- Email: [email protected]
Isolation and mapping of ribosomal RNA genes of Caulobacter crescentus
./. Mol. Biol. (1981) 153, 291-303
Isolation and Mapping of Ribosomal RNA Genes of Caulobacter crescentus NORIKO OHTA AND AUSTIN NEWTON Princeton (Received
Department of Biology University, Princeton, N.J. 08544,
18 March
1981, and in revised form
U.S.A.
30 July
1981)
Ribosomal DNA fragments of 1.0,3.4,3.7 and 6.1 kbt produced by EcoRI digestion of the Caulobacter crescentus genome were identified by hybridization to a labeled ribosomal RXA probe. These genomic sequences were further characterized by the isolation of 13 hybrid h Charon 4 phages with rDNA inserts, and two of the recombinant phages, Ch4Cc773 and Ch4Ccl880, were examined extensively. The Cc773 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 3.7 kb and the Cc1880 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 6.1 kb that hybridized to 32Plabeled rRNA. Thus, the two clones contain different DNA insertswhich together account for all of the rDNA fragments detected in digests of the C. crescentus genome. Hybridization with isolated transfer RNA and individual rRNA species indicated that the arrangement of genes in both units is 16 S-spacer tRNA(s)-23 S5 S, tRNA(s). Homology between the DNA inserts is largely restricted to the rRNA coding regions, which suggests that the two rDNA units are located in different regions of the chromosome. Results of quantitative hybridization experiments are most consistent with a single Cc1880 and Cc773 unit per genome equivalent of 2.7 x 10’ daltons. The relatively simple organization of rDNA sequences in t,he C. crescentus chromosome compared to Escherichia coli is discussed.
1. Introduction The ribosomal RNA genesof vertebrates, amphibians, Drosophila and many lower eukaryotes, like Physarum, Tetrahymena and yeast, have been studied extensively (for a review, see Long & Dawid, 1980). Work on the ribosomal RNA genes of prokaryotes, however, has been largely limited to Escherichia coli (Kenerley et al.. 1977) and more recently Bacillus subtilis (Moran & Bott, 1979). Analysis of E. coli ribosomal DNA in hybrid h transducing phages and plasmids has shown that the ribosomal genes are organized into a unit of 16 S, 23 S and 5 S genes and that the haploid genome contains seven rDNA units (Kenerley et al., 1977). All of the ribosomal RNA units contain transfer RNA genesin the 16 S-23 S spacer and three of them also contain tRNA genes on the 23 S RNA distal side of the 5 S rDNA (Morgan et al., 1978). The B. subtilis chromosome also contains seven to ten copies of the rRNA genes (Smith et al., 1968), and a preliminary restriction analysis of t Abbreviation 0022-2836/N/340291-13
used: kb, 10” bases. $02.00/O
291 0
1981 Academic
Press Inc. (London)
Ltd.
292
N. OHTA
AND
A. NEWTON
genomic sequences suggests that the arrangement of the rRNA genes is similar to that in E. coli (Moran & Bott, 1979). We have examined the organization of the ribosomal RNA genes in the prokaryotic organism, Caulobacter crescentus strain CB15, as part of a more general study of gene expression and development (seeNewton, 1972; Sheffery & Newton, 1981). These prosthecate (stalked) bacteria are gram negative with a genome of about the samesize as that reported for E. coli (Wood et al., 1976), but the life cycle of Caulobacter differs significantly from that of other bacteria. C. crescentusdivides asymmetrically to produce a swarmer cell and a stalked cell with different cell cycles; the swarmer ceil cycle is described by Gl (presynthetic gap), S (DNA replication) and G2 (postsynthetic gap) periods, while the stalked cell cycle contains only an S and G2 periods (Degnen & Newton, 1972). Since the Gl period of the swarmer cell is retained even in rapidly growing cultures (Iba et al. 1977), the two progeny cells always initiate chromosome replication at different times after division. Consistent with this pattern of DNA synthesis, the organization of cell cycle steps in C. crescentus apparently precludes dichotomous chromosome replication (Osley & Newton, 1980; Nathan, Osley & Newton, unpublished results) and, perhaps as a consequence, the maximum growth rate in rich medium is significantly slower than that observed in E. wli. The level of rRNA synthesis in bacteria is closely co-ordinated with cell growth (Schaechter et al., 1958), and these observations suggested the possibility of an unusual arrangement or number of ribosomal RNA genes in the genome of these cells. The results presented below reveal a relatively simple restriction pattern for the rDNA sequencesin C. crescentus genome compared to the patterns obtained from other bacterial genomes (Moran & Bott, 1979; and this paper). Analysis of hybrid Charon 4 phages that carry CB15 rDNA inserts suggeststhat all of the 16 S, 23 S and 5 S genes are contained in two rDNA units designated Cc773 and Cc1880, and that the units are at different locations on the C. crescentus chromosome. The organization of the rRNA genes and the tRNA genes in the Cc773 and Cc1880 inserts
has also been determined.
2. Materials
and Methods
(a) Bacterial and phagestrains C. crescentus strain CB15 (ATCC 19089) was normally grown in peptone-yeast extract (PYE) medium (Poindexter, 1964) and E. wli K12 strains C600 (thr-leu-) and K802 (hr-h.sm+ gaZK SuII la-met; obtained from P. Schedl)were grown in L broth (Lennox, 1955). Strain K802 was the host for h Charon 4 phage and its derivatives. The growth and use of h Charon 4 phage ax a vector for cloning have been described by Blattner et al. (1977). (b) Preparation of DNA and RNA DNA was prepared from C. crescentus cells according to Nakamura et al. (1979) with slight modifications. Several steps in the purification, including incubation with RNase, phenol extraction, chloroform/iso-amylalcohol extraction and ethanol precipitation, were normally repeated twice. DNA from E. wli strain C600 was purified by the same procedure. Total rRNA was prepared by phenol extraction of C. crescentus or E. coli cell extracts (Ohta et al., 1975) and then sedimented on 5% to 30% sucrose density gradients by
rDNA
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centrifugation at 39,000 revs/min for 14 h in a Beckman SW41 rotor. RNA peaks corresponding to the 16 S and 23 S RNA were pooled and precipitated with ethanol at -20°C. When separated 16 S or 23 S RNA probes were required, RNA was purified from isolated 30 S or 50 S ribosomal subunits. (c) Enzymes and incubation conditio?zs T4 polynucleotide kinase was obtained from P. L. Biochemicals. Endonucleases BornHI, EcoRI and Hind111 were purchased from Miles Biochemicals. BgEII, KpnI, SmaI and XhoI were purchased from Bethesda Research Laboratories. T4 ligase and DNA polymerase I were a gift of P. Schedl. Incubation conditions for restriction endonucleases were those recommended by vendors. (d) Preparation of [y-“*P]ATP and in vitro ‘2P-labeling of RNA [y3’P]ATP (> 9000 Ci/mmol) was prepared from carrier-free [‘*P]orthophosphate (New England Nuclear) and ADP (Sigma) by the method of Johnson & Walseth (1979). rRNA (16 S, 23 S or the mixture) was labeled with [Y-~*P]ATP at the 5’ end according to Pirtle et al. (1978). (e) Preparation of in vitro J”P-labeled tRNA and 5 S RNA A culture of C. crescentus was grown overnight to a density of approximately 6 x 10s cells/ml in 01 medium (Schmidt & Stanier, 1966) that contained 0.5 mM- hosphate and then diluted B-fold in Gl medium with 0.1 mM-phosphate. Carrier-free H, P‘PO, (5 mCi; New England Nuclear) was added to the 5 ml culture and incorporation was allowed to continue for 5 to 7 h. RNA was purified and then fractionated by 10% polyacrylamide gel electrophoresis according to Ikemura & Nomura (1977). Sections of the gel that contained the tRNAs and the 5 S RNA were visualized by autoradiography, cut out and then finely minced using razor blades. RNA was eluted from gel pieces by suspending them in 10 mMTris/lO mM-EDTA/03 ru-NaCl, pH 8.0 overnight at room temperature. The eluates were phenol extracted several times and RNA was precipitated by 2.5 vol. ethanol. electrophuresis, Southern transfer and hybridization DNA restriction fragments were fractionated by horizontal agarose gel electrophoresis (McDonell et al., 1977) using E buffer (Childs et al., 1977). Procedures described by Moran et al. (1979) were followed for the transfer of the DNA fragments from gels to nitrocellulose filters (Schleicher and Schuell BA85) and for hybridization to 32P-labeled RNA except that hybridization was carried out at 37°C for 48 h. (f) Agarose
gel
3. Results (a) Restriction
mapping
of genomic
rDNA
Genomic DNA was purified from C. crescentus and E. coli cells, digested to completion with EcoRI and Hind111 and the restriction fragments were separated by electrophoresis on 0.7% agarose gels. The DNA fragments containing ribosomal genes were identified by filter hybridizations (Southern, 1975) using as a probe either C. crescentus (Fig. l(a), lanes 1 to 4) or E. coli (Fig. l(a), lanes 5 to 8) rRNA that had been labeled in vitro with [y-32P]ATP (Pirtle et al., 1978). CB15 DNA gave relatively simple restriction patterns compared to E. coli DNA. Fragments of 6.1 kbt. 3.7 kb, 3.4 kb and 1.0 kb were produced by EcoRI digestion and fragments t See footnote
to p. 291.
