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Microbiology
(1997), 143,3313-3328
Printed in Great Britain
The
Bacillus
subtilis
genome from
gerBC
(31
1
")
to
lid?
(334")
E.
Presecan,'
I.
Moszer,'
L.
Boursier,l
H.
Cruz Ramos,'
V.
de
la
Fuente,2
M.-F. Hullo,'n2
C.
Lelong,'
5.
Schleich,'
A.
Sekowska,'
B.
H.
Song,'t
G.
Villani,'
F.
Kunst,*
A.
Danchin' and
P.
Glaser'
Author for correspondence: Philippe Glaser. Tel:
+33
1
45
68
84
41.
Fax:
+33
1
45
68
89
48.
e-mail
:
pglaser@pasteur.fr
~~
1~
Unite de Regulation de
"Expression
Genetiquel,
and Unite de Biochirnie
Microbienne2.
lnstitut
As
part
of
the international project
to
sequence the
Bacillus
subtilis
genome,
the DNA region located between
gerBC
(31
1
")
and
licR
(334")
was assigned
to
the lnstitut Pasteur.
In
this paper, the cloning and sequencing
of
176
kb
Of
DNA
Pasteur,
2s'
rue
Docteur Roux, 75724
Cedex 15, France
du
Paris
and the analysis
of
the sequence
of
the entire
271
kb
region
(65%
of
the
B.
subtilis
chromosome) is described;
273
putative coding sequences were
identified. Although the complete genome sequences
of
seven other
organisms (five bacteria, one archaeon and the yeast
Saccharomyces
cerevisiae)
are available
in
public databases,
65
genes
from
this region
of
the
B.
subtilis
chromosome encode proteins
without
significant similarities
to
other
known
protein sequences. Among the
208
other genes,
115
have
paralogues
in
the currently
known
B.
subtilis
DNA sequences and the products
of
178
genes were
found
to
display similarities
to
protein sequences
from
public databases
for
which a
function
is
known.
Classification
of
these genes
shows a
high proportion
of
them
to
be involved
in
the adaptation
to
various
growth
conditions (non-essential cell wall constituents, catabolic and
bioenergetic pathways); a small
number
of
the genes are essential
or
encode
anabolic enzymes.
Keywords
:
genome sequencing,
Bacillus subtilis,
intergenic repeated element,
orthologues/paralogues, spore coat
INTRODUCTION
The systematic sequencing of the chromosome of the
Gram-positive bacterium
Bacillus subtilis
is being con-
ducted by an international consortium of European and
Japanese laboratories (Kunst
et al.,
1995;
Ogasawara
&
Yoshikawa,
1996).
By April
1997,95
%
of the sequence
was determined, and the analysis of several long regions
has been published (e.g. Ogasawara
et al.,
1994;
Albertini
et al.,
1995;
Mizuno
et al.,
1996;
Soldo
et al.,
1996;
Sorokin
et al.,
1996;
Wipat
etal.,
1996;
Yoshida
et
al.,
1996).
The completion of this genome sequence will
be the first complete genetic determination of a sporulat-
ing
Bacillus
sp. with a high level
of
competence and
recombination potential. As for the other completely
t
Present address:
Department
of
Biology, Teachers College, Kyung-
Pook
University, TAEGU,
702-701,
South Korea.
Abbreviation
:
PTS,
phosphotransferase system.
The EMBUGenBanWDDBJ accession numbers for the sequence reported in
this paper are
249782, 249792, 249884, 280355, 282987, 283337, 292952,
292953,292954
and
293767.
known genomes, the Gram-negative bacteria
Haemo-
philus influenzae
(Fleischmann
et al.,
1995)
and
Escheri-
chia coli
(O'Brien,
1997),
the mycoplasmas
Mycoplasma
genitalium
(Fraser
et al.,
1995)
and
Mycoplasma pneu-
moniae
(Himmelreich
et al.,
1996),
the cyanobacterium
Synechocystis
PCC
6803
(Kaneko
et al.,
1996),
the
archaeon
Methanococcus jannaschii
(Bult
et al.,
1996)
and the yeast
Saccharomyces cereuisiae
(Goffeau
et al.,
1996),
about one-third
of
the genes in the
B. subtilis
chromosome are 'unknown' genes (Moszer
et al.,
1996),
i.e. they encode proteins without similarities to proteins
with a known function. To elucidate their role, an
original international research project
of
systematic
functional analysis was set up, using the approach of
reverse genetics for which
B.
subtilis
is particularly
suited (Harwood
&
Wipat,
1996).
Accessibility of the
whole genetic information
of
an organism associated
with the knowledge
of
other genome sequences opens
new possibilities for sequence analysis, for example
classification
of
genes according to their codon usage
(Moszer
et al.,
1995).
It becomes possible
to
study the
distribution
of
nucleotide motifs along the chromosome
0002-1 790
0
1997
SGM
3313
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E. PRESECAN
and
OTHERS
333" 330" 326" 324"
and thereby get insights into regulatory processes
(HCnaut
et
al.,
1996), and to compare chromosomal
gene clusters conserved during evolution in different
organisms. These analyses may reveal functions for
unknown genes and new functions for already known
genes (Danchin, 1996). For this kind
of
analysis ap-
propriate computer tools will be required.
Several lines
of
investigation have been opened or
speeded up as a direct consequence of the recent
availability of
B.
subtilis
sequence data. An interesting
area
of
research in
B.
subtilis
is the sporulation pathway
(for a recent review see Stragier
&
Losick, 1996). The
knowledge
of
the whole genome sequence will con-
tribute to the discovery
of
new genes involved in this
bacterial cell differentiation process. In particular, sev-
eral important sporulation loci were discovered in the
described region (Perego
et
al.,
1994, 1996; Londoiio-
Vallejo
&
Stragier, 1995; Karow
et
al.,
1995).
This paper describes the features of the
B.
subtilis
chromosomal region initially assigned
to
our group
(Kunst
et
al.,
1995). This 271 kb region between
gerBC
(311") and
licR
(334") covers
6.5
YO
of the chromosome.
It includes a region
of
97 kb which has been sequenced
and published (Glaser
et
al.,
1993).
METHODS
Bacterial strains.The same isolate
of
B.
subtilis
strain 168
provided by
C.
Anagnostopoulos is used by all the participant
groups in the European
B.
subtilis
genome sequencing project.
This strain was the source for the chromosomal DNA used to
construct a lambda library (Kunst
&
Devine, 1991) and for all
the other cloning methods. Direct cloning by plasmid rescue
was performed in the
E.
coli
pcnB
strain TP611 (Glaser
et
af.,
1993). Strain P2392 was used for propagation
of
the lambda
library (Silhavy
et al.,
1984).
E.
coli
strains used for DNA
sequencing were XL-1 Blue (Bullock
et al.,
1987) for dsDNA
preparations and TG1 for ssDNA preparations (Gibson,
1984).
Media. Luria-Bertani (LB) medium was used for standard
cultures of
B.
subtilis
and
E.
coli
(Miller, 1972). 2YT medium
was used for M13 recombinant phage preparation. Lambda
stocks were prepared from LB plates
(15
g agarose
1-')
using
LB overlays containing
7
g agarose
1-l.
Antibiotics were added
to the following final concentrations, when necessary: for
E.
coli,
100
mg ampicillin
1-1
;
for
B.
subtilis,
5
mg chlorampheni-
col l-', 10 mg erythromycin
1-l.
Phages and plasmids. Gene libraries of
B.
subtilis
DNA were
constructed in Lambda FIX I1 (Stratagene) (Kunst
&
Devine,
1991). DNA sequences were determined from subclones in the
phage M13mp8 (Messing
&
Vieira, 1982) or in pUC18
(Yanisch-Perron
et
af.,
1985). The plasmid rescue method was
performed after an initial cloning step in pDIA5304 (Glaser
et
al.,
1993) or pDIA5305. pDIAS305 was obtained by insertion
of the pC194
cat
gene (Horinouchi
&
Weisblum, 1982) into
the unique NaeI restriction site
of
pMTL22 (Chambers
et
al.,
1988). The high-copy-number
B.
subtilis
replicon pMTL5OOE
(Swinfield
et
al.,
1990) was used for marker rescue in
B.
subtilis.
DNA
manipulations.
E.
coli
transformation was generally
performed as described by Chung
&
Miller (1988). Shotgun
libraries in M13mp8 or in pUC18 were used
to
transform
E.
coli
XL-1 Blue
as
described by Hanahan (1983). Recombinant
plasmids were transferred to
E.
coli
TP611 by the calcium
chloride transformation method (Sambrook
et
al.,
1989).
B.
subtilis
cells were transformed as described by Kunst
&
Rapoport (1995). Southern blots, and plaque and colony
transfers were performed as described by Sambrook
et
af.
(1989). Membranes were further hybridized with non-radio-
actively labelled probes (Boehringer DIG-dUTP labelling).
-
-
-
(Glaser
et
ul.,
1993)
111111111111111111111111111-111-1111-
5348
5363 5364 5365
narA glyC
spoIID
gerB
spoIZZD
316" 314" 312
140 160 180 200 220 240 260 280
NotI
,
NotI NotI
I
sp1
I
Sfi'
pfi1
I
I
I
5353 5354 5329
hnarA
pPP41 5355 5366 5367 5368 5369 5370 5371 5372
..................
