Download Unique genetic arrangement in the dnaA region of the Borrelia

FEMS Microbiology Letters 111 (1993) 109-114
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
109
FEMSLE 05523
Unique genetic arrangement in the dnaA region
of the Borrelia burgdorferi linear chromosome:
Nucleotide sequence of the dnaA gene
Iain G. Old, Danielle Margarita
and Isabelle Saint Girons
Unit~ de Bact~riologie Mol~culaire et M~dicale, lnstitut Pasteur, Paris, France
(Received 8 April 1993; revision received 28 April 1993; accepted 29 April 1993)
Abstract." The complete nucleotide sequence of the Borrelia burgdorferi dnaA gene (encoding the initiator protein of chromosome
replication) and its flanking regions was determined. The putative DnaA polypeptide exhibited 29-42% identity with those of other
eubacteria. The gene order in the dnaA region at the centre of the B. burgdorferi linear chromosome is rnpA-rpmH-dnaN-dnaAgyrB-gyrA in contrast to the consensus eubacterial order of rnpA-rprnH-dnaA-dnaN-recF-gyrB, suggesting a rearrangement during
the evolution of the Borrelia chromosome. We did not detect the multiple 9-nucleotide repeats known as DnaA boxes, which
characterise origin of replications, in the dnaA-gyrB and dnaA-dnaN intergenic regions. In addition B. burgdorferi DnaA protein
differs considerably from those of other eubacteria in a normally highly conserved region at the C-terminus of the polypeptide
which may be involved in DNA binding.
Key words."Borrelia burgdorferi; Linear chromosome; dnaA; dnaN; gyrB
Introduction
T h e dnaA g e n e p r o d u c t (the i n i t i a t o r p r o t e i n
for c h r o m o s o m e r e p l i c a t i o n ) is o n e o f several
p r o t e i n s which a r e essential for t h e r e p l i c a t i o n of
t h e b a c t e r i a l c h r o m o s o m e . It is also t h e only
p r o t e i n which is r e q u i r e d for initiation f r o m b a c t e r i a l origins a n d is n o t r e q u i r e d for D N A replic a t i o n events after initiation. D n a A b i n d s specifi-
Correspondence to: I.G. Old, Unit6 de Bact6riologie Mol6culaire et M6dicale, D6partement de Bact6riologie et Mycologie, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex
15, France.
cally to 9 b p n u c l e o t i d e r e p e a t s k n o w n as D n a A
boxes. O r i g i n r e g i o n s a r e c h a r a c t e r i s e d by nont r a n s l a t a b l e r e g i o n s c o n t a i n i n g at least 4 D n a A
boxes in a c h a r a c t e r i s t i c a r r a n g e m e n t [1,2]. T h e
c o n s e n s u s s e q u e n c e for a D n a A box is 5'T T A T C C A C A - 3 ' a n d a s e q u e n c e differing by up
to 2 b a s e s while r e t a i n i n g t h e fourth, s e v e n t h a n d
e i g h t h n u c l e o t i d e s m a y be d e f i n e d as such [3]. In
G r a m - p o s i t i v e b a c t e r i a a n d p s e u d o m o n a d s , origins a r e f o u n d next to t h e g e n e s dnaA a n d gidA
(Fig. 1) while in Escherichia coli t h e origin is
f o u n d next to gidA, a n d a single D n a A box,
essential for dnaA a u t o r e g u l a t i o n , is f o u n d in t h e
dnaA p r o m o t e r r e g i o n [1,2].
T h e g e n e t i c o r g a n i s a t i o n of t h e dnaA r e g i o n of
t h e c h r o m o s o m e has b e e n s t u d i e d in several eu-
110
bacteria: for reviews see [1,2]. The consensus is
gid B-gidA - 5 OK-6O K-rnpA -rm p H-dnaA -dna NrecF-gyrB [4] (Fig. 1B,C) and between Pseudomonas putida and Bacillus subtilis a total of 12
genes in a 15 kb region are similar [4]. In E. coli
there is less similarity as gyrA is elsewhere on the
chromosome while gidB, gidA and the origin
have been translocated by the inversion of a 40
kb region [4] (Fig. 1A). Due to the highly conserved nature of dnaA region of chromosome, it
has been proposed that this was the replication
origin of ancestral bacteria and that its organisation has been conserved in most eubacteria [1].