N. OHTA 1234
5678
AND
A. NEWTON I
2
3
4
5
6
6.1 -
I.0
16s
23s
(b) FIG. 1. (a) Restriction analysis of rDNA regions of C. crescentus and E. c&i. Genomic DNA from C. crescentus (lanes 1, 2, 5 and 6) and E. coli (lanes 3, 4, 7 and 8) was digested to completion with either EcoRI (lanes 1, 3, 5 and 7) or with Hind111 (lanes 2, 4,6 and 8) and the DNA fragments separated on 67% agarose gel. The fragments were transferred to nitrocellulose filters and hybridized to labeled, total rRNA isolated from C. crescentus (lanes 1 to 4) or from E. coli (lanes 5 to 8) as described in Materials and Methods. (b) Mapping of 16 S and 23 S genes in Ch4Cc773 and Ch4Cc1880 DNA. Fragments produced by EcoRI digestion of Ch4Cc773 DNA (lanes 1 and 3) and Ch4Cc1880 DNA (lanes 2 and 4) were se rated on 1.0% agarose and examined for hybridization to 32P-labeled 16 S RNA (lanes 1 and 2) and p”‘P-labeled 23 S RNA (lanes 3 and 4). Ethidium bromide-stained agarose gels of EcoRI digests of Ch4Cc773 (lane 5) and Ch4Cc1880 (lane 6) are shown for comparison. The sizes of the restriction fragments were estimated using h DNA and pBR322 DNA digested with several restriction enzymes, including EcoRI and HindIII, as standards (Sutcliffe, 1978). The molecular weights of the fragments in this and other Figures are given in the margin in kb.
of 195 kb, 9.8 kb, 2.6 kb and 24 kb were produced by Hind111 digestion of CB15 DNA. EcoRI digestion of E. coli DNA produced more than 11 fragments that hybridized. There are seven different rRNA regions in the E. coli (Kenerley et al., 1977) and these results suggested that there are significantly fewer rDNA units in the C. crescentus genome. Identical hybridization patterns could be observed after longer exposures of
rDNA
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CAULOBACTER
IN
autoradiograms whether the rRNA probe was from C. crescentusor from E. coli cells (data not shown). The intensity of the bands was always stronger with the homologous probe, however, as indicated by the failure to detect some of the expected bands at shorter exposures when the heterologous probes were used (cf. Fig. l(a)). Thus, there is significant but not complete homology between the rDNA sequencesof the two bacteria.
(b) Construction of C. crescentus gene clone bank in /\ Charon 4 The ribosomal regions of the C. crescentuschromosome were characterized in more detail by cloning the rDNA units in h Charon 4. To obtain a random distribution of EcoRI fragments in the desired size range for cloning, purified CB15 DNA was digested to different extents using serially diluted enzyme; the digests were pooled and then fractionated by sucrose gradient centrifugation. DNA fragments of 10 to 20 kb were collected, ligated to Charon 4 DNA end pieces as described by Maniatis et al. (1978) and the hybrid DNAs were packaged in vitro (Hohn & Murray, 1977). Individual plaques were picked, amplified on lawns of E. coli strain K!302, transferred with toothpicks to wells of microtiter dishes and stored at - 18°C in 50% glycerol. The average size of inserts in these clones is 10 kb, and if the C. crescentusgenome size is taken to be equivalent to that of E. coli. or 2.7 x lo9 dalton (Britten & Kohne, 1968), then any gene should be present in a bank of 2300 with a 99% probability (Clarke & Carbon, 1976). (c) Identi$cution of rDNA clones Although rRNA from C. crescentuscross hybridizes to E. coli DNA (Fig. l), the E. coli host DNA did not give a positive responseusing 32P-labeled rRNA in in situ plaque hybridization (Benton & Davis, 1977). Thus, the h Charon 4 library was
TABLE
1
EcoRI inserts in rDNA clonesof Charon 4 Class
Charon
4 clones
Inserted
12
II
b
Cc3651,
c
cc3966
a
Cc1880,
Cc4636,
Cc5507 +
Cc3666,
fragments
1.0
(kb)
3.7
3.4 +
+
+
+
+
+
+
+
+
3.4
1.0
6.1
+
+
+
+
+
+
cc773
IEI
EcoRI
42
Cc4084
7.8
Cc4095. 0~4204, Cc5274 b
Cc4681
+ Indicates
the presence
of the EcoRI
fragment
in the CB15
DNA
insert.
+
296
N. OHTA
AND
A. NEWTON
screened directly with in vitro labeled, C. creacentus rRNA. A total of 13 positive clones of independent origin were identified among approximately 4500 clones tested, and 12 of these were examined by EcoRI restriction analysis (Table 1). All of the clones contained either a Cc773 type DNA insert (class I) or a Cc1880 type DNA insert (class II). The hybrid clones containing these two inserts were characterized in detail. (d) Restriction
mapping
of CB15 rDNA
inserts of Ch4Cc773 and Ch4Cc1880
As shown in Table 1, the Cc773 insert of 81 kb produces EcoRI fragments of 1.0 kb, 3.4 kb and 3.7 kb, while the Cc1880 insert of 168 kb also produces the EcoRI fragments of 1.0 kb and 3.4 kb, plus a 6.1 kb fragment instead of the 37 kb fragment. Consequently, all of the genomic rDNA fragments identified in Figure 1 are contained in Ch4Cc773 and Ch4Cc1880. To determine the arrangement of ribosomal RNA genes in the cloned sequences, 16 S RNA and 23 S RNA probes were prepared by isolating RNA from separated 30 S and 50 S ribosomal subunits, labeled with 32P in vitro and then hybridized to EcoRI digests of DNA from the hybrid phages (Fig. l(b)). The 16 S RNA hybridized only to the 3.4 kb fragment produced by the Cc773 and Ccl880 inserts, while 23 S RNA hybridized to the 3.4 kb, 1.0 kb and 3.7 kb fragments of the Cc773 insert and the 3.4 kb, 1.0 kb and 6.1 kb fragments of the Cc1880 insert. We also observed this same pattern of hybridization when these two probes were hybridized to EcoRI fragments of genomic CB15 DNA (data not shown). Since the 3.4 kb and 1.0 kb fragments are common to both rDNA units, these results suggest that the 3.7 kb and 6.1 kb fragments contain the 23 S RNA flanking sequences and that the arrangement of EcoRI fragments is 3.4 kb, 1.0 kb, 3.7 kb in Ch4Cc773 (Fig. 2(a) and (b)) and 3.4 kb, 1.0 kb, 6.1 kb in Ch4Cc1880 (Fig. 2(b) and (c)). This map order was confirmed by rRNA hybridization to DNA fragments produced by digestion with other restriction enzymes. These experiments also indicate thet one end of the 16 S gene lies very close to the left of the 3.4 kb fragment. The 14.6 kb fragment produced by BumHI digestion of Ch4Cc773 DNA extends into the left arm of Charon 4 and covers less than 63 kb of the 3.4 kb EcoRI fragment, but it hybridized with the 16 S RNA probe (data not shown). The orientation of the CB15 DNA inserts in the hybrid clones was determined from the results of digestions with BamHI, BglII, HindIII and double digestions with EcoRI and each of these enzymes (Fig. 2(b) and (c) and unpublished data). The results show that two inserted segments have opposite orientations with the 16 S rDNA region joined to the long arm of Charon 4 in Ch4Cc773 and to the short arm of Charon 4 in Ch4Cc1880. The only difference in the restriction maps of the rDNA coding regions of the two clones was the position of the BgZII sites that are adjacent to the spacer tRNA; the maps of the flanking sequences adjacent to the 23 S gene differ substantially, however (Fig. 2 and below). (e) Mapping of tRNA and 5 S RNA genes The presence of tRNA and 5 S RNA genes in the Cc773 and Cc1880 inserts was examined by hybridization using in viva 32P-labeled 5 S RNA or tRNA as a probe
I-DNA
RI ,/’ ( b 1 Ch4Cc
773
I
ORGANIZATION
IN
Xh Sm Sm Xh HI11 RI I
I I
RI
CAULBACTOR
\
HIII Sm XhSm II
I
Bo
297
I
RI y
R
i3g
L.,i
w-2.07
I
16s
I I
(cl
Ch4CclBBO
RI l I I ~,KP &y4+
ItI
I I
123s
!!