*
...........
......
.....................................................
...
..........
.
............
.
...............
.
.......................
.
.....................
...
................
.
.......
.
.......
.
...................
.
....................................
.
................................
...
Fig.
1.
Cloning
of
the
licR-gerBC
region. The thin upper line represents the genetic map with divisions representing
chromosomal degrees. Relevant genetic markers are indicated. Underneath this line, the physical map is represented by a
thicker line. Restriction site positions are deduced from the sequence and are in good agreement with the physical
mapping
of
ltaya
&
Tanaka
(1991).
The thick dotted line represents the
97
kb region previously sequenced (Glaser
et
a/.,
1993).
The short dotted line represents the single
B.
subtilis
plasmid derivative. Numbers refer
to
the
f.
coli
pDlA plasmids
used
for
sequencing the newly sequenced region.
3314
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The
311"-334"
B.
subtilis
chromosomal region
Table
I.
Boundaries of the
17
cloned fragments
Plasmid/phage Beginning' End'
pDIA
5363
97 kb"
pDIA
5364
pDIA 5365
pDIA
5348
pDIA
5353
pDIA 5354
pDIA 5355
pDIA
5329
harA
pDIA 5366
pDIA 5367
pDIA 5368
pDIA 5369
pDIA 5370
pDIA 5371
pDIA
5372
pPP4lb
1
(XbaI)
6 543
101
448
(EcoRV)
107788 (NotI)
126 849 (XbaI)
142069 (KpnI)
151 210 (BglII)
156207 (PstI)
162280 (PstI)
167912
(NotI)
177261 (Sau3A)
194576 (HincII)
204509 (PstI)
214619 (SzuaI)
227 320 (SacII)
240 641 (BglII)
253 248 (KpnI)
263 330 (SacII)
7851 (EcoRV)
103 557
108016 (HindIII)
128 777 (EcoRV)
142330 (SspI)
152 949 (EcoRI)
157 5
18
(BglII)
166327
(SalI)
168 828 (EcoRV)
178 399 (HindIII)
195 141 (Sau3A)
205 685 (SacI)
216491 (KpnI)
228 235 (XbaI)
241 670 (ApaI)
255 853 (SacI)
263 953 (EcoRI)
271 513 (PstI)
*First
base
of
the
restriction site.
References: "Glaser
et
al.
(1993); bTrach
et
al.
(1988).
Chromosome walking experiments involved either a plasmid
rescue method, using integrative plasmids (Niaudet
et
al.,
1982), or a marker rescue method after chromosomal in-
tegration of a selective marker (Cm") (Glaser
et
al., 1993).
Fifteen walking steps (Fig.
1
and Table
1)
were performed as
follows.
A
single walking step was performed from the
ywaA
end of the 97 kb contig. The resulting plasmid (pDIA5363)
contains the whole lic operon (Tobisch
et
al., 1997). From
plasmid pPP41 containing the SPOOF gene (Trach
et
al., 1988),
four successive walking steps were performed in the direction
of the pta gene, leading to plasmids pDIA5353, pDIA5348,
pDIA5365 and pDIA5364. pDIA5364 has a 2.1 kb overlap with
the 97 kb contig. In the other direction a single walking step
was possible (pDIA5354). From InarA, only a single step
could be performed in the direction of the pPP41 insert
(pDIA5329). The region (only 1.6 kb long) between the
pDIA5354 and pDIA5329 inserts could not be cloned into
E.
coli
by
this method. It was therefore cloned in
B.
subtilis using
the pMTL5OOE plasmid (pDIA.5355). From the other end of
IZnarA,
five successive walking steps were performed, giving
plasmids pDIA5366-5370. Finally, two walking steps were
performed from the cloned
gerBC
gene, giving plasmids
pDIA5372 and pDIA5371. Plasmids pDIA5370 and pDIA5371
overlap by 2.6 kb.
Shotgun cloning and
DNA
sequencing.
Fifteen shotgun
sequencing experiments were performed independently. The
corresponding strategy has already been described (Moszer
et
al., 1991
;
Glaser
et
al., 1993).
Data handling and computer analysis.
DNA sequences were
compiled using the
XBAP
program of R. Staden (Dear
&
Staden, 1991). Sequences were analysed using the DNA Strider
1.1
software (Marck, 1988). The
BLAST
program (Altschul
et
al.,
1990) was used to search for similar sequences in the non-
redundant database
(5
April 1997) at the Institut Pasteur
computer centre. The
CLUSTAL
w
program was used for the
multiple alignment of the repeated DNA element (Higgins
et
al., 1992).
B.
subtilis sequences were handled using the
SubtiList
database (Moszer
et
al., 1995).
RESULTS
AND
DISCUSSION
Cloning and sequencing
of
the
gerBC-licR
region
We have previously described the cloning
of
a
97
kb
region from
ywaA
to
ywf0
as four lambda clones, three
E.
coli
plasmids and two
B.
subtilis
plasmids (Glaser
et
al.,
1993).
For the remaining part
of
the assigned region,
one fragment was cloned as a lambda insert isolated
using the cloned
narA
gene as a probe
(AnarA)
(Santana
et al.,
1994).
To
obtain the rest
of
the region, a plasmid
rescue walking strategy was used (Glaser
et al.,
1993).
Successive walking steps were performed, starting from
ends
of
the cloned fragments
of
the region as described
in Methods. The new regions were obtained as
15
E.
coli
plasmids, one lambda clone and one
B.
subtilis
plasmid
(Fig.
1
and Table
1).
The sequences
of
15
fragments were
determined after shotgun subcloning. Only the
1.6
kb
region
of
plasmid pDIA53.55 was sequenced
by
directed
subcloning and primer walking. The extensively studied
SPOOF
region (Trach
et al.,
1988)
was only partially
resequenced. The chromosomal region from
licR
to
gerBC
is
271518
bp long and overlaps the regions
sequenced in Dr
Y.
Fujita's laboratory in Hiroshima
(Japan) (Yoshida
et al.,
1996)
and in Dr
D.
Karamata's
laboratory in Lausanne (Switzerland)
(Soldo
et al.,
1996).
Identification of coding sequences and general
analysis
The putative coding sequences in the six reading frames
were identified according
to
previously described rules
(Glaser
et al.,
1993),
and
273
coding sequences were
predicted. They were
39
codons
(phrF)
to
1228
codons
(narG)
long with a mean length
of
875
bp (Fig.
2).
Compared
to
other systematic sequencing projects, a
relatively large number
of
small genes was predicted
:
19
genes were shorter than
100
codons.
Of
these,
10
encode
proteins with known function
(dltC, rpmE, atpE,
spoZZZD,
sbo)
or
similar
to
known
proteins
(ywbE,
ywhB, phrF, ywmG, ywql),
and for nine
(ywcE, ywdA,
ywhR, ywjc,
ywkF,
ywmE, ywq0, ywsA, ywtC)
no
counterpart could be identified in the databases.
A
majority
of
the genes are transcribed in the direction
of
the replication fork movement
(189
of
273, 69%),
but
their distribution is not uniform. Indeed, three regions
with a majority
of
genes in the opposite direction could
be defined (from
ywaA
to
ywbH,
from
ywhB
to
sbo,
and
from
ywqM
to
rsbR;
see Fig.
2).
Because
of
the limited
size
of
the region, the significance
of
this observation
could not be assessed and analysis awaits the complete
genome sequence.
Operon organization and non-coding regions
The
273
coding sequences were grouped into
147
putative transcription units by analysis
of
the intergenic
regions, mainly by the identification
of
the putative
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E. PRESECAN
and
OTHERS
a
WE4
Fig.
2.
Coding sequence organization in the
B.
subtilis
licR-gerBC
chromosomal region.
A
number was assigned to each
gene corresponding to
its
rank in the sequence. Genes are represented by arrows with different fill-in motifs, to
distinguish them according to their classification (see Table
2):
.,
cell
envelope and cellular processes; M,
intermediary meta bolism;
,
information pathways;
m,
other functions;
m,
miscellaneous;
m,
unknown genes similar to
other genes of unknown function;
0,
unknown genes without similarities. The direction of the replication complex
is
from left to right. Putative transcription terminators are indicated by
9.
3316
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The
311'-334'
B.
subtilis
chromosomal region
transcription terminators and by taking into account
their length. Eighty-two of these transcription units
were predicted to consist of a single gene. Fifty-seven
may be involved in translational coupling as assessed
from the relative positions of the ribosome-binding site
and the upstream gene stop codon. The longest operon
is the
sps
operon, which is comprised of
11
genes. In the
region, several operons of known function, including
the
atp
(Santana
et al.,
1994),
the
narGHJZ
(Cruz Ramos
et al.,
1995)
and the
qox
(Santana
et al.,
1992)
operons,
have a conserved organization in a broad range
of
bacteria. However, in the case of the urease operon, only
the structural genes
ureABC
were found, and genes
encoding nickel accessory proteins, required for nickel
incorporation into the enzyme, are missing (Cruz Ramos
et al.,
1997).