The spirochaete B. burgdorferi is an exception
to the normal organisation in the dnaA region of
the chromosome. While the rpmH, gyrB and
gyrA genes of B. burgdorferi mapped to the centre of the 1 Mb linear chromosome, gidA was
located between 293 and 395 kb from these genes
[5-7]. In this study we present the sequence of
the B. burgdorferi dnaA gene and demonstrate
that the genetic organisation in this normally
highly conserved region is unique among the eubacteria.
Materials and Methods
Bacterial strains and media
E. coli strains XLl-blue and DH5a were grown
as previously described [6].
Cloning of B. burgdorferi genes
The dnaA clone, pB22, was isolated from a
previously constructed library of EcoRI-partially
digested B. burgdorferi 212 DNA in pUC18 [6].
Pulsed field gel electrophoresis (PFGE) and DNA
hybridisations
These were carried out as previously described
[6].
DNA sequencing and computer analysis of sequences
Deletions of pB22 were constructed with Exonuclease III using an Erase-a-base kit (Promega).
DNA sequencing was carried out as previously
described [6]. The GCG computer package [8]
was used to carry out Fasta analyses and multiple
sequence alignments.
Results and discussion
Sequencing of randomly chosen clones from
our B. burgdorferi library allowed us to identify a
clone, pB3, with a 3.5 kb insert, which carried
gyr'BA' (DNA gyrase a and /3 subunits) [6]. We
isolated a further clone, pB22, with a 4.8 kb
insert which carried rpmH (ribosomal protein
L34) [7]. Further sequence analysis revealed pB22
also carried dnaA, dnaN (DNA polymerase III /3
subunit), rnpA' (Ribonuclease P, protein component) and gyrB', the insert being contiguous with
gyr'BA' carried by pB3 (Fig. 1D). The identification of these genes was inferred from Fasta analyses of their deduced amino acid sequences
against the SWISS-PROT database [8].
The B. burgdorferi dnaA gene and its flanking
regions were sequenced (Genbank accession
number L14948). The 1458 nucleotide open reading frame encodes a 486 amino acid polypeptide
of 56.8 kDa. The open reading frame is preceded
by a possible ribosome binding site A G G A while
a possible promoter, TFCACA-17N-CCTAAT is
located 60 nucleotides upstream of the start
codon. The GC % of B. burgdorferi is one o f the
lowest in eubacteria (28-31%) and the codon
usage is heavily biased to use A and T: 78.6% of
codons in dnaA have A or T in the 3rd position.
The deduced DnaA polypeptide of B. burgdorferi exhibits 29-42% identity with those from
other eubacteria (Fig. 2). DnaA can be divided
into three domains [9]: I, with moderate similarity; II (which may act as a hinge), with little
similarity; and III, with high similarity. It has
been noted that a remarkably conserved stretch
found in the C-terminal region may be involved
in DNA binding (unpublished data in [1]). The
sequence G G R D H T T V is almost invariably present towards the C terminus of eubacterial DnaA
polypeptides (SGRDHTTV is found in Buchnera
aphidicola [10]), whereas in B. burgdorferi only 5
of the 8 amino acids are conserved (Fig. 2C). If
this conserved stretch does represent a DNA-binding region of DnaA, this would suggest that B.
111
burgdorferi DnaA boxes, to which DnaA protein
binds, may differ from those found in other eubacteria.
Further sequencing of pB22 revealed an open
reading frame 177 bp upstream of dnaA corresponding to the 5' end of gyrB (Fig. 1D). Like
DnaA, GyrB is highly conserved and the corresponding region of the deduced polypeptide exhibited between 45 and 54% identity with those
of other eubacteria. 242 bp downstream of dnaA
was an open reading frame that would encode a
polypeptide with 19-21% identity to the S-subunit of DNA polymerase III encoded by dnaN.