1
I/
1
\ \
r------4 HI11 L-----g+---4
Xh Sm Xh I
I Bo
d1 )
I Kp
L
/ /’
&-z~
/' rC3.4+
‘,I+1 (d)
I
I
eI.35J \ \
t,5s I I
p+2+je----4.9RI HIII Xh
Xh SmlSm XhHIIIRI ” I’ ‘1 ’ ’ 1 I Bg ~0 \
I
Xho ’ KPn
IO Ic-dr FiI .I-f HI11
/'
6-l
,’
4+7.0+?
iI/’ 9’ HIII
Kpn
HI11
L---64‘1
FIG. 2. Restriction
maps of X Ch4Cc773 and h Ch4CclSSO inserts and genomic arrangement of rDNA units. (b) and (c) Restriction maps of h Ch4Cc773 and A Ch4Cc18SO DNA inserts. (a) and (d) Arrangement of Cc773 and Cc1880 rDNA units in the C. creaeentwr chromosome. Only those restriction sites which are relevant in placing various fragments are given in (a) and (d). Regions of the map shown in a broken line were deduced from genomic restriction patterns and those in an unbroken line were obtained by restriction patterns of cloned inserts (Table 1). Heavy, unbroken lines in (a) and (d) indicate the maximum extent of rDNA coding region. Distances are given in kb. Abbreviations are: Ba, BumHI: Bg, &$II; RI, EcoRI, HIII, HindIII; Kp.Kpn, ZfpnI; Sm, SmaI; and Xh,Xho, XM.
(Materials and Methods). A lOO-fold excess of unlabeled 16 S and 23 S RNA was included to prevent background hybridization from contaminating 16 S and 23 S RNA breakdown products in the radioactive probes. Hybridization of 5 S RNA to the 3.7 kb EcoRI fragment of the Cc773 insert and to the 6.1 kb EwRI fragment of the Cc1880 insert (Fig. 3) places the 5 S gene at the end of the rDNA unit adjacent, to the 23 S gene. The 5 S DNA sequence was further localized within the 6.1 kb fragment by showing that the 5 S RNA probe hybridized only to 1.3 kb EcoRI-BamHI fragment, the 2.2 kb EwRI-BglII fragment (Fig. 3) and the 1.2 kb EwRI-Hind111 fragment of the Cc1880 insert (data not shown). Hybridization with double digest fragments from Ch4Cc773 DNA placed the boundary of the 5 S genes in this rDNA insert in a similar position (data not shown, see Fig. 2). When total, labeled tRNA from C. crescentuscells was used to probe restriction fragments from the hybrid clones, the 3.4 kb fragments of both clones hybridized, as well as the 3.7 kb fragment of Ch4Cc773 and the 6.1 kb fragment of Ch4Cc1880 (Fig. 3). Double enzyme digests of Ch4Cc1880 DNA allowed more accurate mapping of the tRNA genes. The tRNA hybridized to the same 1.3 kb EwRIBamHI fragment and EwRI-BglII fragment, as did the 5 S RNA probe, which
298
N. OHTA I
3
2
AND 4
A. NEWTON 6
5
7
8
6.1 -
-
-
tRNA
55
2.2
I.3
RNA
FIG. 3. Mapping of 5 S and tRNA
genes in rDNA units. Ch4Cc773 (lanes 1 and 5) and Ch4Cc1880 (lanes 2 and 6) DNA were completely digested with EcoRI. Double digests of Ch4Cc1880 DNA were also made with EcoRI and BumHI (lanes 3 and 7) and with EcoRI and @#II (lanes 4 and 8). DNA fragments were separated and analyzed by hybridization to labeled tRNA (lanes 1 to 4) and 5 S RNA (lanes 5 to 8) as described in Fig. 1 and text.
places both of the genes adjacent to the end of the 23 S RNA. In addition, the tRNA hybridized to a 2.1 kb BarnHI fragment and a 1.35 kb EcoRI-BgZII fragment of Cc1880. Since the only overlap between the two fragments is less than O-35 kb long (see Fig. 2(b) and (c)), this places the “spacer” tRNA gene(s) within this region. Hybridization of the tRNA probe to double digests of the Ch4Cc773 gene gave comparable restriction patterns (data not shown). The approximate locations of the 5 S and tRNA genes in the Cc773 and Cc1880 rDNA units are shown above in Figure 2. Genomic DNA from C. crescentus was also probed with the labeled 5 S and tRNA species. When 5 S RNA was the probe, the only EcoRI fragments to hybridize were 3.7 kb and 6-l kb (data not shown), suggesting that the 5 S genes in these cells are
I-DNA
ORGANIZATION
IN
CAULOBACTh’R
299
located exclusively in the Cc773 and Cc1880 rDNA units. Use of the tRNA probe. however, revealed higher molecular weight fragments that hybridized in addition to the 3.4 kb, 3.7 kb fragments. Thus, a number of tRNA genes in C. crescentm ohromosome are located outside of the rDNA regions. (f) Gen,omicorganization of rDNA units The restriction patterns produced by the DNA sequencesflanking the 23 S RNA gene of Ch4Cc773 and Ch4Cc1880 suggest that the two rDNA inserts arise from different regions of the CB15 genome (Fig. 2). This conclusion was confirmed by using the nick translated, 3.7 kb EcoRI fragment from Ch4Cc733 to probe EcoRIHind111 digests of Ch4Cc1880. Of the two fragments produced by the 6.1 kb, EcoRI fragment of the Cc1880 insert (see Fig. 2(c)), the 1.2 kb fragment hybridized while the 4.9 kb fragment did not (data not shown). Thus, homology between these two cloned sequencesappears to be limited to the rDNA units themselves. We have also compared genomic restriction patterns with those from rDNA clones like Ch4Cc3966 and Ch4Cc4681 (Table 1) in order to examine the flanking sequenceson both the 16 S and 23 S sidesof the Cc773 and the Cc1880 rDNA units. Hind111 digestion of genomic DNA produces a 195 kb and a 9.8 kb fragment that’ hybridize to 16 S rRNA (Fig. 1 and data not shown). Since the Cc3966 DNA insert does not contain a Hind111 site in the 12 kb and 4.2 kb fragments that flank the I6 S RNA gene, the 195 kb genomic Hind111 fragment can be tentatively assigned t,o the Cc773 region and the 9.8 kb fragment to the Cc1880 region. An additional 23 S rDNA flanking sequence of 7.8 kb in the Cc1880 region is present in the (‘h4Cc4681 insert (Table l), and another flanking sequence on the same side of the (‘~773 unit has been identified by KpnI digestion of genomic DNA (data now shown). The genomic maps constructed from these results (Fig. 2(a) and (d)) confirm that the two rDNA units are from different regions of the C. crescentus chromosome. They also show that the rDNA units are not present’ as closely spaced, tandem repeat units, since the Cc773 unit is present only once in the 28.2 kb sequenceexamined in these st,udiesand the Cc1880 unit is present only once in t’he 22.6 kb sequence.