In the thermophilic
Bacillus
sp. strain TB-
90,
these genes
(ureEFGDH)
map downstream from the
ureC
gene in the
ure
operon (Maeda
et al.,
1994).
In
some cases, such as
ywbHG
in
B.
subtilis
and
yohJK
in
E.
coli,
the transcriptional organization of unknown
genes is also conserved. Three genes with unknown
function,
ywqH, ywqI
and
ywqJ,
are similar to three
other
B.
subtilis
genes,
yxiB,
yxiC
and
yxiD,
respectively,
and in both cases the three genes seem to be organized in
an operon.
As for other long segments of the
B.
subtilis
chromo-
some, the overall coding capacity of the region is high
(88
%
)
. Only
10
intergenic regions are longer than
500
bp. In seven cases they are located between divergent
coding sequences:
dltA
and
ywaB
(613
bp),
ywcB
and
ywcC
(957
bp),
upr
and
ywcZ
(530
bp),
ywfN
and
ywf0
(601
bp),
ywmA
and
ywmB
(529
bp),
ywqB
and
ywqC
(581
bp), rbsR and
ywsC
(784
bp), and in three cases
between genes in the same orientation
:
galT
and
qoxA
(835
bp),
mmr
and
thrZ
(595
bp),
ywmG
and
ureA
(1000
bp). Most of these regions may be regulatory.
Alternatively, they may contain short genes devoid of
typical ribosome-binding site sequences, or may be
transcribed as non-translated
RNA.
Systematic comparison with known nucleotide se-
quences using the intergenic regions as query sequences
revealed that two intergenic regions
(ywmE-narQ
and
ywbN-ywbO)
contained a repeated element. This se-
quence was found six times in the publicly available
B.
subtilis
sequences (Fig.
3).
This element, named Bs-rep
was described in
1994
as being repeated three times in
the sequences available at that time (Popham
&
Setlow,
1994).
The six repeats are located in less than one-third
of the chromosome (from position
3230
kb
to position
513
kb) and are all in the same orientation with respect
to the replication fork movement. Comparisons with
sequence databases show that this element is extremely
well conserved in two sequences from the phylogen-
etically closely related bacterium
:
Bacillus licheni-
formis
(Fig.
3).
Furthermore, two classes could be
distinguished on the basis
of
sequence similarities
:
ydbT-ydcA
and
ywmE-narQ
in one group and the
remaining six sequences including the two
B.
Zicheni-
formis
sequences in the other. The nature (IS element
or scar of a mobile element) and the possible role of this
repeated sequence are unknown.
Gene function analysis
At the beginning
of
the sequencing project, only eight
genes were known in this region of the
B.
subtilis
chromosome. Since then, independent of our sequencing
project,
25
other genes have been characterized. Most
are involved in two aspects
of
bacterial life
:
carbon and
nitrogen source utilization and sporulation.
sacXY
(Zukowski
et al.,
1990)
and
sacTPA
(Fouet
et al.,
1986,
1987)
encode proteins involved in saccharose metab-
olism. The
rbs
operon is involved in ribose utilization
(Woodson
&
Devine,
1994).
Two exocellular proteases
are encoded by the
epr
(Bruckner
et al.,
1990)
and
upr
(Sloma
et al.,
1991)
genes. The
als
operon
(alsRSD;
Renna
et
al.,
1993)
encodes proteins required for the
synthesis
of
acetoin as a by-product, and the
nrgAB
operon (Wray
et al.,
1994)
encodes an ammonium
permease (NrgA). Seven sporulation genes have been
analysed:
SPOOF
(Yoshikawa
et
al.,
1986),
spoZZQ
Fig.
3.
Alignment of Bs-rep repeats. Gene
names indicate the genes bracketing each of
the repeated elements.
BI,
B.
licheniformis.
8.
subtilis sequences were extracted from
the SubtiList database (Moszer et
a/.,
1995)
and those of
B.
lichenifomis
from EMBL
(accession nos
280222
and
D31955).
Strictly
conserved nucleotides found in at least four
sequences are represented as black boxes.
3317
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E.
PRESECAN
and
OTHERS
(a) Cots
(381
aa) CotG
(195
aa)
100 200 300
1
1
0
-1
-2 -2
-2
-2
-3
-3 -3
-3
-4
-4-4
-4
100 200 300 100
100
(b)
A A A A
B
B
B B
100 200
300
100
(c)
253
SSKSYSSSKSSKR SLKSSDDQSSKSGR
SSRSKSSSKSSKR SLKSSDYQSSKSGR
SSRSKSSSKSSKR SLKSSDYQSSK
SSKR SPRSSDYQSSR345
36
HKKSHRTHKKSRS HKKSYCSHKKSRS
HKKSFCSHKKSRS HKKSYCSHKKSRS
HKKSYRSHKKSRS YKKSYRSYKKSRS
YKKSCRSYKKSRS YKKSYCSHKKKSRS
YKKSCRTHKKSYRS
54
Consensus sequence: Consensus sequence:
KK
LK
D
SSRSySSSKSSKR
S
pRSSDyQSSKSGR
Fig.
4.
Sequence comparisons between the
cotB
and the
cotG
genes. (a) Kyte-Doolittle plots
of
the amino acid
hydropathy (Kyte
&
Doolittle,
1982).
(b) Distribution
of
the acidic (A) and basic
(B)
amino acids. (c) Alignments
of
the
13
or
14
amino acid repeated sequences
of
each protein and the corresponding consensus sequences.
(Londoiio-Vallejo
et
al.,
1997),
spollD (Lopez-Diaz
et
al.,
1986),
spolIlD (Kunkel
et
al.,
1989),
cotG
(Sacco
et
al.,
1995),
cotH
(Naclerio
et
al.,
1996)
and
cotB
(Donovan
et
al.,
1987).
Only the first
72
codons
of
cot&
which encodes a
protein located in the outer coat of the spore, were
known. The analysis
of
its complete sequence (Fig.
4)
revealed that this protein may have two domains. The
150
residues C-terminal domain is hydrophilic and has a
low complexity organization (two types of repeated unit
of
13
and
14
amino acids, respectively), similar
to
that
of
CotG (Sacco
et
al.
,
1995
;
Fig.
4).
Although the sequence
identity is low, both types
of
repeated unit are serine-
rich and highly charged. The similar organization may
reflect a conserved structural domain in both coat
proteins. It has been reported that the CotB protein is
absent from a
cotG
mutant strain (Sacco
et
al.,
1995).
It
is therefore possible that both proteins are part
of
a
complex or molecular assembly involving these repeated
structures.
Eight other genes with various activities from the
ZicR-gerBC
region have been described by other groups.
They determine or encode methylenomycin A resistance
(mmr;
Putzer
et
al.,
1992),
one
of
the two fructose-
bisphosphate aldolases (fbaA
;
Trach
et
al.,
1988
;
Mitchell
et
al.,
1992),
the minor threonine-tRNA syn-
thetase
(thrZ;
Putzer
et
al.,
1990),
the non-essential
RNA polymerase
6
subunit (rpoE; Lampe
et
a!.,
1988),
the transcription termination factor Rho
(rho
;
Quirk
et
al.,
1993),
the CTP synthase (ctrA; Trach
et
al.,
1988),
the thymidine kinase
(tdk;
Trach
et
al.,
1988)
and a
morphogene gene
(mbl;
Abhayawardhane
&
Stewart,
1995).
Fifty-four genes discovered during the systematic se-
quencing of this part
of
the chromosome were analysed
either in our laboratory, or in collaboration with other
groups. Several loci are involved in energy conversion
:
the atp operon (encoding the nine subunits
of
the F,F,
ATPase
;
Santana
et
al.,
1994),
the
qox
operon (encoding
the four subunits
of
the major aa,-type terminal oxidase
;
Santana et al.,
1992),
the narGHJI, narK and narA genes
(involved in the utilization
of
nitrate as an alternative
terminal electron acceptor; Cruz Ramos
et
al.,
1995;
Glaser
et
al.,
1995),
and
fnr
(a regulatory gene for
anaerobic respiration; Cruz Ramos
et
al.,
1995).
Five
loci are involved in carbon and nitrogen source utiliza-
tion: lichenan degradation (the
lic
operon; Tobisch
et
al.,
1997),
fatty acid utilization (acyl-CoA dehydro-
genase, acdA; V. de la Fuente and others, unpublished),
acetate production as a by-product (phosphotrans-
acetylase, pta
;
E.
Presecan and others, unpublished)
,
urea utilization (the
ure
operon; Cruz Ramos
et
al.,
1997),
and arginine, ornithine and proline degradation
(the
roc
operon
;
Calogero
et
al.,
1994).
The
dlt
and the
sps
operons are involved in the synthesis
of
cell wall
components
:
D-alanylation
of
cell wall lipoteichoic and
3318
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The
311"-334"
23.
subtilis
chromosomal region
Table
2.
Classification of the genes of the
B.
subtilis
licR-gerBC
region
Gene Product Total no.
BLAST
Genes encoding similar protein/similar gene Function/activity
of
the gene product
or
Gene
name size paralogues score products* similar protein
n0.t
(a4
1.