DnaN is less well conserved than GyrB and DnaA
and this low identity is in the order of that found
between other eubacteria [11]. We also found an
open reading frame following rpmH which, when
translated, exhibited 35% identity with RnpA of
Bacillus subtilis (results not shown). In Gramnegative bacteria the dnaA-dnaN intergenic region is a few nucleotides and the two genes form
a single operon, while in Gram-positive bacteria
the intergenic region is larger [1,2]. B. burgdorferi
is more like Gram-positive bacteria in that the
dnaA-dnaN intergenic region is 242 bp and the
gyrBA genes are in tandem.
The arrangement of the genes in the B.
burgdorferi dnaA region is shown in Fig. 1D. We
A E s c h e r i c h i a coli
oriC
--~¢
~g/dA
~(40kb)~¢--.-.4~---- .~.~
g/d.B
rnpA
60K rpmH
50K
B P s e u d o m o n a s putida
oriCll
g/dB
g/dA
~
dnaA
.~
dnaN
recF
gyrB
dnaN
recF
gyrB
oriCl
rnpA
60K rpmH
50K
dnaA
C B a c i l l u s subtilis
orilll
oril
orUI
i
gidB
gidA
50K
mpA
60K rpmH
dnaA
dnaN
recF
gyrB
gyrA
D BorreUa burgdorferi
(293-395kb) - . 4 ~
~
r
m
gidA
rnpA
rpmH
dnaN
dnaA
gyrA
gyrB
y
pB4
pB22
pB3
Fig. 1. Genetic organisation in the dnaA regions of four different eubacteria, adapted from [1,2,4]. The open reading frames (boxed
regions) and intergenic regions (gaps) are drawn approximately to scale. The direction of transcription is shown by single headed
horizontal arrows while origin regions are marked with vertical arrows. The genes shown are: gidA, gidB (glucose inhibited division
proteins); 50K, 60K (proteins of undetermined function); rnpA (ribonuclease P, protein component); rpmH (ribosomal protein
L34); dnaA (chromosome replication initiation protein); dnaN (DNA polymerase III, /~-subunit); recF (inducer of SOS DNA
repair); gyrB, gyrA (DNA gyrase, /3 and a subunits). (.4,) Escherichia coli: gyrA is located over 1500 kb distance on the
chromosome; (B) Pseudomonas putida: The region corresponding to gyrA has not been sequenced; (C) Bacillus subtilis: A fourth
origin is located between gyrA and rrnO; (D) Borrelia burgdorferi: The extent of the inserts of pB3 [6], pB4 [6] and pB22 (this work)
are indicated and the sequence data described in this paper (Genbank accession number L14948) is marked by a dashed line with
double headed arrows. The direction of transcription of gidA relative to dnaA is not known, while B. burgdorferi homologues of
gidB and the 50K and 60K genes have not been isolated.
B.subtilis
E.coli
M.luteus
P.putida
B.burgdorferi
Domain I->l<-Domain
II
M _ _ _ E N i L D L W N Q A L A Q i E K K L S - K P S F E T W M K S T K A H S L Q G D T L T I T A P N E F A R D W L E S R Y L H L I A D T I YE L T G E E L S I K F V I P Q N Q D V E D F M P K . . . . . . . . . .
MS ..... LSLWQQCLARLQDELPATE-F
S M W i Rp L Q A E - L S D N T L A L Y A P N R F V L D W V R D K Y L N N
INGLLT S-FCGRI APQLRFEVGTKPVTQ---TP
QAAVTSNV
M V A D Q A V L S S W R S V V G S L E D D A R V S A R L M G F V Y L A Q P Q G L I GN T L L L A V P N E T T R E T L Q G T - - - Q V A D A L T D A L T Q E F R E E
I L L A I S I D A N LQP P R TP S S E A R R S S
MS ..... VELWQQCVELLRDELPAQQ_FNTWI
Rp L Q V E - A E G D E L R V Y A P N R F V L D W V N E K Y L G R L L E L L G E - N G S G
IA P A L S L L I G S R R S S A P R A A P N A P V S A A V
MEKSKN I___WSLILTEIKKELS-EEEFYVWFENLCFLE
S I G D N IKI S T P N L F H K N Q I E K R F T K K I K E ILI K N G Y N N I V I V F . . . . . . . . T N Q P P K T H S N K . . . . .
