcl/otlaotiicroi V.PI.I. 5482 (NCI'C 10852) was obtainedil frotii the \tAacrohe lla)oratorv, Virgitiia Polvtethn4ic' IlnstitLIte anId Sla ,Ite t niversitv, B3lackshurg. I'roceduies for the cLtltivattiot ain(d miainiteniarmce of a'naerohic b)ac'teria (2, 1)W were oLsedC throughout this stoLdV. t 'less niotedi ill figUre legenc(ls, the (defiinecl, hasal iiie(dio of Varel andl Bryant (21 wcaS Llused for all experfimlct',s, with i.-m-ethioninie (75 mg/liter) replacing vitantini B, atid( an atimosphere of 1001()' argon. Growth (:17C( of the cultUres Was m11eaISUre(l tusinig either a Klett-Sttntmersoni colorimeter (re(d filter) or a Bausch ani(d Lomb Spectronic 20 (660 nn10) LIsing test tubes 1:3 by 196 tnt))i. Radiolabeling of ppGpp and pppGpp. To label cultures of B. thetaiota(omticron undem atlaerohiC ((0t)(litiorls, 2)))) /Wi of carrier-free [;'2 lorthophosphate (specific ra(lioactivity, 100 Ci/ mg) was adcledi to culttore tcheS (1:3 by0 166 m) anI(d (rliecd uic(ler a streattl
%7oi,. 13 7, 1979
ppGpp ANI) pppGpp IN BACTEROIDES
of argon gas. A 2-mi sample of a log-phase culture (100 to 150 Klett units; 1 Klett unit equals approximately 2.8 [tg of whole cell protein) was added under anaerobic conditions to each of these tubes, and incubation was continued for 10 min at 37°C. At this point, one of the tubes was exposed to air and agitated (air-shift), while the other tube was maintained under anaerobic conditions (anaerobic control). At selected time points, 50-1l portions of the culture were removed, added to 5 t1 of ice-cold 88%;- formic acid (final concentration of formic acid, 2 M) and frozen at -70°C. Acidified samples were thawed and centrifuged for 5 min in an Eppendorf centrifuge, and the supernatant fluid was used to spot polyethyleneimine (PEI)-cellulose thinlayer chromatograms. For certain experiments, ppGpp and pppGpp were labeled with ['Hlguanosine, as described for [32Plorthophosphate above. 'H-labeled ppGpp and pppGpp were produced by aerating the culture for 60 min. A culture sample (1.0 ml) was then centrifuged in an Eppendorf centrifuge for 5 min, the supernatant fluid was removed, and the cells were suspended in 100 gi of 2 M formic acid, frozen at -70°C, thawed, centrifuged, and chromatographed as above. Separation and quantitation of ppGpp and pppGpp. Separation of ppGpp and pppGpp was carried out by PEI-cellulose thin-layer chromatography. Baker-flex PEI-cellulose sheets (J. T. Baker Chemical Co., Phillipsburg, N.J.) were prewashed once with deionized water and dried at room temperature. For one-dimensional separation of "2P-labeled ppGpp and pppGpp, 10 or 15 1l of sample was spotted and chromatographed in 1.5 M potassium phosphate (pH 3.4) (6). "2P-labeled nucleotides were detected by radioautography using Kodak XR-5 X-ray film exposed for 24 to 72 h. The radioactivitv in "2P-containing nucleotides was deternmined by a previouslv described procedure (18). For two-dimensional separation and qualitative identification of ppGpp and pppGpp, two different solvent systems (2 and 3) were used (8). A 25-/il sample of 'P-labeled or ['H]guanosine-labeled material was spotted with 5 tig each of authentic unlabeled ppGpp and pppGpp. "2P-labeled chromatograms were visualized by radioautography. [3H]guanosine-labeled ppGpp and pppGpp were located by the UV absorbance of the cochromatographed authentic standards, ppGJpp and pppGpp. The radioactivity in the 3H-labeled hyperphosphorylated guanosine spots was determined by cutting out the appropriate spots, scraping off the PEI-cellulose, and eluting with 1.5 M LiCl. A 10-,ul portion of the eluate was counted as describe(d previously (18). Purification and chemical analysis of :12P-labeled ppGpp and pppGpp. A 4-ml sample of a logphase culture of B. thetaiotaomicron (absorbancy at 660t nm = 0.6) was transferred to a tube containing 400 1Ci of [j'Porthophosphate undler anaerobic conditions. T'he tube was allowed to incubate for another 3(1 min, then aerated for 60 min. The culture was centrifuged, and the supernatant fluid was discarded. T'he sedimented cells were suspended in 0.2 ml of 2 M formic acid and subjected to three freeze-thaw cycles at -70'C. The cell suspension was centrifuged, and the supernatant was applied to a PEI-cellulose sheet