(g) Estimation
of rDNA copy number
We also examined the possibility that the rDNA units in C. crescentuscells are present in the genome as widely spaced, redundant copies. The number of rDNA units was estimated by quantitative hybridization of labeled rRNA to complete EcoRI digests of Ch4Cc773 DPL’A, Ch4Cc1880 DNA and genomic DNA4 that had been separated on agarose gels (Fig. 4). Assuming one genome equivalent of CB15 DNA as 2.7 x 10’ daltons (Wood et al., 1976) the results are most consistent with the presence of two copies per genome of the 3.4 kb EcoRI fragment, which is generated by the Cc773 and the Cc1880 rDNA units, and one copy per genome of the 3.7 kb fragment and 6.1 kb fragment, which are generated by Cc773 and the Cc1880 units. respectively. Quantitative densitometry measurements that were made on the results from six independent, experiments gave average values that were somewhat
300
N. OHTA
I
2
AND
3
A. NEWTON
4
5
6
7
6
FIG. 4. Estimation of rDNA copy number in C. crescentzls genome. Ch4Cc773 DNA, Ch4CclS80 and CM5 DNA were digested to completion with BcoRI and applied to agarose gel in 1, 2 or 3 genomic equivalents (Ch4Cc773 and Ch4Cclt3SO) or 06 or 1 genomic equivalent (CB15 DNA). DNA fragments were transferred to nitrocellulose filters, hybridized to labeled 23 S RNA ae in Fig. 1 and analyzed IM described in the text.
CA UL OBACTER
301
higher, however, with 2.1 copies for the 3.4 kb fragments, fragment and 1.2 for the 6.1 kb fragment.
1.3 for the 3.7 kb
rDNA
ORGANIZATION
IN
4. Discussion Restriction analysis of genomic DNA from C. crescentus and the characterization of Charon 4 phage clones that contain CB15 rDNA inserts are consistent in showing that the chromosome of these cells contains only two different rDNA units. All of the 12 rDNA clones examined contain inserts of either the Cc773 or the Cc1880 type (see Table l), and together these two inserts account for all rDNA fragments detected in the genomic digests (Fig. 1). Thus, the organization of ribosomal sequences in C. crescentus is relatively simple compared to E. co& which contains seven different rDNA units (Kenerley et d., 1977). The use of individual RNA species as probes indicates that the 16 S, 23 S and 5 S genes of the C. crescentus genome are located exclusively in the Cc773 and the Cc1880 rDNA units, while tRNA genes map both within the rDNA units and at other sites on the chromosome. The arrangement of genes in both the Cc773 and Cc1880 inserts is 16 S-tRNA(s)-23 S-5 S,tRNA(s); the experiments do not indicate the relative order of the 5 S and flanking tRNA(s) gene or the number of tRNA genes present in the spacer and 23 S flanking regions, however. These maps are similar to those reported for one group of the rDNA units in E. coli that contains tRNA genes in the 23 S flanking sequence, as well as in the 16 S-23 S spacer, e.g. the map of the E. coli rrnC operon is 16 S-tRNAZG”-23 S-5 S,tRNAT’p,tRNAAap (Morgan et al., 1978). While the Cc773 and the Cc1880 regions share this general gene arrangement and considerable homology with the rDNA coding regions of E. co& (Fig. l), the rDNA restriction maps of the two organisms show little resemblance (cf. Fig. 2 and deBoer et al., 1979). We have attempted to identify the tRNA species present in the Ch4Cc773 and Ch4Cc1880 phages in in wivo transcription experiments (Lund et al., 1976), but the CB15 DNA inserts were not expressed. The EcoRI site that defines the junction of the Charon 4 left arm and the Cc773 insert and Charon 4 right arm and Cc1880 insert lies very close to the presumptive 5’ end of the 16 S RNA gene, and these rDNA inserts may lack the promoter regions. Transcription of the rRNA genes may be possible in a clone like Ch4Cc5507 (Table l), which contains an additional flanking fragment adjacent to the end of the 16 S gene. The maximum size of the C. crescentus rDNA unit can be calculated as 5.6 kb from the position of the EcoRI site flanking the 16 S RNA gene and the Hind111 site in the 6.1 kb fragment of the Cc1880 insert; as shown in Figure 2, only the 1.2 kb fragment produced by EcoRI-Hind111 double digest of the 6.1 kb fragment hybridized to the rRNA probe. The results obtained to date indicate that the rDNA coding sequences in the Cc773 and Cc1880 units are highly conservative, as observed in other systems. With the exception of a BglII site in the 3.4 kb fragment that is located closer to the tRNA spacer in Ch4Cc773 (Fig. 2(b) and (c)), the restriction maps of the two rDNA units are identical. This sequence homology does not extend outside of the coding regions, however. There is a similar clustering of BamHI, BglII and Hind111 sites in the 23 S RNA flanking sequences, but the
302
N. OHTA
AND A. NEWTON
positions of these sites are not identical, and there is no indication of homology between these sequences.The genomic map in Figure 2(a) and (d) shows that the Cc773 and Cc1880 are in fact located in different regions of C. crescentus chromosome. The quantitative reconstruction experiments are subject to large systematic errors, but these results (Fig. 4) and the genomic maps constructed above (Fig. 2(a) and (d)) are consistent in suggesting that a C. crescentus genome contains no more than a single copy of the two rDNA units. Although this conclusion should be confirmed by other experiments, the organization of the ribosomal sequencesin these cells is clearly simpler than that in many other bacteria. Since the level of rRNA synthesis is tightly coupled to cell growth (Schaechter et al., 1958), the small number of rRNA genes in C. crescentus may only reflect the relatively slow growth rate of these bacteria. C. crescentus cultures reach a maximum doubling time of approximately 90 minutes compared to approximately 20 minutes for E. coli and R. subtilis, which have seven to ten rDNA copies per genome (Kenerley et al., 1977 ; Smith et al., 1968). These results suggest that bacteria like C. crescentus, which do not replicate their chromosomes dichotomously (Nathan, Osley & Newton, unpublished
results)
and grow
slowly,
have been under
relatively
less evolutionary
pressure to duplicate genomic rDNA units. We thank Paul Schedl for his advice during the course of this work and JoAnn Mermelstein and Ling-Sing Chen for technical assistance. The research was supported by Public Health Service grants GM25644 and GM22299 from the National Institutes of Health and by the Whitehall Foundation. Note added in proof: Feimgold and Shapiro have independently cloned rRNA genes of C. crescentus strain CBIB (personal communication).
REFERENCES Benton, W. D. & Davis, R. W. (1977). Science, 199, 180-182. Blattner, F. R., Williams, B. G., Blechl, A. E., Denniston-Thompson, K., Faber, H. E., Furlong, L., Grunwald, D. J., Kiffer, D. O., Moore, D. D., Schumm, J. W., Sheldon, E. L. & Smithies, 0. (1977). Science, 196, 161-169. Britten, R. J. $ Kohne, D. E. (1968). Science, 161, 529-540. Childs, G. J., Ohtsubo, H., Ohtsubo, E., Sonnenberg, F. & Freundlich, M. (1977). J. Mol. Biol. 117, 175-193. Clarke, L. & Carbon, J. (1976). Cell, 9, 91-99. deBoer, H. A., Gilbert, S. F. & Nomura, M. (1979). Cell, 17, 201-209. Degnen, S. T. & Newton, A. (1972). J. Mol. BioE. 64, 671-680. Hohn, B. & Murray, K. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 3259-3263. Iba, H., Fukuda, A. & Okada, Y. (1977). J. Bacterial. 129, 1192-1197. Ikemura, T. & Nomura, M. (1977). Cell, 11, 779-793. Johnson, R. A. & Walseth, J. F. (1979). Advances in Cyclic Nucleotide Research, 10, 135-167. Kenerley, M. E., Morgan, E. A., Post, L., Lindahl, L. & Nomura, M. (1977). J. Bacterial. 132, 931-949. Lennox, E. S. (1955). Virology, 1, 1%206. Long, E. 0. & Dawid, I. B. (1980). Annu. Rev. B&hem. 49, 727-764. Lund, E., Dahlberg, J. E., Lindahl, L. S., Jaskunas, S. R., Dennis;P. P. & Nomura, M. (1976). Cell, 7, 165-177.