Cell envelope and cellular processes
1.1.
Cell wall components
dltE
252 9 24e-21
7.5e
-
18
dltD
392
0
1.6e
-
42
dltC
78
0
1.6e
-
3
1
dltB
39s
0
3.Se
-
159
dltA
503 10 2.3e
-
153
ywhK
45
1
1
4*4e
-
20
1.3e
-
137
ywhL
458
1
6.9e
-
20
7.4e- 138
murA
436
1
54e
-
139
mbl
313 2 94e- 182
YWqC
248
1
1.0e
-
56
YW9D
237
1
20e
-
57
YW9E
254
0
26e
-
62
ywsc
247
0
2.4e
-
184
ywtA
149
0
45e
-
84
ywtB
380
0
1.6e
-
124
ywtD
390
5
29e
-
21
9.8e -20
1.2.
Transpodbinding proteins and lipoproteins
licB
102
1
28e
-
37
licC
452 2 7.8e
-
2.53
60e
-
221
licA
110
1
49e-38
ywbF
399
0
2Se
-
108
ywcA
516 2 75e
-
174
YWCI
256
1
4Se -42
sacP
460
4
1.8e-
171
rocC
470 9 3.9e -215
YWfA
412 13 1.2e
-
26
ywhE
647 3 2Se
-
143
YWhQ
239 23 68e
-
33
narK
39s
1
7.4e
-
43
1.2e
-
66
ywiA
575 26 3-7e
-
136
ywkB
319
0
3.9e
-
37
ywoA
193
0
1.4e
-
25
nrgA
404
0
l.le
-
128
ywoD
452 7 24e
-
28
1.7e -36
ywoE
490
1
1.4e
-
79
93e
-
135
ywoG
396 9 27e
-
40
ywrA
178
1
6Se
-
16
ywrB
197
1
70e
-
16
ywrK
442
1
28e-79
rbsB
305
0
21e
-
96
rbsC
322
0
2.8e
-
117
rbsA
493 9 1.0e
-
149
rbsD
131
0
52e
-
39
ywtG
457 S.3e
-
143
ydfG, Escherichia coli
(sw
:
P39831)
trn2, Hyoscyamus niger
(sw
:
P50164)
dltD, Staphylococcus aureus
(embl
:
D86240)
dltC, Staphylococcus aureus
(embl
:
D86240)
dltB, Staphylococcus aureus
(embl
:
D86240)
dltA, Lactobacillus casei
(sw
:
P35854)
orf492, Methanosarcina mazeii
(embl
:
X84710)
ywhL, Bacillus subtilis
(embl
:
280360)
orf375, Methanosarcina mazeii
(embl
:
X84710)
ywhK, Bacillus subtilis
(embl
:
280360)
murZ, Synechocystis
sp. PCC 6803 (embl
:
D6400)
mbl, Bacillus cereus
(sw
:
P32444)
capA, Staphylococcus aureus
(sw
:
P39850)
capB, Staphylococcus aureus (sw
:
P39851)
capC, Staphylococcus
aureus
(sw
:
P39852)
capB, Bacillus anthracis
(sw
:
P19580)
capC, Bacillus anthracis
(sw
:
P19581)
capA, Bacillus anthracis
(sw
:
P19579)
spy, Escherichia coli
(embl
:
D86610)
nlpC, Haemophihs influenzae
(sw
:
P45296)
cdA, Bacillus stearothermophilus
(pir
:
B49898)
ywbA, Bacillus subtilis
(sw
:
P39584)
celB, Bacillus stearothermophilus
(pir
:
C49898)
celD, Bacillus stearothermophilus
(pir
:
E49898)
malA, Bacillus stearothermophilus
(pir
:
S43915)
dhlc, Xanthobacter autotrophicus
(pir
:
S52839)
nirC, Escherichia coli
(sw
:
P11097)
pts3, Klebsiella pneumoniae
(sw
:
P27219)
rocE, Bacillus subtilis
(sw
:
P39137)
PID
:
g1742740,
Escherichia coli
(embl
:
D90810)
mrcA, Synechocystis
sp. PCC 6803 (embl
:
D90913)
ecsA, Bacillus subtilis
(sw
:
P55339)
nasA, Bacillus subtilis
(sw
:
P42432)
MTCY04C12.22c,
Mycobacterium tuberculosis
ygaD, Bacillus subtilis
(embl: 282044)
mdcE, Klebsiella pneumoniae
(embl
:
U56096)
bcrC, Bacillus licheniformis
(sw
:
P42334)
amt, Mycobacterium tuberculosis
(sw
:
410968)
qacA, Staphylococcus aureus
(sw
:
P23215)
MTCY3G12.01,
Mycobacterium tuberculosis
fur4, Schizosaccharomyces pombe
(embl
:
X98696)
PID
:
gl773191,
Escherichia coli
(embl: U82664)
norA, Synechocystis
sp. PCC 6803 (embl
:
D90909)
chrA, Pseudomonas aeruginosa (sw
:
P14285)
(embl
:
281360)
(embl
:
279702)
chrA, Pseudomonas aeruginosa
(sw
:
P14285)
arsB, Staphylococcus xylosus
(sw
:
QOl2.55)
rbsB, Escherichia coli
(sw
:
P0292.5)
rbsC, Haemophilus influenzae (sw
:
P44736)
xylC, Escherichia coli (sw
:
P37388)
rbsD, Haemophilus
influenme
(sw
:
P44734)
yxbc, Bacillus subtilis
(sw
:
P46333)
D-Alanine esterification of lipoteichoic acid
D-Alanine esterification of lipoteichoic acid
D-Alanine esterification
of
lipoteichoic acid
D-Alanine esterification of lipoteichoic acid
D-Alanine esterification of lipoteichoic acid
Surface antigen
(carrier)
Surface antigen
UDP-N-acetylglucosamine 1-carboxyvinyl
Rod-shape-determining protein
Required for the biosynthesis of type
1
capsular
Required for the biosynthesis of type
1
capsular
Required for the biosynthesis of type
1
capsular
Synthesis of the polyglutamate capsule
Synthesis of the polyglutamate capsule
Synthesis of the polyglutamate capsule
Lipoprotein precursor
transferase
polysaccharide
polysaccharide
polysaccharide
B-Glucoside permease IIB component (PTS)
B-Glucoside permease IIC component (PTS)
8-Glucoside permease IIA component (PTS)
Maltose permease
Na+-dependent symport
Nitrite transporter
Sucrose permease IIBC component (PTS)
Arginine utilization
;
permease
Arabinose polymer transport
Penicillin-binding protein
ABC-type transporter
Nitrite extrusion protein
ABC-type transporter
Unknown transporter
Bacitracin transport permease protein
Ammonium transporter
Drug-resistance protein
Uracil permease
Quinolone-resistance protein
Essential
for
chromate resistance chromate
Essential for chromate resistance chromate
Arsenical pump membrane protein
Ribose transport operon
;
D-ribose-binding protein
Ribose transport operon
;
D-ribose-binding protein
Ribose transport operon
;
ATP-binding protein
Ribose transport operon
;
D-ribose-binding protein
Metabolite transporter
transport
transport
7
8
9
10
11
114
115
181
215
230
23
1
232
266
267
268
270
2
3
4
27
37
54
55
84
85
108
120
126
135
154
204
206
209
210
212
243
244
2.53
260
261
262
263
273
3319
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E. PRESECAN
and
OTHERS
Table
2
(cont.)
Gene Product Total no.
BLAST
Genes encoding similar protein/similar gene Function/activity
of
the gene product
or
Gene
name size paralogues score products" similar protein n0.t
(4
1.4.
Membrane bioenergetics
;
electron-transport chain
;
ATP
synthase
qoxA
qoxB
qoxc
qoxD
ywcc
narc
narH
nar]
322
649
204
124
249
1228
487
184
narl 223 0
atpl 126
0
atpB 244 0
atpE 70 0
atpF 170
0
atpH 181
0
atpA 502
0
atpG 287 0
atpD 473 0
atpC 132 0
narQ 262
0
ywrO 175 2
1.5.
Mobility and chemotaxis
flh0 270 3
flhP 268 2
ywcF 393 2
1.8.
Sporulation
spsA 2.56
1
spsB
472
0
spsc
389 2
spsD 289
0
spsE 373 0
spsF
239
0
spsc 222
0
spsH 117
0
spsl 246 3
1.7.