along with 5 gg each of unlabeled authentic ppGpp and pppGpp, and then chromatographed in 1.5 M potassium phosphate (pH 3.4). The UV-absorbing areas corresponding to ppGpp and pppGpp were cut out, scraped, and eluted with 0.5 ml of 1.5 M LiCl. The lithium salts of the "2P-labeled ppGpp and pppGpp were then precipitated with 20 volumes of ice-cold 95% ethanol. The precipitate was centrifuged, suspended in deionized water, and stored at -70°C. Alkaline hydrolysis and periodate treatment of purified "2P-labeled ppGpp and pppGpp from B. thetaiotaomicron were performed according to the procedure of Cashel and Kalbacker (6). Effect of aeration on macromolecular synthesis. I'he effect of aeration on macromolecular synthesis was examined by labeling logarithmically growing cultures of B. thetaiotaomicron with either I"[35S]methionine, [;H]thymidine, ['H]guanosine, or [ 'C]succinate and measuring the rates of incorporation of isotope into cold trichloroacetic acid-insoluble material. For the experiments with ['Hlthymidine, ['H]guanosine, and [4Clsuccinate, 10 ml of a 35-ml logphase culture (absorbancy at 660 nm, 0.08 to 0.1) was added to each tube, mixed, and incubated for 20 min at 37°C. L-[35S]methionine was added directly to 10 ml of culture and incubated for 10 min. Each tube was then subdivided by adding 4.0 ml of labeled culture to a pair of previously sterilized culture tubes (13 by 100 mm) under anaerobic conditions (2). Culture samples (0.1 ml) were withdrawn periodically during growth of the culture until the absorbancy reached approximately 0.4. At this time, one of the tubes was vigorously aerated, and the other was left anaerobic. Direct chemical analysis of protein and total nucleic acid synthesis was also performed by the method of Lowry et al. (11) and the orcinol procedure (17), respectively. Chemicals. Carrier-free ["P]orthophosphate (100 Ci/mg), [8- 'Hlguanosine (7.1 Ci/mmol), [ sH-CH 3]thNrmidine (23 Ci/mmol), and L,-['5S]methionine (835 Ci/ mmol) were obtained from Amersham-Searle Corp. (Arlington Heights, Ill.). I,-[U,1-''C]glutamate (260 mCi/mmol), a-[ U-14C]ketoglutarate (277 mCi/mmol), and [2,3-'XClsuccinate (18.3 mCi/mmol) were purchased from New England Nuclear Corp. (Boston, Mass.). pppGpp heptalithium salt and ppGpp hexalithium salt were obtained from ICN Pharmaceuticals, Inc. (Cleveland, Ohio). All other chemicals were of the highest grade commercially available.
957
RESULTS Initially, experiments were carried out to characterize hyperphosphorylated nucleotides formed during response to molecular oxygen. Cultures of B. thetaiotaomicron growing exponentially in the presence of [3P]orthophosphate were exposed to air as outlined in Fig. 1. Samples of the cultures were then treated and chromatographed as indicated in Materials and Methods. Figure 2 illustrates the results of two-dimensional cochromatography of authentic ppGpp and pppGpp and formic acid extracts of cells exposed to air. It was shown that two of the 32p-
958
J. BACTERIOL.
GLASS ET AL.
beled nucleotides were hydrolyzed by base, and dephosphorylated derivatives comigrated with those produced by hydrolysis of authentic ppGpp and pppGpp present in the same reaction mixture, as judged by appearance of counts in appropriate products separated by one-dimensional PEI-cellulose thin-layer chromatography.
B
F
C
E
FIG.
tion
1.