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Maniatis, T., Hard&on, R. C., Lacy, E., Lauer, T., O’Connell, C., Quon, D., Sim, G. K. & Efstratiadis, A. (1978). Cell, 15, 687-701. McDonell, M. W., Simon, M. N. & Studier, F. W. (1977). J. Mol. Biol. 110, 119-146. Moran, C. P. & Bott, K. F. (1979). J. Bacterial. 140, 9S105. Moran, L., Mirault, M., Tissieres, A., Lis, J., Schedl, P., Artavanis-Tsakonas, S. & Gehring. W. J. (1979). Cell, 17, 1-8. Morgan, E. A., Ikemura, T., Lindahl, L., Fallon, A. M. & Nomura, M. (1978). Cell, 13, 335 344. Nakamura, K., Pirtle, R. M. & Inouye, M. (1979). J. Bacterial. 13’7, 595-604. Newton, A. (1972). Proc. Nat. Acud. Sd., U.S.A. 69, 447-451. Ohta, N., Sanders, M. & Newton, A. (1975). Proc. Nat. Acud. Sci., U.S.A. 72, 2343-2346. Osley, M. A. & Newton, A. (1980). J. Mol. BioZ. 138, 109-128. Pirtle, R. M., Pirtle, I. L. & Inouye, M. (1978). Proc. Nat. Acud. Sci., U.S.A. 75, 21992194. Poindexter, J. S. (1964). Bacterial. Rev. 28, 231-295. Schaechter, M., Maalee, 0. & Kjeldgaard, N. 0. (1958). J. Gen. Microbial. 19, 592-606. Schmidt, *J. & Stanier, R. Y. (1966). J. Cell BioZ. 28, 423436. Sheffery, M. & Newton, A. (1981). CeZZ,24, 49-57. Smith, I., Dubnau, D., Morrell, P. & Marmur, J. (1968). J. Mol. BioZ. 33, 123-140. Southern, E. M. (1975). J. Mol. BioZ. 98, 503-517. Sutcliffe, ,J. G. (1978). ,VucZ. Acids Res.5, 2721-2728. Wood, N. B.. Rake, A. 1’. & Shapiro, L. (1976). J. Bacterial. 126, 13051315.
Edited by M. Gotteswmn
Isolation and Mapping of Ribosomal RNA Genes of Caulobacter crescentus NORIKO OHTA AND AUSTIN NEWTON Princeton (Received
Department of Biology University, Princeton, N.J. 08544,
18 March
1981, and in revised form
U.S.A.
30 July
1981)
Ribosomal DNA fragments of 1.0,3.4,3.7 and 6.1 kbt produced by EcoRI digestion of the Caulobacter crescentus genome were identified by hybridization to a labeled ribosomal RXA probe. These genomic sequences were further characterized by the isolation of 13 hybrid h Charon 4 phages with rDNA inserts, and two of the recombinant phages, Ch4Cc773 and Ch4Ccl880, were examined extensively. The Cc773 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 3.7 kb and the Cc1880 insert contains EcoRI fragments of 1.0 kb, 3.4 kb and 6.1 kb that hybridized to 32Plabeled rRNA. Thus, the two clones contain different DNA insertswhich together account for all of the rDNA fragments detected in digests of the C. crescentus genome. Hybridization with isolated transfer RNA and individual rRNA species indicated that the arrangement of genes in both units is 16 S-spacer tRNA(s)-23 S5 S, tRNA(s). Homology between the DNA inserts is largely restricted to the rRNA coding regions, which suggests that the two rDNA units are located in different regions of the chromosome. Results of quantitative hybridization experiments are most consistent with a single Cc1880 and Cc773 unit per genome equivalent of 2.7 x 10’ daltons. The relatively simple organization of rDNA sequences in t,he C. crescentus chromosome compared to Escherichia coli is discussed.
1. Introduction The ribosomal RNA genesof vertebrates, amphibians, Drosophila and many lower eukaryotes, like Physarum, Tetrahymena and yeast, have been studied extensively (for a review, see Long & Dawid, 1980). Work on the ribosomal RNA genes of prokaryotes, however, has been largely limited to Escherichia coli (Kenerley et al.. 1977) and more recently Bacillus subtilis (Moran & Bott, 1979). Analysis of E. coli ribosomal DNA in hybrid h transducing phages and plasmids has shown that the ribosomal genes are organized into a unit of 16 S, 23 S and 5 S genes and that the haploid genome contains seven rDNA units (Kenerley et al., 1977). All of the ribosomal RNA units contain transfer RNA genesin the 16 S-23 S spacer and three of them also contain tRNA genes on the 23 S RNA distal side of the 5 S rDNA (Morgan et al., 1978). The B. subtilis chromosome also contains seven to ten copies of the rRNA genes (Smith et al., 1968), and a preliminary restriction analysis of t Abbreviation 0022-2836/N/340291-13
used: kb, 10” bases. $02.00/O
291 0
1981 Academic
Press Inc. (London)
Ltd.
292
N. OHTA
AND
A. NEWTON
genomic sequences suggests that the arrangement of the rRNA genes is similar to that in E. coli (Moran & Bott, 1979). We have examined the organization of the ribosomal RNA genes in the prokaryotic organism, Caulobacter crescentus strain CB15, as part of a more general study of gene expression and development (seeNewton, 1972; Sheffery & Newton, 1981). These prosthecate (stalked) bacteria are gram negative with a genome of about the samesize as that reported for E. coli (Wood et al., 1976), but the life cycle of Caulobacter differs significantly from that of other bacteria. C. crescentusdivides asymmetrically to produce a swarmer cell and a stalked cell with different cell cycles; the swarmer ceil cycle is described by Gl (presynthetic gap), S (DNA replication) and G2 (postsynthetic gap) periods, while the stalked cell cycle contains only an S and G2 periods (Degnen & Newton, 1972). Since the Gl period of the swarmer cell is retained even in rapidly growing cultures (Iba et al. 1977), the two progeny cells always initiate chromosome replication at different times after division. Consistent with this pattern of DNA synthesis, the organization of cell cycle steps in C. crescentus apparently precludes dichotomous chromosome replication (Osley & Newton, 1980; Nathan, Osley & Newton, unpublished results) and, perhaps as a consequence, the maximum growth rate in rich medium is significantly slower than that observed in E. wli. The level of rRNA synthesis in bacteria is closely co-ordinated with cell growth (Schaechter et al., 1958), and these observations suggested the possibility of an unusual arrangement or number of ribosomal RNA genes in the genome of these cells. The results presented below reveal a relatively simple restriction pattern for the rDNA sequencesin C. crescentus genome compared to the patterns obtained from other bacterial genomes (Moran & Bott, 1979; and this paper). Analysis of hybrid Charon 4 phages that carry CB15 rDNA inserts suggeststhat all of the 16 S, 23 S and 5 S genes are contained in two rDNA units designated Cc773 and Cc1880, and that the units are at different locations on the C. crescentus chromosome. The organization of the rRNA genes and the tRNA genes in the Cc773 and Cc1880 inserts
has also been determined.