Cell division
SPSl
315 2
spsK 432
0
phrF
rapF
spoOF
spoIlR
spollD
rapB
spollQ
spolllD
rapD
cotc
cotH
cotB
39
381
124
224
343
377
283
93
354
195
362
380
1
8
8
0
1
8
0
0
8
0
0
0
2.2e
-
40
75e
-
247
91e-70
3.0e
-
09
1.4e
-
67
00
2.9e
-
229
3.3e
-
15
4.3e
-
11
1.8e
-
31
1.3e
-
62
27e
-
38
1.2e
-
128
20e
-
30
3.4e
-
67
1.5e
-
59
1.3e
-
300
1.4e
-
143
l5e
-
288
3.7e
-
60
1.2e-36
6.3e
-
71
7.8e
-
20
8.4e
-
45
4.5e
-
39
8-1e-69
3.4e
-
155
3.le
-
82
1.2e
-
46
7.9e
-
14
3.5e -05
48e
-
65
75e-115
25e
-
66
2.3e
-
08
61e--118
6.1e-62
5.3e
-
180
1.4e
-
137
1-3e
-
37
qox-2, Acetobacter aceti (sw
:
P50653)
cyoB, Escherichia coli (sw
:
P18401)
cox-3, Thermophilic bacterium PS-3 (sw
:
403439)
cyoD, Escberichia coli (sw
:
P18403)
frp,
Vibrio harueyi (embl
:
U08996)
narc, Escherichia coli (sw
:
P09152)
narH, Escherichia coli (sw
:
P11349)
narJ, Thiosphaera pantotropha (pir
:
S61308)
narJ, Escherichia coli (sw
:
P11351)
narV, Escherichia coli (sw
:
P19316)
MTCY04C12.21c, Mycobacterium tuberculosis
atpl, Bacillus megaterium (sw
:
P20598)
atpB, Bacillus megaterium (sw
:
P20600)
atpE,
Bacillus megaterium (sw
:
P20603)
atpF, Bacillus megaterium (sw
:
P20601)
atpH, Bacillus stearothermophilus (sw
:
P42008)
atpA, Bacillus megaterium (sw
:
P17674)
atpC, Bacillus megaterium (sw
:
P20602)
atpD, Bacillus caldotenax (sw
:
P41009)
atpC, Bacillus megaterium (sw
:
P12699)
fdhD, Wolinella succinogenes (sw
:
P28181)
yrkL, Bacillus subtilis (sw
:
P54439)
(embl: 281360)
flgG, Caulobacter crescentus (sw
:
406172)
flgG, Bacillus subtilis (sw
:
P23446)
rodA, Haemophilus influenzae (sw
:
P44468)
cgeD, Bacillus subtilis (sw
:
P42092)
MJ1066, Methanococcus jannaschii (pir
:
A64433)
MJ1065, Methanococcus jannaschii (pir
:
H64432)
MJ1063, Methanococcus jannaschii (pir
:
F64432)
MJ1062, Methanococcus jannaschii (pir
:
E64432)
flaR,
Caulobacter crescentus (embl
:
U27302)
rhsA, Sphingomonas
S88
(embl: U51197)
No significant similarities
No significant similarities
rfbB,
Leptospira interrogans (embl
:
U61226)
rfbD2, Synechocystis sp. PCC 6803
phr, Bacillus
subtilis
(embl: D50453)
rapB, Bacillus subtilis (embl
:
281356)
SPOOF,
Bacillus thuringiensis (sw
:
P52942)
No significant similarities
SPZD, Bacillus amyloliquefaciens (sw
:
P132.51)
rapA, Bacillus subtilis
(sw
:
400828)
No significant similarities
No significant similarities
rapC, Bacillus subtilis (embl
:
D50453)
No significant similarities
No significant similarities
No significant similarities
(embl
:
D90899)
2.
Intermediary metabolism
2.1.
Metabolism
of
carbohydrates, sugar and amino sugars
licH 442 2 1.4e
-
147 celF, Escherichia cola (sw
:
Pi741
1)
@PA 286
0
3.0e-20 lgtA, Rhizobium leguminosarum (embl
:
X94963)
ywbA
444
2 8.9e-2.54 licB, Bacillus subtilis (sw :P46317)
ywbC 126
1
28e
-
10 lguL, Lycopersicon esculentum (tomato)
(sw
:
442891)
Quinol oxidase polypeptide
I1
precursor
Quinol oxidase polypeptide
I
Quinol oxidase polypeptide
111
Quinol oxidase polypeptide
IV
NADPH
:
flavin oxidoreductase
Nitrate reductase
a
subunit
Nitrate reductase
/3
subunit
Nitrate reductase protein J
Nitrate reductase subunit
y
ATP synthase
I
subunit
ATP synthase a subunit
ATP synthase c subunit
ATP synthase b subunit
ATP synthase
S
subunit
ATP synthase
a
subunit
ATP synthase
y
subunit
ATP synthase
B
subunit
ATP synthase
E
subunit
Unknown
Hypothetical NAD(P)H oxidoreductase
Unknown
;
similar to flagellar hook-basal body
Unknown
;
similar to flagellar hook-basal body
Rod-shape-determining protein
Spore-coat polysaccharide
Spore-coat polysaccharide
Spore-coat polysaccharide
Spore-coat polysaccharide
Spore-coat polysaccharide
Spore-coat polysaccharide
Spore-coat polysaccharide
Spore-coat polysaccharide
Spore-coat polysaccharide (glucose-1-phosphate
Spore-coat polysaccharide (UDP-N-
Spore-coat polysaccharide (dTDP-C-deoxy-~-
Phosphatase regulator peptide
Response regulator aspartate phosphatase
Sporulation
Sporulation
Sporulation
Response regulator aspartate phosphatase
Sporulation
Sporulation
Response regulator aspartate phosphatase
Spore-coat protein
G
Spore-coat protein
H
Spore-coat protein
B
thy midyly ltransferase)
acetylglucosamine epimerase)
mannose-deh ydrogenase)
6-Phospho-B-glucosidase
Galactosyltransferase
Cellobiose permease IIC component (PTS)
Lactoylglutathione lyase
43
44
45
46
49
130
131
132
133
170
171
172
173
174
175
176
177
178
186
257
216
217
48
69
70
71
72
73
74
75
76
77
78
79
112
113
145
161
182
188
202
214
218
249
250
25
1
5
18
22
24
3320
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IP: 45.45.132.90
On: Thu, 25 Aug 2016 08:45:06
The
31 lo-334'
B.
subtilis
chromosomal
region
Table
2
(cont.)
Gene Product Total no.
BLAST
Genes encoding similar protein/similar gene Function/activity
of
the gene product
or
Gene
name sue paralogues score products* similar protein
n0.t
(4
390
513
480
255
259
323
62
705
285
186
429
321
582
149
189
440
571
255
293
0
0
2
19
0
0
0
0
1
0
1
0
1
0
0
0
3
0
0
6.0e
-
122
20e
-
122
65e
-
177
1.6e
-
123
3.7e
-
69
73e
-
36
1.le-28
2.7e
-
97
3.6e
-
12
5.le
-
30
2.7e
-
111
998e
-
72
3.9e
-
128
73e-112
5.8e
-
183
2.0e
-
192
74e-44
1.0e
-
12
93e -52
49e
-
137
6.0e
-
203
2.0e
-
66
3.7e
-
69
gal-1, Lactobacillus helveticus
(sw
:
Q00052)
gal-7, Butyrivibrio fibrisolvens
(sw
:
P45981)
surA, Bacillus stearothermophilus
(embl
:
U34872)
scrB, Salmonella typhimurium
(sw
:
P37075)
dhg-2, Bacillus megaterium
(sw
:
P39483)
MTCY09F9.36,
Mycobacterium tuberculosis
pbbB,
Alcaligenes eutropbus
(sw
:
P14697)
pta, Clostridium acetobutylicum
(embl
:
U38234)
dmpl, Pseudomonas putida
(pir
:
S24422)
MJ0092,
Methanococcus jannaschii
(pir
:
D64311)
f.aB,
Bacillus subtilis
(sw
:
P42420)
M 50960,
Methanococcus jannaschii
murZ, Synechocystis
sp. PCC 6803 (embl
:
D6400)
(embl
:
284498)
(embl: H64419)
Galactokinase
Galactose-1-phosphate uridyltransferase
Sucrose-6-phosphate hydrolase
41
42
56
Glucose dehydrogenase
88
Short-chain alcohol dehydrogenase family 92
Acetoacetyl-CoA reductase
Phosphate acetyltransferase 94
4-Oxalocrotonate tautomerase 105
Fumarate reductase 140
Fructose-l,6-diphosphate
aldolase 146
Transaldolase 147
signature
UDP-N-acetylglucosamine
1-
Glycerol metabolism
Malate oxidoreductase
carboxyvinyltransferase
148
149
153
glpX,
Escherichia coli
(sw
:
P28860)
maoX, Saccharomyces cerevisiae
(sw
:
P36013)
MTCY3G 12.02c,
Mycobacterium tuberculosis
rpiB, Escherichia coli
(sw
:
P37351)
dhbB, Bacillus subtilis
(sw
:
P45747)
yecD, Escherichia coli
(sw
:
P37347)
slr1299,
Synechocystis
sp. PCC 6803
als,
Lactococcus lactis
(embl
:
L16975)
aldC, Enterobacter
aerogenes
(sw
:
P05361)
rbsK, Haemophilus
influenme
(sw
:
P44331)
(embl
:
279702)
(embl
:
D90916)
Ribose 5-phosphate isomerase B
Isochorismatase
166
208
UDPglucose dehydrogenase 233
Acetolactate synthase (pH 6)
Acetolactate decarboxylase
Ribose transport operon
;
ribokinase
255
256
264
2.2.