~
Kinetics
B.
in
~
~
of ppGpp
and pppGpp
jotaomicron.
theta
A
culture
accumula-
was
labeled
anaerobically for 10 min with f32 P]orthophosphate and exposed to air as indicated. Samples were taken and chromatographed in one dimension with 1.5 M 3.4,). '3Plabeled spots potassium phosphate buffer were visualized by radioautography, cuit out, and
(pH
counted
the
as
described
in
the text. The control culture
maintained under anaerobic conditions
was
period.
time
same
Control
cutlture (01);
during
air-shifted
culture (0).
labeled spots indeed ppGpp
and
pppGpp.
comigrated An
with authentic
unknown,
2p -labeled
compound migrating between the hyperphosphorylated nucleotides ppGpp and pppGpp was also observed (Fig. 3); however, it has not yet been characterized. Additional experiments carried
were
compounds labeled sional
out
to
determine
with
[:3H]guanosine,
chromatography
scribed
in
whether
these
contained guanosine. Cultures
was
and
were
two-dimen-
carried out
as
de-
Materials and Methods; substantial
amounts of labeled
guanosine
were
found
comi-
grating with authentic ppGpp and pppGpp (550 cpm
of
ppGpp
and
approximately
120 cpm
of
pppGpp). Other experiments were also devised to establish the phosphate configuration of these compounds using criteria orginallv used by Cashel and Kalbacker to characterize ppGpp and A 2p-lapppGpp produced in E. coli (6). First, beled hyperphosphorylated guanosine nucleotides, which had previously been shown to co-
migrate with authentic ppGpp and pppGpp, isolated as outlined Materials and Meth-
were
ods
in
subjected to treatment with sodium and
alkaline
hydrolysis and metaperiodate. Both la-
FIG. 2. Taao-dimensional chromatography of 32plabeled ppGpp and pppGpp from B. thetaiotaomicron. (A) 232P-labeled culture sample at 40 min postair shift; solvent .system 2 (8). (B) T2P-labeled culture sample at 60 min post-air shift; soli-ent system .3 (8). Both samples were obtained from the same culture. Both ppGpp and pppGpp cochromatographed aith un labeled authentic ppGpp and pppGpp. The chromatograms were developed after 24 h of exposure. GTP. Guanosine 5'-triphosphate.
VOL. 137, 1979
ppGpp AND pppGpp IN BACTEROIDES
959
FIG. 3. Effect of rifampin and tetracycline on ppGpp and pppGpp accumulation in B. thetaiotaomicron. Three tubes, each containing 2 ml of log-phatse culture, u-ere labeled anaerobically for 10 min wlith [23P/orthophosphate. Two of the tubes were then treated w!ith rifampin (20 yig/ml,) or tetracycline (50 ,ug/ml,) and incubated for another 10 min. At this time all tubes w!ere air shifted, and samples w!ere taken for analy.sis by one-dimensional PEI-cellulose thin-layer chromatography. The chromatograms were expo.sed to) X-ray film for 72 h. C indicates the control culture with samples taken at 0 min (immediately prior to air shift) and 3'0 and 60 mmn post-air shift. Tet, Tetracycline-treated culture weith samples taken at 30 and 60 min po.st-air shift. Rif, Rifampin-treated culture with samples taken at 30 and 60 min post-air shift. UNK, Unknown 2~P-labelecd compound migrating in the region of the hyperphotsphorylated guanosine nucleotides.. In the case of ppGpp, two major spots were seen,
corresponding to undergraded ppGpp and a guanosine triphosphate (probably ppGp), and a third minor spot appeared which may be cyclic 2',3'-ppGp. Only two spots were seen in the case of pppGpp and were probably the undegraded pentaphosphate and pppGp. Free orthophosphate was seen to be released as a function of time. Both ppGpp and pppGpp were resistant to periodate treatment, as indicated again by onedimensional PEI-cellulose thin-layer chromatography. Experiments were then performed to assess the kinetics of accumulation of ppGpp and pppGpp in response to molecular oxygen, which (Fig. IA) inhibits the growth of B. thetaiotaomicron. By 30 min both ppGpp and pppGpp levels increased to a level at least 10-fold and 2fold above basal levels, respectively. After 60
min of aeration, there was an apparent decrease in ppGpp and pppGpp levels (Fig. 1B and C). There was no apparent increase (