B.subtilis
E.coli
M. luteus
P.putlda
B.burgdorferi
Domain II->L<-Domain
III
.......... PQV ........... KKAVKEDTSDFP ...................................
QNMLNPK Y TFDTFVI GS GNRFAHAASLAVAEAP AK
AAPAQVAQTQPQRAAP ...................
STRSGWDNVPAPA ........... EPTYRSNVNVKHT ........ FDNFVEGK SNQLARAAARQVADNP GG
LAGGP SGAAAP DVELPPAATAAT SRRAVAEELPGFR IEPPADVVPAANAAPNGNG .... KPTPAPp STSAETSRLNDRYHFETFVI
GS SNRFAHAAANAVAEAPAK
A A S _ _ L A Q T Q A H K T A P A A A V E P V A V A A A E P V L V E T S S R D S F D A M A E P A A A P P S G G G R A E Q R T V Q V E G A L K H T SY L N R T F T F D T F V E G K S N Q L A R A A A W Q V A D N P K H
.................... QETKNPALNETFSKFDKLKEKTT
S K E A I QN I Q D R I K M Y I K K E E E E P T N F K N - P F L K K R Y T F E N F I I G P N N K L A Y N A S L S I S K N P G K
*. *. *
*
*
*.
..
*
B.subtilis
E.coli
M. luteus
P.putida
B.burgdorferi
I<-ATP Binding->1
A Y N P LF I Y G G V G L G K T H L M H A I G H Y V I D H N P S A K V V Y L S S E K F T N E F INS I R D N K A V D F R N R Y R N V D V L L
A Y N P LF L Y G GT G L G K T H L L H A V G N G I M A R K P N A K V V Y M H S E R F V Q D M V K A L Q N N A I E E F K R Y Y R S V D A L L
A Y N P LF I Y G E S G L G K T H L L H A I G H Y A R R L Y P G L R V R Y V N S E E F T N D F INS I R H D E G A S F K Q V Y R N V D ILL
G Y N P LF L Y G G V G L G K T H L M H A V G N H L L K K N P N A K V V Y L H S E R F V A D M V K A L Q L N A I N E F K R F Y R S V D A L L
K Y N P C L I Y G G V G L G K T H L L Q S I G N K T E E L H H N L K I L Y V T A E N F L N E F V E S IK T H E T K K F K K K Y R Y L D M L L
B.subtilis
E.coli
M. luteus
P.putlda
B.burgdorferi
Rpp KE i p T L E D R L R S R F E W G L I TD I T P P D L E T R I A
RYPKE INGVEDRLKSRFGWGLTVA IEPPELETRVA
LPPKQLSGFEDRLRSRFEWGL
i TD I Q P P D L E T R I A
RYPKE IEGLEERLKSRFGWGLTVAVEPPELETRVA
RSP S E L T N F T D R L K S R F T R G L N V D
ISKPNFE LRAA
B.subtilis
E. coli
M. luteus
P.putida
B.burgdorferl
S K P K V I T IKE I Q R V V G Q Q F N I K L E D F K A K K R T K S V A F P R Q
I A M Y L S R E M T D S SLP
Q E - K L V T i DN i Q K T V A E Y Y K I K V A D L L S K R R S R S V A R P R Q M A M A L A K E L T N H
SLP
E T A H E iT P E L I L H A T G E Y F N L T L E E L T S K S R T R T L V T A R Q I A M Y L L R E L T E M S L P
Q D - K L V S V D N i Q R T V A E Y Y K IK I S D L L S K R R S R S V A R P R Q V A M A L S K E L T N
HSLP
E P N N K I N IENI K K I L L R E L K I T H K D I E G H S K K P E I T K A R H I Y A Y L L R N F T E L S T V E
,
*
,
,
,
I D D I QF L A G K E Q T Q E E F F H T F N T L H E E S K Q
IV I S SD
I D DI Q F F A N K E R S Q E E F F H T F N A L L E G N Q Q I I L T SD
I DDI Q F L A D K E A T V E E F F H T F N T L Y N N N K Q V V IT SD
I D DI QF F A R K E R S Q E E F F H T F N A L L E G G Q Q V I LT SD
ID D IHDLQKKEG IQEELF HTFNALYEDNKQLVFTCD
I L R K K A K A E G L D I P N E V M L Y I A N Q I D S N I R E L E G A L I R V V A Y S S L I N K D I N A D L A A E A L K D I I . . . . . . PS
I L M K K A D E N D IR L P G E V A F F I A K R L R S N V R E L E G A L N R V
I A N A N F T G R A IT I D F V R E A L R D L L . . . . . . A L
I L R K K A E A E G L V A P P E A L E Y I A S R I S T N I R E L E G A L I R V T A F A S L N R Q T V D IE L A E H V L K D LI . . . . . . TD
ILMKKADQAKVELPHDAAFF
I A Q R I R S N V R E L E G A L K R V I A H S H F M G R D IT IE LI R E S L K D LL . . . . . . A L
IV E K K A E E D G I N V P K N IL N L V A Q K V T T N V R D L E A A V T K L K A Y
I D L D N I E ID IE I V E K I I K E I I I Y E K E T T N
DNA Binding?