Metabolism
of
amino acids and related molecules
ywaA
ywaD
epr
qr
yweB
rocA
rocB
YWfG
ywbF
ywhG
glYA
ureA
ureB
ureC
nrgB
ywrD
363
455
645
806
425
515
566
399
276
290
415
105
124
569
116
525
0
0
3
6
1
8
1
4
0
2
0
0
0
0
0
1
1.5e-111
53e- 15
1.7e
-
91
26e
-
57
25e
-
148
7-3e-231
1.9e
-
166
5.2e
-
259
6.1e-66
3.9e
-
97
3.0e
-
96
2.4e
-
78
1.5e
-
220
8.0e
-
46
24e-33
8.0e
-
272
3.8e -28
1.2e
-
87
7.5e
-
114
ilvE, Mycobacterium tuberculosis
(sw
:
P10399)
ape3, Saccharomyces cerevisiae
(sw
:
P37302)
aprM, Bacillus
sp. strain B18' (pir
:
A48373)
subT,
Bacillus amyloliquefaciens
(sw
:
P00782)
dhe-2, Clostridium dificile
(sw
:
P27346)
ypcA, Bacillus subtilis
(sw
:
PSO73S)
putA, Synechocystis
sp. PCC 6803 (embl
:
D90913)
ycgN, Bacillus subtilis
(embl
:
D50453)
yqjN,
Bacillus subtilis
(sw
:
P54551)
aspC, Synechocystis
sp. PCC 6803 (embl
:
D90913)
MJ0313,
Methanococcus jannaschii
(pir
:
B64339)
MJ0309,
Methanococcus jannaschii
(pir
:
F64338)
glyA, Hyphomicrobium methylovorum
ureA, Bacillus pasteurii
(sw
:
P41022)
ureB,
Bacillus pasteurii
(sw
:
P41021)
ureC,
Bacillus pasteurii
(sw
:
P41020)
glnB, Klebsiella pneumoniae
(sw
:
P11671)
ggt,
Escherichia coli
(sw
:
P18956)
MTCY369.18,
M ycobacterium tuberculosis
(pir
:
S30382)
(embl
:
280226)
Branched-chain amino acid aminotransferase 6
Proteinase precursor 14
Minor extracellular serine protease 21
Minor extracellular serine protease 51
Glutamate dehydrogenase (NAD) 81
1-Pyrroline-5-carboxylate
dehydrogenase 82
Unknown
Aspartate transaminase
Spermidine synthase
Agmatinase
Serine
hydroxymethyltransferase
83
91
109
110
168
Urease
y
subunit
Urease subunit
Xrease
CI
subunit
Nitrogen-regulated PI1 protein
y-Glutamyltranspeptidase
precursor
191
192
193
205
246
2.3.
Metabolism
of
nucleotides and nucleic acids
ctrA
535
0
41e-243
tdk
195
0
1.3e-54
UPP
209
0
45e
-
125
2.4.
Metabolism
of
lipids
ywdH
457
8
1.4e
-
144
MJ1174,
Methanococcus jannaschii
(pir
:
E64446)
kitH, Mycoplasma genitalium
(sw
:
P47280)
upp, Bacillus caldolyticus
(embl
:
X99545)
CTP synthase
Thymidine kinase
Uracil
phosphoribosyltransferase
143
152
169
sIr0091,
Synechocystis
sp.
PCC 6803
cis, Escherichia coli
(sw
:
P31071)
ywnE, Bacillus subtilis
(embl
:
Y08559)
ywnE, Bacillus subtilis
(embl
:
YOSSSS)
cls, Escherichia coli
(sw
:
P31071)
acdS, Clostridium acetobutylicum
(sw
:
P52042)
cls, Escherichia coli
(sw
:
P31071)
ywiE, Bacillus subtilis
(sw
:
P45860)
(embl
:
D64004)
Aldehyde dehydrogenase 64
ywiE
500
2
48e
-
120
WE
398 2 26e
-
55
25e
-
78
8.3e
-
45
acdA
379 3 2.3e
-
157
ywnE
482 2 1.2e-61
2.9e
-
120
Cardiolipin synthase
Cardiolipin synthase
134
139
Acyl-CoA dehydrogenase
Cardiolipin synthetase
141
198
3321
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E.
PRESECAN
and
OTHERS
Table
2
(cont.)
Gene Product Total no.
BLAST
Genes encoding similar proteinhimilar gene Function/activity
of
the gene product
or
Gene
name size paralogues score products* similar protein n0.t
(a4
YWPB
132 0 1.3e -43 fabZ, Neisseria meningitidis (embl
:
U79481)
2.5.
Metabolism of coenzymes and prosthetic groups
ywaB 311
0
l.le -21 menA, Haemophilus influenzae
(sw
:
P44739)
YWbl 272
0
20e
-
27
41e-74
ywbK 222
1
1.2e-31
l.le -53
ywiB 174
1
5.Oe
-
09
1.2e
-
34
ywkE 288
0
1.2e
-
26
narA
341
0
46e
-
67
thi6,
Saccharomyces cerevisiae
(sw
:
P41835)
HI0417, Haemophilus influenzae (pir
:
A64151)
thi4, Schizosaccharomyces pombe
(sw
:
P40386)
HI0415, Haemophilus
influenzae
(pir
:
A64152)
dyrA, Thermotoga maritima (pir
:
JC4116)
yyaP, Bacillus subtilis
(sw
:
P37508)
hemK, Escherichia coli (pir
:
183570)
moaA, Arthrobacter nicotinovorans (pir
:
139637)
3. Information pathways
3.1.
DNA replication
YWPH
113
1
3-8e-45 ssb, Bacillus subtilis (sw
:
P37455)
3.2.
DNA
restriction/modification
and repair
ung 225
0
2.4e -84 ung, Escherichia coli
(sw
:
P12295)
YwiD 320
0
8.4e
-
35
uvl
,
Neurospora crassa (pir
:
S55262)
3.5.
RNA synthesis
;
initiation
;
regulation
;
elongation
;
termination
IicR
ywaE
sacY
sacX
ywbl
ywcc
sacT
YWfK
ywhA
ywhH
fnr
rpoE
rho
ywnD
ywoH
YWPl
YwqA
YwqM
ywrc
alsR
rbsR
ywtF
641
171
280
459
301
223
276
299
139
157
238
173
427
257
137
258
922
293
158
302
326
322
2
13
2
4
15
0
2
15
11
1
0
0
0
8
9
3
1
15
4
15
11
2
23e
-
48
1-le-05
9.2e
-
107
1-0e
-
98
4.2e
-
172
1.2e-47
3.3e
-
07
6.5e
-
120
2.2e
-
97
62e
-
44
1.8e
-
09
3.9e
-
07
22e
-
16
1.k
-
170
1.7e
-
60
65e
-
07
1.2e-42
1-6e
-
66
1.4e
-
156
34e-38
8-3e
-
23
1-3e
-
56
3-le-58
61e-50
mtlR, Bacillus stearothermophilus (embl
:
U18943)
slyA, Salmonella typhimurium
(sw
:
P40676)
surT,
Bacillus stearothermophilus (embl
:
U34873)
sacT, Bacillus subtilis
(sw
:
P26212)
sacP, Bacillus subtilis
(sw
:
P05306)
cynR, Escherichia coli
(sw
:
P27111)
arpA, Streptomyces griseus (embl: D49782)
SUTT,
Bacillus stearothermophilus (embl
:
U34873)
sacY, Bacillus subtilis
(sw
:
P15401)
rbcR, Synechocystis sp. PCC 6803 (embl
:
D64004)
ysmB, Bacillus subtilis (embl: 275208)
orfl,
Pseudomonas aeruginosa (embl
:
U49666)
ntcA, Synechococcus sp. PCC 7942
(sw
:
P29283)
No significant similarities
rho,
Escherichia coli
(sw
:
P03002)
tipA, Streptornyces lividans
(sw
:
P32184)
slyA, Escherichia coli
(sw
:
P55740)
agaR, Escherichia coli
(sw
:
P42902)
motl, Saccharomyces cerevisiae
(sw
:
P32333)
~111366, Synechocystis sp. PCC 6803
gltR, Bacillus subtilis (embl: U79494)
MJO151, Methanococcus jannaschii (pir
:
H64318)
xapR, Escherichia coli
(sw
:
P23841)
scrR, Pediococcus pentosaceus
(sw
:
P43472)
lytR, Bacillus subtilis
(sw
:
402115)
(embl
:
D90916)
(3R)-Hydroxymyristoyl-(acyl
carrier protein)
dehydratase saturated fatty acid biosynthesis
Menaquinone biosynthesis 1,4-dihydroxy-2-
Thiamin biosynthetic bifunctional enzyme
naphthoate octaprenyltransferase
Thiamin-phosphate pyrophosphorylase
Dihydrofolate reductase
Protoporphyrinogen oxidase
Nitrate assimilation and anaerobic respiration
ssDNA-binding protein
Uracil-DNA glycosylase
UV-endonuclease
Transcriptional regulator
of
the lic operon
Transcriptional regulator MarR family
Transcriptional regulator BglG family
Transcriptional regulator of sacY
Transcriptional activator LysR family
Transcriptional regulator (A factor receptor)
Transcriptional regulator BglG family
Transcriptional regulator LysR family
Transcriptional regulator MarR family
Transcriptional regulator
Anaerobic regulatory protein
RNA polymerase
6
subunit
Transcription termination factor
Transcriptional activator MerR family
Transcriptional regulator MarR family
Transcriptional regulator DeoR family
Probable helicase of the Snf2/Rad54 family
Transcriptional regulator LysR family
Transcriptional regulator AsnC family
Transcriptional regulator LysR family
Transcription regulator Lac1 family
Attenuator for itself and lytABC operon
3.7.