2. Materials
and Methods
(a) Bacterial and phagestrains C. crescentus strain CB15 (ATCC 19089) was normally grown in peptone-yeast extract (PYE) medium (Poindexter, 1964) and E. wli K12 strains C600 (thr-leu-) and K802 (hr-h.sm+ gaZK SuII la-met; obtained from P. Schedl)were grown in L broth (Lennox, 1955). Strain K802 was the host for h Charon 4 phage and its derivatives. The growth and use of h Charon 4 phage ax a vector for cloning have been described by Blattner et al. (1977). (b) Preparation of DNA and RNA DNA was prepared from C. crescentus cells according to Nakamura et al. (1979) with slight modifications. Several steps in the purification, including incubation with RNase, phenol extraction, chloroform/iso-amylalcohol extraction and ethanol precipitation, were normally repeated twice. DNA from E. wli strain C600 was purified by the same procedure. Total rRNA was prepared by phenol extraction of C. crescentus or E. coli cell extracts (Ohta et al., 1975) and then sedimented on 5% to 30% sucrose density gradients by
rDNA
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centrifugation at 39,000 revs/min for 14 h in a Beckman SW41 rotor. RNA peaks corresponding to the 16 S and 23 S RNA were pooled and precipitated with ethanol at -20°C. When separated 16 S or 23 S RNA probes were required, RNA was purified from isolated 30 S or 50 S ribosomal subunits. (c) Enzymes and incubation conditio?zs T4 polynucleotide kinase was obtained from P. L. Biochemicals. Endonucleases BornHI, EcoRI and Hind111 were purchased from Miles Biochemicals. BgEII, KpnI, SmaI and XhoI were purchased from Bethesda Research Laboratories. T4 ligase and DNA polymerase I were a gift of P. Schedl. Incubation conditions for restriction endonucleases were those recommended by vendors. (d) Preparation of [y-“*P]ATP and in vitro ‘2P-labeling of RNA [y3’P]ATP (> 9000 Ci/mmol) was prepared from carrier-free [‘*P]orthophosphate (New England Nuclear) and ADP (Sigma) by the method of Johnson & Walseth (1979). rRNA (16 S, 23 S or the mixture) was labeled with [Y-~*P]ATP at the 5’ end according to Pirtle et al. (1978). (e) Preparation of in vitro J”P-labeled tRNA and 5 S RNA A culture of C. crescentus was grown overnight to a density of approximately 6 x 10s cells/ml in 01 medium (Schmidt & Stanier, 1966) that contained 0.5 mM- hosphate and then diluted B-fold in Gl medium with 0.1 mM-phosphate. Carrier-free H, P‘PO, (5 mCi; New England Nuclear) was added to the 5 ml culture and incorporation was allowed to continue for 5 to 7 h. RNA was purified and then fractionated by 10% polyacrylamide gel electrophoresis according to Ikemura & Nomura (1977). Sections of the gel that contained the tRNAs and the 5 S RNA were visualized by autoradiography, cut out and then finely minced using razor blades. RNA was eluted from gel pieces by suspending them in 10 mMTris/lO mM-EDTA/03 ru-NaCl, pH 8.0 overnight at room temperature. The eluates were phenol extracted several times and RNA was precipitated by 2.5 vol. ethanol. electrophuresis, Southern transfer and hybridization DNA restriction fragments were fractionated by horizontal agarose gel electrophoresis (McDonell et al., 1977) using E buffer (Childs et al., 1977). Procedures described by Moran et al. (1979) were followed for the transfer of the DNA fragments from gels to nitrocellulose filters (Schleicher and Schuell BA85) and for hybridization to 32P-labeled RNA except that hybridization was carried out at 37°C for 48 h. (f) Agarose
gel
3. Results (a) Restriction
mapping
of genomic
rDNA
Genomic DNA was purified from C. crescentus and E. coli cells, digested to completion with EcoRI and Hind111 and the restriction fragments were separated by electrophoresis on 0.7% agarose gels. The DNA fragments containing ribosomal genes were identified by filter hybridizations (Southern, 1975) using as a probe either C. crescentus (Fig. l(a), lanes 1 to 4) or E. coli (Fig. l(a), lanes 5 to 8) rRNA that had been labeled in vitro with [y-32P]ATP (Pirtle et al., 1978). CB15 DNA gave relatively simple restriction patterns compared to E. coli DNA. Fragments of 6.1 kbt. 3.7 kb, 3.4 kb and 1.0 kb were produced by EcoRI digestion and fragments t See footnote
to p. 291.
N. OHTA 1234
5678
AND
A. NEWTON I
2
3
4
5
6
6.1 -
I.0
16s
23s
(b) FIG. 1. (a) Restriction analysis of rDNA regions of C. crescentus and E. c&i. Genomic DNA from C. crescentus (lanes 1, 2, 5 and 6) and E. coli (lanes 3, 4, 7 and 8) was digested to completion with either EcoRI (lanes 1, 3, 5 and 7) or with Hind111 (lanes 2, 4,6 and 8) and the DNA fragments separated on 67% agarose gel. The fragments were transferred to nitrocellulose filters and hybridized to labeled, total rRNA isolated from C. crescentus (lanes 1 to 4) or from E. coli (lanes 5 to 8) as described in Materials and Methods. (b) Mapping of 16 S and 23 S genes in Ch4Cc773 and Ch4Cc1880 DNA. Fragments produced by EcoRI digestion of Ch4Cc773 DNA (lanes 1 and 3) and Ch4Cc1880 DNA (lanes 2 and 4) were se rated on 1.0% agarose and examined for hybridization to 32P-labeled 16 S RNA (lanes 1 and 2) and p”‘P-labeled 23 S RNA (lanes 3 and 4). Ethidium bromide-stained agarose gels of EcoRI digests of Ch4Cc773 (lane 5) and Ch4Cc1880 (lane 6) are shown for comparison. The sizes of the restriction fragments were estimated using h DNA and pBR322 DNA digested with several restriction enzymes, including EcoRI and HindIII, as standards (Sutcliffe, 1978). The molecular weights of the fragments in this and other Figures are given in the margin in kb.
of 195 kb, 9.8 kb, 2.6 kb and 24 kb were produced by Hind111 digestion of CB15 DNA. EcoRI digestion of E. coli DNA produced more than 11 fragments that hybridized. There are seven different rRNA regions in the E. coli (Kenerley et al., 1977) and these results suggested that there are significantly fewer rDNA units in the C. crescentus genome. Identical hybridization patterns could be observed after longer exposures of
rDNA
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CAULOBACTER
IN
autoradiograms whether the rRNA probe was from C. crescentusor from E. coli cells (data not shown). The intensity of the bands was always stronger with the homologous probe, however, as indicated by the failure to detect some of the expected bands at shorter exposures when the heterologous probes were used (cf. Fig. l(a)). Thus, there is significant but not complete homology between the rDNA sequencesof the two bacteria.
(b) Construction of C. crescentus gene clone bank in /\ Charon 4 The ribosomal regions of the C. crescentuschromosome were characterized in more detail by cloning the rDNA units in h Charon 4. To obtain a random distribution of EcoRI fragments in the desired size range for cloning, purified CB15 DNA was digested to different extents using serially diluted enzyme; the digests were pooled and then fractionated by sucrose gradient centrifugation. DNA fragments of 10 to 20 kb were collected, ligated to Charon 4 DNA end pieces as described by Maniatis et al. (1978) and the hybrid DNAs were packaged in vitro (Hohn & Murray, 1977). Individual plaques were picked, amplified on lawns of E. coli strain K!302, transferred with toothpicks to wells of microtiter dishes and stored at - 18°C in 50% glycerol. The average size of inserts in these clones is 10 kb, and if the C. crescentusgenome size is taken to be equivalent to that of E. coli. or 2.7 x lo9 dalton (Britten & Kohne, 1968), then any gene should be present in a bank of 2300 with a 99% probability (Clarke & Carbon, 1976). (c) Identi$cution of rDNA clones Although rRNA from C. crescentuscross hybridizes to E. coli DNA (Fig. l), the E. coli host DNA did not give a positive responseusing 32P-labeled rRNA in in situ plaque hybridization (Benton & Davis, 1977). Thus, the h Charon 4 library was
TABLE
1
EcoRI inserts in rDNA clonesof Charon 4 Class
Charon
4 clones
Inserted
12
II
b
Cc3651,
c
cc3966
a
Cc1880,
Cc4636,
Cc5507 +
Cc3666,
fragments
1.0
(kb)
3.7
3.4 +
+
+
+
+
+
+
+
+
3.4
1.0
6.1
+
+
+
+
+
+
cc773
IEI
EcoRI
42
Cc4084
7.8
Cc4095. 0~4204, Cc5274 b
Cc4681
+ Indicates
the presence
of the EcoRI
fragment
in the CB15
DNA
insert.