KIGEEFGGRDHTTVI HAHEKI SKLLADDEQLQQHVKE IKEQLK ......
E IGDAFGGRDHTTVLHACRK
I E Q L R E E S H D I K E D F SN LI R T L . . . . . SS
KIGQVLGGRDHTTVI HADRKIRELMAERRT IYNQVTELTNE IKRKQRGA
E IGDMFGGRDHTTVLHACRK
I N E L K E S D A D I R E D Y K N L L R T L . . . . . TT
IGKI IGGKTHSTVLYS INKIDRDRNNDKE INNLI TELMNKIKKN ....
**
**
, **
**
. . . . .
B
C
B.subtilis
B.aphidicola
E.coll
M. luteus
M. caprlcolum
P.mirabilis
P.putida
S.marcescens
S. typhimurium
S.coellcolor
I<-ATP B i n d i n g - > l
IYGGVGLGKTHLMHAI
LYGGTGLGKTHLLHAI
LYGGTGLGKTHLLHAV
IYGESGLGKTHLLHAI
IYGESGMGKTHLLKAA
LYGGTGLGKTHLLHAV
LYGGVGLGKTHLMHAV
LYGGTGLGKTHLLHAV
LYGGTGLGKTHLLHAV
IYGESGLGKTHLLHAI
.**
*.*****. ,
DNA Binding?
IGEEFGGRDHTTVIHAHEKI
IGDAFSGRDHTTVLHACRKI
IGDAFGGRDHTTVLHACRKI
IGQVLGGRDHTTVIHADRKI
IGEEFGGRDHTTVINAERKI
IGDAFGGRDHTTVLHACRKI
IGDMFGGRDHTTVLHACRKI
IGDAFGGRDHTTVLHACRKI
IGDAFGGRDHTTVLHACRKI
IGALFGGRDHTTVMHADRKI
B.burgdorferi
IYGGVGLGKTHLLQSI
.**
******.
.
IGKIIGGKTHSTVLYSINKI
**
*
*.**. .
**
Fig. 2. Alignment of DnaA sequences. The amino acid sequences, presented in the one letter code, were aligned using Clustal V. Identical residues are marked with
an asterisk ( * ) while similar sequences are denoted by a point (.) below the alignment. (A) Alignment of 5 different D n a A proteins from representative eubacteria:
Bacillus subtilis, and Micrococcus luteus, respectively low and high GC% Gram-positive bacteria, Escherichia coli and Pseudomonas putida, Gram-negative bacteria,
and Borrelia burgdorferi. The three different domains (1, 1I, III) are marked above the alignment as is the ATP binding site and a possible DNA binding site. (B)
Conservation of the ATP binding site of DnaA. The relevant regions of 11 different DnaA proteins are aligned. The amino acid sequences are from Bacillus subtilis,
Buchnera aphidicola, Escherichia coli, Micrococcus luteus, Mycoplasma capricolum, Proteus mirabilis, Pseudomonas putida, Salmonella typhimurium, Serratia marcescens,
Streptomyces coelicolor [1,10,14-16] and Borrelia burgdorferi (this work). For further references see [1]. (C) Lack of conservation in a possible D N A binding site of
~o
113
found no O R F on pB22 with similarity to recF
(data not shown). The order dnaN-dnaA-gyrBgyrA is in agreement with that presented by others at the Fifth International Conference on Lyme
borreliosis [12]. This is in contrast to the usual
eubacterial arrangement dnaA-dnaN-recF-gyrB
(Fig. 1A-C). It would appear that, in addition to
a relative translocation of gidA [6], the B.