Protein synthesis
;
ribosomal proteins
;
aminoacyl-tRNA synthetases
;
initiation
;
elongation
;
termination
tyrz 413
1
3.9e
-
135 tyrS, Haemophilus influenzae
(sw
:
P43836) Minor tyrosyl-tRNA synthetase
thrZ
638
1
1.8e-241 thrS, Bacillus subtilis (sw
:
P18255) Minor threonyl-tRNA synthetase
argS 556
0
6.7e
-
161 argS, Brevibacterium lactofermentum (sw
:
P41253) Arginyl-tRNA synthetase
rpmE 66 1 54e-26 rpmE, Mycobacterium leprae
(sw
:
P45834) Ribosomal protein L31
PrfA 356
1
3.7e
-
130 prf, Mycoplasma capricolum (embl
:
50100) Peptide-chain-releasing factor
4.
Other functions
4.1.
Adaptation to atypical conditions
YWPC 130
0
1.4e-48 mscL, Escherichia coli
(sw
:
P23867) Large conductance mechanosensitivity channel
4.3.
Antibiotic production
sbo 43
0
No significant similarities Antibiotic peptide, subtilosin A
4.4.
Drug/analogue sensitivity
mmr
466 3 28e- 151 mmr, Streptomyces coelicolor
(sw
:
P11545) Methylenomycin A resistance protein
219
12
31
32
136
158
187
225
63
138
1
16
19
20
30
39
53
95
104
111
127
142
150
197
213
226
228
240
245
254
265
272
15
103
125
151
157
220
123
102
3322
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The
311'-334'
B.
subtilis
chromosomal region
Table
2
(cont.)
~~ ~ ~
Gene Product Total no.
BLAST
Genes encoding similar proteinhimilar gene Function/activity
of
the gene product
or
Gene
name size paralogues score products,
similar
protein n0.t
(a4
ywnH
163
0
5.
Miscellaneous
ywcH
333 3
ywdL
181
0
ywlE
150
0
Genes without known function
ywaC
ywbD
ywbG
ywbH
ywbL
ywbM
ywbN
ywcD
ywdB
ywdF
ywdK
yweA
YW~J
YWFi
YWfO
YwgB
ywhC
ywhN
ywiA
ywiC
YWkD
ywlC
ywlD
ywmC
ywmD
ywmH
ywnG
ywoF
YWPD
YWPJ
ywnc
YWH
YWl
YW~J
YWqL
YWN
ywtE
210
396
241
128
481
385
416
127
271
268
460
113
154
296
433
156
219
426
448
239
128
346
185
227
224
62
127
172
468
278
285
140
86
602
238
181
286
1
0
3
3
0
0
0
0
1
0
0
0
0
2
0
0
0
0
0
0
2
0
0
1
1
0
1
1
0
0
5
1
1
3
0
0
7
l.5e
-
15
94e
-
56
1*4e
-
09
63e-61
1.2e
-
05
1.2e
-
12
9.7e
-
15
1.8e-35
77e-51
24e
-
16
46e
-
09
3.9e
-
67
5Oe
-
40
65e
-
06
1.0e
-
70
1.0e
-
14
7.5e
-
41
3.le
-
19
79e
-
17
1.4e
-
14
1.7e
-
39
2.0e
-
43
24e
-
09
7.5e
-
12
8.8e
-
14
23e-47
3.3e
-
53
1.4e
-
88
6.8e
-
22
1.6e
-
64
1.4e
-
64
41e-07
23e
-
30
43e
-
29
53e
-
06
7.Se
-
29
9.8e
-
15
3.7e
-
34
3.6e
-
30
3.3e
-
26
21e-22
00019
94e
-
10
6.2e
-
143
5.8e
-
09
8.4e
-
19
61e-10
22e
-
66
7*3e
-
31
97e
-
33
2.6e
-
27
9.8e
-
22
bar, Streptomyces coelicolor
(pir
:
JH0246)
~111647,
Synechocystis
sp. PCC 6803
(embl
:
D90903)
lxa-I, Photobacterium leiognati
(sw
:
F99140)
yhbW, Escherichia coli
(sw
:
P45529)
bncl
,
Brassica
napus
(sw
:
P33523)
wafE, Escherichia coli
(embl: U38473)
Gene in
mleR
region
of
Lactococcus factis
PID
:
g1787201,
Escherichia coli
(embl
:
AE000198)
yohK, Escherichia coli
(sw
:
P33373)
yoh
J,
Escherichia coli
(sw
:
P33372)
slr0964,
Synechocystis
sp. PCC 6803
PID
:
g17872.54,
Escherichia coli
(embl
:
AE000203)
PID
:
g1787255,
Escherichia coli
(embl
:
AE000203)
rfbl,
Escherichia coli
(embl
:
D90865)
Y416,
Haemophilus
influenzae
(sw
:
P44697)
HI1696,
Haemophilus influenzae
(pir
:
D64175)
PID
:
g1786723
Escherichia coli
(embl
:
AE000157)
ygdD, Haemophilus influenzae
(sw
:
P45019)
orfl,
upstream
gbsAB
operon
Bacillus subtilis
yicL, Escherichia coli
(sw
:
P31437)
MG461,
Mycoplasma genitahm
(sw
:
P39651)
orfl56, clpA
region
Lactococcus factis
MJ0611,
Methanococcus jannaschii
(pir
:
C64376)
cpn-60, Synechocystis
sp. PCC 6803
MTCY210.10
Mycobacterium tuberculosis
HI1626,
Haemophilus influenzae
(sw
:
P44278)
yaeR, Escherichia coli
(sw
:
P52096)
sua5, Saccharomyces cerevisiae
(sw
:
P32.579)
ywlD, Escherichia coli
(embl
:
D90826)
ywmD, Bacillus subtilis
(embl
:
281356)
ywmC, Bacillus subtilis
(embl
:
281356)
yjbJ, Escherichia coli
(sw
:
P32691)
ywnG, Bacillus subtilis
(embl: Y08559)
ywnC, Bacillus subtilis
(embl
:
Y08559)
MJ1396,
Methanococcus jannaschii
(pir
:
C64474)
yehU, Escherichia coli
(embl: D90868)
lyts, Staphylococcus
aureus
(embl
:
L42945)
ycsE, Bacillus subtilis
(sw
:
P42962)
yxeH, Bacillus subtilis
(sw
:
P54947)
yidA, Escherichia coli
(sw
:
P09997)
ybhA, Escherichia coli
(sw
:
P21829)
yxiB, Bacillus subtilis
(sw
:
P42294)
yxiC, Bacillus subtilis
(sw
:
P42295)
yqcG, Bacillus subtilis
(sw
:
P45942)
yxiD, Bacillus subtilis
(sw
:
P42296)
yjaF, Escherichia coli
(sw
:
P32679)
MJ1083,
Methanococcus jannaschii
(pir
:
B64435)
ywpJ, Bacillus subtilis
(embl
:
283337)
ycsE, Bacillus subtilis
(sw
:
P42962)
yxeH, Bacillus subtilis
(sw
:
P54947)
yidA, Escherichia coli
(sw
:
P09997)
ybhA, Escherichia coli
(sw
:
P21829)
(embl
:
M26949)
(embl
:
U47861)
(em bl
:
L36907)
(embl
:
D64001)
(embl
:
284395)
Phosphinotricine N-acetyltransferase
Alkanal monooxygenase a-chain, bacterial
luciferase
Seed storage protein
Acid phosphatase
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
201
50
68
165
13
25
28
29
33
34
35
40
58
62
66
67
80
97
99
101
106
117
122
128
156
163
164
183
184
190
196
200
211
221
227
235
236
237
2.39
241
271
*Accession numbers are for SWISS-PROT
(sw),
Protein Information Resource (pir) or
EMBL
(embl).
tNumbers refer to Fig.
2.
3323
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E. PRESECAN
and
OTHERS
teichoic acids
(dlt
operon; Perego
et
al.,
1995)
and
synthesis of a spore-coat polysaccharide
(sps
operon
;
M. F. Hullo and others, unpublished). Two sporulation
loci have also been characterized:
spollR
(Karow
et
al.,
1995
;
Londoiio-Vallejo
&
Stragier,
1995)
and
rapB
(Perego
et
al.,
1996).
Finally, the uracil phosphoribosyl-
transferase involved in the pyrimidine salvage pathway
(upp;
Martinussen
et
al.,
1995)
and a minor alternative
tyrosine-tRNA synthetase
(tyrZ
;
Glaser
et
a[.,
1991)
of
this
B.
subtilis
chromosomal region have been studied.