+
296
N. OHTA
AND
A. NEWTON
screened directly with in vitro labeled, C. creacentus rRNA. A total of 13 positive clones of independent origin were identified among approximately 4500 clones tested, and 12 of these were examined by EcoRI restriction analysis (Table 1). All of the clones contained either a Cc773 type DNA insert (class I) or a Cc1880 type DNA insert (class II). The hybrid clones containing these two inserts were characterized in detail. (d) Restriction
mapping
of CB15 rDNA
inserts of Ch4Cc773 and Ch4Cc1880
As shown in Table 1, the Cc773 insert of 81 kb produces EcoRI fragments of 1.0 kb, 3.4 kb and 3.7 kb, while the Cc1880 insert of 168 kb also produces the EcoRI fragments of 1.0 kb and 3.4 kb, plus a 6.1 kb fragment instead of the 37 kb fragment. Consequently, all of the genomic rDNA fragments identified in Figure 1 are contained in Ch4Cc773 and Ch4Cc1880. To determine the arrangement of ribosomal RNA genes in the cloned sequences, 16 S RNA and 23 S RNA probes were prepared by isolating RNA from separated 30 S and 50 S ribosomal subunits, labeled with 32P in vitro and then hybridized to EcoRI digests of DNA from the hybrid phages (Fig. l(b)). The 16 S RNA hybridized only to the 3.4 kb fragment produced by the Cc773 and Ccl880 inserts, while 23 S RNA hybridized to the 3.4 kb, 1.0 kb and 3.7 kb fragments of the Cc773 insert and the 3.4 kb, 1.0 kb and 6.1 kb fragments of the Cc1880 insert. We also observed this same pattern of hybridization when these two probes were hybridized to EcoRI fragments of genomic CB15 DNA (data not shown). Since the 3.4 kb and 1.0 kb fragments are common to both rDNA units, these results suggest that the 3.7 kb and 6.1 kb fragments contain the 23 S RNA flanking sequences and that the arrangement of EcoRI fragments is 3.4 kb, 1.0 kb, 3.7 kb in Ch4Cc773 (Fig. 2(a) and (b)) and 3.4 kb, 1.0 kb, 6.1 kb in Ch4Cc1880 (Fig. 2(b) and (c)). This map order was confirmed by rRNA hybridization to DNA fragments produced by digestion with other restriction enzymes. These experiments also indicate thet one end of the 16 S gene lies very close to the left of the 3.4 kb fragment. The 14.6 kb fragment produced by BumHI digestion of Ch4Cc773 DNA extends into the left arm of Charon 4 and covers less than 63 kb of the 3.4 kb EcoRI fragment, but it hybridized with the 16 S RNA probe (data not shown). The orientation of the CB15 DNA inserts in the hybrid clones was determined from the results of digestions with BamHI, BglII, HindIII and double digestions with EcoRI and each of these enzymes (Fig. 2(b) and (c) and unpublished data). The results show that two inserted segments have opposite orientations with the 16 S rDNA region joined to the long arm of Charon 4 in Ch4Cc773 and to the short arm of Charon 4 in Ch4Cc1880. The only difference in the restriction maps of the rDNA coding regions of the two clones was the position of the BgZII sites that are adjacent to the spacer tRNA; the maps of the flanking sequences adjacent to the 23 S gene differ substantially, however (Fig. 2 and below). (e) Mapping of tRNA and 5 S RNA genes The presence of tRNA and 5 S RNA genes in the Cc773 and Cc1880 inserts was examined by hybridization using in viva 32P-labeled 5 S RNA or tRNA as a probe
I-DNA
RI ,/’ ( b 1 Ch4Cc
773
I
ORGANIZATION
IN
Xh Sm Sm Xh HI11 RI I
I I
RI
CAULBACTOR
\
HIII Sm XhSm II
I
Bo
297
I
RI y
R
i3g
L.,i
w-2.07
I
16s
I I
(cl
Ch4CclBBO
RI l I I ~,KP &y4+
ItI
I I
123s
!!
1
I/
1
\ \
r------4 HI11 L-----g+---4
Xh Sm Xh I
I Bo
d1 )
I Kp
L
/ /’
&-z~
/' rC3.4+
‘,I+1 (d)
I
I
eI.35J \ \
t,5s I I
p+2+je----4.9RI HIII Xh
Xh SmlSm XhHIIIRI ” I’ ‘1 ’ ’ 1 I Bg ~0 \
I
Xho ’ KPn
IO Ic-dr FiI .I-f HI11
/'
6-l
,’
4+7.0+?
iI/’ 9’ HIII
Kpn
HI11
L---64‘1
FIG. 2. Restriction
maps of X Ch4Cc773 and h Ch4CclSSO inserts and genomic arrangement of rDNA units. (b) and (c) Restriction maps of h Ch4Cc773 and A Ch4Cc18SO DNA inserts. (a) and (d) Arrangement of Cc773 and Cc1880 rDNA units in the C. creaeentwr chromosome. Only those restriction sites which are relevant in placing various fragments are given in (a) and (d). Regions of the map shown in a broken line were deduced from genomic restriction patterns and those in an unbroken line were obtained by restriction patterns of cloned inserts (Table 1). Heavy, unbroken lines in (a) and (d) indicate the maximum extent of rDNA coding region. Distances are given in kb. Abbreviations are: Ba, BumHI: Bg, &$II; RI, EcoRI, HIII, HindIII; Kp.Kpn, ZfpnI; Sm, SmaI; and Xh,Xho, XM.
(Materials and Methods). A lOO-fold excess of unlabeled 16 S and 23 S RNA was included to prevent background hybridization from contaminating 16 S and 23 S RNA breakdown products in the radioactive probes. Hybridization of 5 S RNA to the 3.7 kb EcoRI fragment of the Cc773 insert and to the 6.1 kb EwRI fragment of the Cc1880 insert (Fig. 3) places the 5 S gene at the end of the rDNA unit adjacent, to the 23 S gene. The 5 S DNA sequence was further localized within the 6.1 kb fragment by showing that the 5 S RNA probe hybridized only to 1.3 kb EcoRI-BamHI fragment, the 2.2 kb EwRI-BglII fragment (Fig. 3) and the 1.2 kb EwRI-Hind111 fragment of the Cc1880 insert (data not shown). Hybridization with double digest fragments from Ch4Cc773 DNA placed the boundary of the 5 S genes in this rDNA insert in a similar position (data not shown, see Fig. 2). When total, labeled tRNA from C. crescentuscells was used to probe restriction fragments from the hybrid clones, the 3.4 kb fragments of both clones hybridized, as well as the 3.7 kb fragment of Ch4Cc773 and the 6.1 kb fragment of Ch4Cc1880 (Fig. 3). Double enzyme digests of Ch4Cc1880 DNA allowed more accurate mapping of the tRNA genes. The tRNA hybridized to the same 1.3 kb EwRIBamHI fragment and EwRI-BglII fragment, as did the 5 S RNA probe, which
298
N. OHTA I
3
2
AND 4
A. NEWTON 6
5
7
8
6.1 -
-
-
tRNA
55
2.2
I.3
RNA
FIG. 3. Mapping of 5 S and tRNA
genes in rDNA units. Ch4Cc773 (lanes 1 and 5) and Ch4Cc1880 (lanes 2 and 6) DNA were completely digested with EcoRI. Double digests of Ch4Cc1880 DNA were also made with EcoRI and BumHI (lanes 3 and 7) and with EcoRI and @#II (lanes 4 and 8). DNA fragments were separated and analyzed by hybridization to labeled tRNA (lanes 1 to 4) and 5 S RNA (lanes 5 to 8) as described in Fig. 1 and text.