burgdorferi dnaA and dnaN genes have been inverted and recF has been either lost or transposed elsewhere during the evolution of the B.
burgdorferi linear chromosome. The proximity of
dnaA to gyrBA suggested the gene lay between
457 and 488 kb on the B. burgdorferi genetic map
[6] and this was confirmed by Southern hybridisations (data not shown).
As replication origins are found next to dnaA
in Gram-positive bacteria and Pseudomonads
(Fig. 1B,C), we searched the gyrB-dnaA and
dnaA-dnaN intergenic regions for DnaA boxes.
We were unable to detect clusters of DnaA boxes
characteristic of an oriC region [2] in the gyrBdnaA intergenic region while there was only one
DnaA box (AAATCCACA) in the dnaA-dnaN
intergenic region. The absence of any DnaA box
in the dnaA upstream region might suggest that
B. burgdorferi dna,4 is not autoregulated. As the
dnaA and dnaN genes have been inverted relative to rprnH and gyrB, another possible origin
location is between dnaN and rpmH. Initial analysis of 500 bp of sequence upstream of rpmH
have revealed only two DnaA boxes (data not
shown). Although a central location for the origin
of replication on a linear chromosome is appealing, we cannot preclude the possibility that an
origin is next to gidA (Fig. 1D).
Location of the origin of replication was also
hampered in Buchnera aphidicola, a Gram-negative intracellular symbiont of aphids with 28-30%
GC. As in B. burgdorferi there is no recF gene,
no DnaA boxes were detected in the dnaA region
[10], and the D N A binding region may be atypical
(Fig. 2C). It is not known whether the origin is
located elsewhere, as in E. coli and Proteus
mirabilis, or whether the DnaA boxes are atypical
in this organism [10].
A feature of the oriC region of E. coli is the
presence of some 20 G A T C sites. The state of
methylation of G A T C sites in the origin region is
an important factor in the regulation of chromosome replication in enteric bacteria such as E.
coli and P. mirabilis, which possess an adenine
methylation system, while organisms lacking the
Dam system do not appear to be regulated in this
manner [2]. Strain 212 (our unpublished results),
like the majority of Borrelia burgdorferi (sensu
lato) strains, does not possess an adenine methylation system [13]. It is therefore unlikely that B.
burgdorferi uses the adenine methylation system
for control of chromosome replication. Indeed,
we found only 5 G A T C sites in 4.7 kb of sequence in the B. burgdorferi dnaA region.
We conclude that B. burgdorferi is unique
amongst the eubacteria: it has a linear chromosome, the arrangement of genes in the dnaA
region is atypical, as is a possible D N A binding
motif in the C-terminus of the DnaA protein and
no arrangements of DnaA boxes characteristic of
a eubacterial ori region were found in the dnaAdnaN or dnaA-gyrB intergenic regions. We are
unable to conclude whether this is because the B.
burgdorferi origin is also atypical or because it is
located elsewhere, perhaps beside gidA, as in E.
coli (Fig. 1A). We are currently examining these
possibilities.
Acknowledgements
We are grateful to Barrie Davidson for helpful
discussions and to Guy Baranton for his continued interest in this work. This work was supported by grants from the Gould Foundation to
the Institut Pasteur.
References
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Microbiol. 5, 2589-2597.
2 Smith, D.W., Yee, T.W., Baird, C. and Krishnapillai, V.
(1991) Pseudomonad replication origins: a paradigm for
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3 Holz, A., Schaefer, C., Gille, H., Jueterbock, W.R. and
Messer, W. (1992) Mutations in the DnaA binding sites of
14
4
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6
7
8
9
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Physical map of the linear chromosome of the bacterium
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and localization of rDNA genes. J. Bacteriol. 174, 37663774.
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