In the latest version
of
the
B.
subtilis
genetic map, there
are
96
markers located between
gerBC
and
licR (celR)
(Biaudet
et
al.,
1996).
The relative positions of the
markers determined by genetic methods are in good
agreement with the position of the corresponding genes
on the chromosome. Only eight genetic markers could
not be unambiguously associated to a gene.
furC
and
furE
(resistance to 5-fluorouracil) may both correspond
to
upp.
Alternatively, one of these loci and
axpB
(resistance to azopyrimidines) may lie in
ywoE,
which
encodes a protein similar
to
uracil permeases. For the
other five loci,
flaC,
outF,
divll, bac
and
gneA,
no
obvious gene could be allotted.
The predicted gene products of the
licR-gerBC
region
were compared with all known protein sequences
available in public sequence libraries
(5
April
1997).
The
273
genes of the region were thereby classified into five
groups: (i) genes with known function (non-y gene)
encoding proteins similar to known proteins
(94
genes)
;
(ii) orphan genes (for which no similar sequence has
been found) with known function
(9
genes); (iii) genes
with unknown function but encoding proteins similar to
proteins with known function
(75
genes)
;
(iv) unknown
genes encoding proteins similar
to
proteins with un-
known function
(39
genes) and
(v)
orphan genes with
unknown function
(56
genes). Although this classifica-
tion is subjective, the proportion of genes in classes (iv)
and
(v)
is similar to that found for other eubacterial
genomes
(35
‘7’0)
using the
BLAST
program. Although the
complete genomes of five bacteria, one archaeon and the
yeast
S.
cerevisiae
are known, no phylogenetically
related genes could be detected
by
this method for more
than
20%
of the genes in this region of the
B.
subtilis
chromosome. In
10
cases
(spsA,
rocB,
phrF, ywmC,
ywmD, ywnC, ywnG, ywqH, ywql, ywq]),
counterparts
were detected only in
B.
subtilis
sequences (paralogue
orphan genes). These genes, together with the class (ii)
and
(v)
genes, may constitute a kind of signature for
B.
subtilis
and related species. It is however not possible
to
know whether the absence of detectable similarities
reflects a recent origin
of
these genes or whether the
relatedness to other proteins has been obscured by
evolution.
The
217
genes in classes (i), (ii), (iii) and (iv) were further
classified according to Moszer
et
al.
(1996)
and are
presented in Table
2
(see also the arrow fill-in motifs in
Fig.
2).
No member was found for
10
categories
[numbers in parentheses represent classes and subclasses
according to Moszer
et
al.
(1996)l:
sensors
(1.3),
Table
3.
Orthologues
of
the
B.
subtilis
genes
from
the
/id?-gerBC
reg ion
Genome Total number Number
of
of
genes orthologues*
Escherichia coli
4285 158
Haemophilus influenzae
1743 92
Synechocystis
PCC
6803 3168 94
Methanococcus jannaschii
1682 59
Saccharomyces cerevisiae
6200 54
My coplasma genitalium
470 27
*
Number
of
genes in the
licR-gerBC
region having an orthologue
in each genome.
secretion
(1.6),
germination
(1.9),
competence
(1. lo),
metabolism
of
phosphate
(2.6)
and of sulphur
(2.7),
DNA recombination
(3.3),
RNA modification
(3.6),
protein modification
(3.8)
and folding
(3.9).
The dis-
tribution of genes into the remaining classes is consistent
with what has been described for the whole genome
(Moszer
et
al.,
1996).
However, there are several striking
features. About half of the genes in the
licR-gerBC
region belong to the ‘cell envelope and cellular pro-
cesses
’
class
of
genes, with a large proportion in the
‘
cell
wall components’ subclass (the
sps
operon in the
sporulation subclass is also involved in the synthesis of
wall components). The region contains very few genes
encoding anabolic enzymes (in the
‘
intermediary metab-
olism’ class)
:
glyA, narA
and the
thiC
operon. The
‘
information pathways
’
class contains mainly regu-
latory genes and very few essential genes. Three amino-
acyl-tRNA synthetase genes are found in this region, but
only
argS
is essential (A. Sekowska and others, un-
published),
thrZ
and
tyrZ
encode minor synthetases of
unknown physiological function (Glaser
et
al.,
1991
;
Putzer
et
al.,
1990).
Other than
argS,
only
rpmE
and
rho,
encoding the ribosomal protein
L31
and a transcription
terminator factor, respectively, are important genes of
this class.
Orthologues and paralogues in the
gerBC-licR
region
We have searched for
B.
subtilis
sequences similar
to
sequences present in six known genomes (orthologues).
The number
of
genes
of
the
B.
subtilis licR-gerBC
region
having an orthologue in each
of
these genomes is
presented in Table
3.
The largest number
of
orthologues
(58%) was with
E.
coli
sequences. Note however that
the
E.
coli
genome contains more than twice as many
genes as the
H.
influenzae
genome. A striking result of
this analysis is the small number
(27)
of
genes in the
licR-gerBC
region having an orthologue in
Mycoplasma
genitalium.
This may be due
to
the information content
of this part of the
B.
subtilis
chromosome (see above).
Indeed mycoplasmas have minimal genomes which
appear to have evolved from Gram-positive bacteria by
elimination of non-essential genes (Maniloff,
1983).
The
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On: Thu, 25 Aug 2016 08:45:06
The
311"-334"
B.
subtilis
chromosomal region
Conclusion
1
0.12
0
500
1000 1500 2000 2500
3000
3500
4000
kb
Fig.
5.
Chromosomal distribution of the paralogues of the
genes in the
/icR-gerBC
region. Bars represent the frequency of
paralogues (number of paraloguedcorresponding number of
genes) found in
100
kb segments of the chromosome. The
darker bars are
in
the
/id?-gerK
region.
number of orthologues with the archaeon
(Methano-
coccus
jannaschii)
and with the eukaryote
(S.
cerevisiae)
genomes are similar
(49
and
54)
but correspond
to
different genes (data not shown).
We systematically compared the
273
putative gene
products with protein sequences deduced from known
B.
subtilis
DNA
sequences.
Of
these,
115
genes
(42
'/o
)
are similar to at least one other
B.
subtilis
gene
(paralogues). Forty-five genes have only one counter-
part. In contrast,
17
genes have more than eight
paralogues (Table
2).
These genes encode proteins which
belong to large families of regulatory or transporter
proteins. For example, three genes in this region
of
the
chromosome encode regulatory proteins of the LysR
family and there are
13
other members of this class in the
remaining known sequences.
We
performed a prelimi-
nary analysis of the distribution of the paralogues along
the genome map, considering only genes which are not
part
of
large families of proteins (Fig.
5).
The dis-
tribution is not uniform, presenting a higher density of
paralogues in or near our
271
kb region (between
3600
and
4100
kb). In several cases paralogues are in tandem
repeat, in the same transcriptional unit
(ywbH
and
yw6G
or
ywhK
and
ywhL),
or in two transcriptional
units
(ywmC
and
ywmD).
Some protein families are
particularly well represented in this part of the chromo-
some. The three known genes putatively encoding
cardiolipin synthase are found in a
70
kb region:
ywiE
and
ywnE
are transcribed in the same orientation and
ywjE
is transcribed in the opposite direction. Three
of
the eight known members of the newly discovered
regulatory aspartyl phosphatase family (Perego
et al.,
1996)
are encoded in this part of the chromosome
(rapA,
rapD, rapF).
The systematic sequencing approach has considerably
accelerated the study
of
the genetic content and the gene
function analysis
of
the
licR-gerBC
region of the
B.
subtilis
chromosome. This region appears to contain
genes mainly involved in adaptation
to
growth condi-
tions. In particular, it carries a high density
of
loci
responsible for the utilization
of
various carbon and
nitrogen sources and alternative terminal electron ac-
ceptors. The synthesis of non-essential cell wall com-
ponents and their modification may reflect adaptation to
physical and chemical variations in
B.
subtilis'
ecological
niche: the soil and the surface of plant leaves (Priest,
1993).
The small number
of
genes having an orthologue
in the
Mycoplasma genitalium
genome, considered as a
minimal genome with very little adaptive capacity,
together with the large number
of
paralogues in this part
of
the chromosome are consistent with this observation.
Our analysis also supports the hypothesis
of
a functional
organization
of
the
B.
subtilis
chromosome. The sys-
tematic analysis
of
the unknown genes will confirm or
invalidate this view.
ACKNOWLEDGEMENTS
We thank Dr
J.
Hoch
for
providing plasmid pPP41. This work
was
supported by
the
Institut
Pasteur, the
European
Com-
mission (Biotechnology contract B102-CT-930272), the Mini-
stkre
de 1'Education Nationale, de 1'Enseignement SupCrieur
et
de
la
Recherche,
the
Groupement de
Recherche
et
d'Etude
sur
les Gknomes (GREG) (grant 1993-no.
20)
and the Centre
National de la Recherche Scientifique (GDR 1029 and URA
1300). H.C.R.
was
supported by
a
fellowship
from
the
Universidad Nacional Autonoma de Mkxico.
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