places both of the genes adjacent to the end of the 23 S RNA. In addition, the tRNA hybridized to a 2.1 kb BarnHI fragment and a 1.35 kb EcoRI-BgZII fragment of Cc1880. Since the only overlap between the two fragments is less than O-35 kb long (see Fig. 2(b) and (c)), this places the “spacer” tRNA gene(s) within this region. Hybridization of the tRNA probe to double digests of the Ch4Cc773 gene gave comparable restriction patterns (data not shown). The approximate locations of the 5 S and tRNA genes in the Cc773 and Cc1880 rDNA units are shown above in Figure 2. Genomic DNA from C. crescentus was also probed with the labeled 5 S and tRNA species. When 5 S RNA was the probe, the only EcoRI fragments to hybridize were 3.7 kb and 6-l kb (data not shown), suggesting that the 5 S genes in these cells are
I-DNA
ORGANIZATION
IN
CAULOBACTh’R
299
located exclusively in the Cc773 and Cc1880 rDNA units. Use of the tRNA probe. however, revealed higher molecular weight fragments that hybridized in addition to the 3.4 kb, 3.7 kb fragments. Thus, a number of tRNA genes in C. crescentm ohromosome are located outside of the rDNA regions. (f) Gen,omicorganization of rDNA units The restriction patterns produced by the DNA sequencesflanking the 23 S RNA gene of Ch4Cc773 and Ch4Cc1880 suggest that the two rDNA inserts arise from different regions of the CB15 genome (Fig. 2). This conclusion was confirmed by using the nick translated, 3.7 kb EcoRI fragment from Ch4Cc733 to probe EcoRIHind111 digests of Ch4Cc1880. Of the two fragments produced by the 6.1 kb, EcoRI fragment of the Cc1880 insert (see Fig. 2(c)), the 1.2 kb fragment hybridized while the 4.9 kb fragment did not (data not shown). Thus, homology between these two cloned sequencesappears to be limited to the rDNA units themselves. We have also compared genomic restriction patterns with those from rDNA clones like Ch4Cc3966 and Ch4Cc4681 (Table 1) in order to examine the flanking sequenceson both the 16 S and 23 S sidesof the Cc773 and the Cc1880 rDNA units. Hind111 digestion of genomic DNA produces a 195 kb and a 9.8 kb fragment that’ hybridize to 16 S rRNA (Fig. 1 and data not shown). Since the Cc3966 DNA insert does not contain a Hind111 site in the 12 kb and 4.2 kb fragments that flank the I6 S RNA gene, the 195 kb genomic Hind111 fragment can be tentatively assigned t,o the Cc773 region and the 9.8 kb fragment to the Cc1880 region. An additional 23 S rDNA flanking sequence of 7.8 kb in the Cc1880 region is present in the (‘h4Cc4681 insert (Table l), and another flanking sequence on the same side of the (‘~773 unit has been identified by KpnI digestion of genomic DNA (data now shown). The genomic maps constructed from these results (Fig. 2(a) and (d)) confirm that the two rDNA units are from different regions of the C. crescentus chromosome. They also show that the rDNA units are not present’ as closely spaced, tandem repeat units, since the Cc773 unit is present only once in the 28.2 kb sequenceexamined in these st,udiesand the Cc1880 unit is present only once in t’he 22.6 kb sequence.
(g) Estimation
of rDNA copy number
We also examined the possibility that the rDNA units in C. crescentuscells are present in the genome as widely spaced, redundant copies. The number of rDNA units was estimated by quantitative hybridization of labeled rRNA to complete EcoRI digests of Ch4Cc773 DPL’A, Ch4Cc1880 DNA and genomic DNA4 that had been separated on agarose gels (Fig. 4). Assuming one genome equivalent of CB15 DNA as 2.7 x 10’ daltons (Wood et al., 1976) the results are most consistent with the presence of two copies per genome of the 3.4 kb EcoRI fragment, which is generated by the Cc773 and the Cc1880 rDNA units, and one copy per genome of the 3.7 kb fragment and 6.1 kb fragment, which are generated by Cc773 and the Cc1880 units. respectively. Quantitative densitometry measurements that were made on the results from six independent, experiments gave average values that were somewhat
300
N. OHTA
I
2
AND
3
A. NEWTON
4
5
6
7
6
FIG. 4. Estimation of rDNA copy number in C. crescentzls genome. Ch4Cc773 DNA, Ch4CclS80 and CM5 DNA were digested to completion with BcoRI and applied to agarose gel in 1, 2 or 3 genomic equivalents (Ch4Cc773 and Ch4Cclt3SO) or 06 or 1 genomic equivalent (CB15 DNA). DNA fragments were transferred to nitrocellulose filters, hybridized to labeled 23 S RNA ae in Fig. 1 and analyzed IM described in the text.
CA UL OBACTER
301
higher, however, with 2.1 copies for the 3.4 kb fragments, fragment and 1.2 for the 6.1 kb fragment.
1.3 for the 3.7 kb
rDNA
ORGANIZATION
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4. Discussion Restriction analysis of genomic DNA from C. crescentus and the characterization of Charon 4 phage clones that contain CB15 rDNA inserts are consistent in showing that the chromosome of these cells contains only two different rDNA units. All of the 12 rDNA clones examined contain inserts of either the Cc773 or the Cc1880 type (see Table l), and together these two inserts account for all rDNA fragments detected in the genomic digests (Fig. 1). Thus, the organization of ribosomal sequences in C. crescentus is relatively simple compared to E. co& which contains seven different rDNA units (Kenerley et d., 1977). The use of individual RNA species as probes indicates that the 16 S, 23 S and 5 S genes of the C. crescentus genome are located exclusively in the Cc773 and the Cc1880 rDNA units, while tRNA genes map both within the rDNA units and at other sites on the chromosome. The arrangement of genes in both the Cc773 and Cc1880 inserts is 16 S-tRNA(s)-23 S-5 S,tRNA(s); the experiments do not indicate the relative order of the 5 S and flanking tRNA(s) gene or the number of tRNA genes present in the spacer and 23 S flanking regions, however. These maps are similar to those reported for one group of the rDNA units in E. coli that contains tRNA genes in the 23 S flanking sequence, as well as in the 16 S-23 S spacer, e.g. the map of the E. coli rrnC operon is 16 S-tRNAZG”-23 S-5 S,tRNAT’p,tRNAAap (Morgan et al., 1978). While the Cc773 and the Cc1880 regions share this general gene arrangement and considerable homology with the rDNA coding regions of E. co& (Fig. l), the rDNA restriction maps of the two organisms show little resemblance (cf. Fig. 2 and deBoer et al., 1979). We have attempted to identify the tRNA species present in the Ch4Cc773 and Ch4Cc1880 phages in in wivo transcription experiments (Lund et al., 1976), but the CB15 DNA inserts were not expressed. The EcoRI site that defines the junction of the Charon 4 left arm and the Cc773 insert and Charon 4 right arm and Cc1880 insert lies very close to the presumptive 5’ end of the 16 S RNA gene, and these rDNA inserts may lack the promoter regions. Transcription of the rRNA genes may be possible in a clone like Ch4Cc5507 (Table l), which contains an additional flanking fragment adjacent to the end of the 16 S gene. The maximum size of the C. crescentus rDNA unit can be calculated as 5.6 kb from the position of the EcoRI site flanking the 16 S RNA gene and the Hind111 site in the 6.1 kb fragment of the Cc1880 insert; as shown in Figure 2, only the 1.2 kb fragment produced by EcoRI-Hind111 double digest of the 6.1 kb fragment hybridized to the rRNA probe. The results obtained to date indicate that the rDNA coding sequences in the Cc773 and Cc1880 units are highly conservative, as observed in other systems. With the exception of a BglII site in the 3.4 kb fragment that is located closer to the tRNA spacer in Ch4Cc773 (Fig. 2(b) and (c)), the restriction maps of the two rDNA units are identical. This sequence homology does not extend outside of the coding regions, however. There is a similar clustering of BamHI, BglII and Hind111 sites in the 23 S RNA flanking sequences, but the
302
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AND A. NEWTON
positions of these sites are not identical, and there is no indication of homology between these sequences.The genomic map in Figure 2(a) and (d) shows that the Cc773 and Cc1880 are in fact located in different regions of C. crescentus chromosome. The quantitative reconstruction experiments are subject to large systematic errors, but these results (Fig. 4) and the genomic maps constructed above (Fig. 2(a) and (d)) are consistent in suggesting that a C. crescentus genome contains no more than a single copy of the two rDNA units. Although this conclusion should be confirmed by other experiments, the organization of the ribosomal sequencesin these cells is clearly simpler than that in many other bacteria. Since the level of rRNA synthesis is tightly coupled to cell growth (Schaechter et al., 1958), the small number of rRNA genes in C. crescentus may only reflect the relatively slow growth rate of these bacteria. C. crescentus cultures reach a maximum doubling time of approximately 90 minutes compared to approximately 20 minutes for E. coli and R. subtilis, which have seven to ten rDNA copies per genome (Kenerley et al., 1977 ; Smith et al., 1968). These results suggest that bacteria like C. crescentus, which do not replicate their chromosomes dichotomously (Nathan, Osley & Newton, unpublished
results)
and grow
slowly,
have been under
relatively
less evolutionary
pressure to duplicate genomic rDNA units. We thank Paul Schedl for his advice during the course of this work and JoAnn Mermelstein and Ling-Sing Chen for technical assistance. The research was supported by Public Health Service grants GM25644 and GM22299 from the National Institutes of Health and by the Whitehall Foundation. Note added in proof: Feimgold and Shapiro have independently cloned rRNA genes of C. crescentus strain CBIB (personal communication).
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Edited by M. Gotteswmn