Download Novel genes involved in the regulation of

Microbiology (2000), 146, 885–891
Printed in Great Britain
Novel genes involved in the regulation of
pathogenicity factor production within the rpf
gene cluster of Xanthomonas campestris
J. Maxwell Dow, Jia-Xun Feng,† Christine E. Barber, Ji-Liang Tang†
and Michael J. Daniels
Author for correspondence : J. Maxwell Dow. Tel : j44 1603 452571. Fax : j44 1603 250024.
e-mail : max.dow!bbsrc.ac.uk
The Sainsbury Laboratory,
John Innes Centre,
Norwich Research Park,
Norwich NR4 7UH, UK
The synthesis of extracellular enzymes and extracellular polysaccharide (EPS)
in Xanthomonas campestris pathovar campestris (Xcc) is subject to co-ordinate
regulation by a cluster of genes called rpf (for regulation of pathogenicity
factors). These genes are located within a 219 kb region of the chromosome
isolated as the cosmid clone pIJ3020. The genes in the left-hand section of this
region of the chromosome have previously been characterized. This paper
reports on the genes in the right-hand section and on the phenotypes of
mutants with transposon insertions in these genes. Sequence analysis
identified eight genes or ORFs with the gene order
rpfD–orf1–orf2–orf3–orf4–recJ–rpfE–greA. RecJ and GreA have established
functions in recombination and transcriptional elongation, respectively. rpfD
encoded a protein with some amino acid sequence relatedness to a
hypothetical protein from Caulobacter crescentus and an autolysin response
regulator in Bacillus subtilis. The predicted protein products of orf1, 2 and 3
were related to each other and had substantial amino acid sequence
relatedness to hypothetical proteins from C. crescentus. Transposon insertions
in orf1, 2 and 3 had no effect on the synthesis of extracellular enzymes or EPS.
The predicted proteins RpfE and Orf4 showed the highest amino acid sequence
relatedness to hypothetical proteins from Bordetella pertussis and Klebsiella
pneumoniae, respectively. Transposon insertions in rpfE led to reduced levels
of some extracellular enzymes (endoglucanase and protease) and increased
levels of others (polygalacturonate lyase). Transposon insertions in orf4 had
no effect on polygalacturonate lyase but led to reduced levels of protease and
endoglucanase. Levels of EPS were reduced in both rpfE and orf4 mutants.
These alterations in the levels of extracellular enzymes, which were relatively
modest (between two- and threefold), did not affect the pathogenicity of Xcc
on turnip. It is proposed that the gene designation should be rpfI for orf4.
Keywords : Xanthomonas campestris, regulation of pathogenicity factors, rpf gene
cluster
INTRODUCTION
Xanthomonas campestris pathovar campestris (Xcc) is
.................................................................................................................................................
† Present address : Molecular Genetics Laboratory, Guangxi University,
Guangxi 530005, PR China.
Abbreviations : DSF, diffusible signal factor ; EPS, extracellular polysaccharide ; PGL, polygalacturonate lyase.
The EMBL accession number for the sequence reported in this paper is
AJ245997.
the causal agent of bacterial black rot of crucifers
(Onsando, 1992). The organism produces extracellular
polysaccharide (EPS) and a range of extracellular
enzymes that contribute collectively to pathogenesis
(Dow & Daniels, 1994). One of the aims of our work on
Xcc is to understand how the synthesis of these
pathogenicity factors is regulated. The synthesis of
extracellular enzymes and EPS is subject to co-ordinate
regulation by a cluster of genes called the rpf cluster, for
regulation of pathogenicity factors (Dow & Daniels,
0002-3758 # 2000 SGM
885
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 10:23:15
J. M. D O W a n d O T H E R S
METHODS
Strains and culture conditions. The Xcc strains used in this
study were 8004, a rifampicin-resistant mutant of a wild-type
isolate (Daniels et al., 1984), and mutant strains carrying Tn5
or Tn5lac within the rpf cluster of genes (Tang et al., 1991).
Strain 8478 carries a Tn5 insertion in rpfE ; strain 8535 carries
a Tn5lac insertion in orf4. All strains were normally grown in
peptone\yeast extract\glycerol medium (NYGB) at 28 mC as
described by Daniels et al. (1984).
.................................................................................................................................................
Fig. 1. Physical map of the Xcc DNA cloned as pIJ3020. The
upper element shows restriction sites for BamHI (B) and EcoRI
(E) and the location of the region analysed in detail here. The
lower element shows the organization of the predicted ORFs
with their protein products. The arrows indicate the direction
of transcription. The predicted start codon for orf2 overlaps
with the stop codon of orf1. orf3 starts at a GTG codon,
although an alternative ATG is present 102 nt downstream. The
predicted stop codon of rpfE overlaps with the predicted start
codon of recJ. The positions of insertion of Tn5 and Tn5lac
transposons are indicated by inverted triangles and flags,
respectively. The direction of the flag indicates the orientation
of the Tn5lac insertions. The positions of restriction sites for
BamHI (B), EcoRI (E) and SalI (S) are also shown ; these were
used to generate the subclones for sequencing.
1994). These genes are located within a 21n9 kb region of
the chromosome which has been isolated as the cosmid
clone pIJ3020 (Tang et al., 1991). Analysis of pIJ3020 by
transposon mutagenesis has demonstrated the existence
of at least eight genes (rpfA–H), insertions in which lead
to a co-ordinate reduction in the levels of all extracellular
enzymes tested and in EPS (Tang et al., 1991). Some of
the rpf genes have been characterized in detail. rpfA
encodes the major aconitase of Xcc and is implicated in
iron homeostasis (Wilson et al., 1998) ; rpfB and rpfF are
involved in the synthesis of a small diffusible signal
molecule (Barber et al., 1997 ; Slater, 1999) ; rpfG, C and
H encode elements of a two-component sensory-transduction system (Tang et al., 1991 ; Slater, 1999). Adjacent to rpfG are two genes with broader regulatory
functions ; prfB encodes peptide release factor 2, and
lysU encodes lysyl-tRNA synthetase (Dow & Daniels,
1994). The genes rpfA, B, F, C, H and G together with
prfB and lysU comprise the left-hand part of the Xcc
DNA within pIJ3020 (Fig. 1). Here we report the
characterization of the genes in the right-hand section,
which includes rpfD and rpfE. This analysis has
identified a further gene (orf4) involved in the regulation
of extracellular enzyme and EPS synthesis, together with
three ORFs (orf1, 2 and 3) of unknown function. In
addition, two genes were identified whose products are
highly related to RecJ and GreA, which in other bacteria
have established functions in recombination and transcriptional elongation, respectively. The predicted protein products of orf1, orf2, orf3, orf4, rpfD and rpfE
show the most significant amino acid sequence similarity
to hypothetical proteins predicted from (as yet unfinished) microbial genome sequencing projects.
Enzyme and EPS assays. For measurements of endoglucanase
activities, Xcc strains were grown in NYGB medium to an
OD of 2. Enzyme activities in cell-free culture supernatants
were'!!measured by radial diffusion assays into substratecontaining agar plates as described by Gough et al. (1988). For
induction of polygalacturonate lyase (PGL), strains were
grown in MMXC medium supplemented with 0n25 % (w\v)
polygalacturonate (Dow et al., 1987) to an OD of 0n5. PGL
activity was measured spectrophotometrically'!!
at 235 nm as
described by Dow et al. (1987). One unit of activity was
defined as a change in optical density of 1 unit min−"
corresponding to the production of 0n5 µmol unsaturated
product. For measurements of EPS production, strains were
grown in NYGB medium supplemented with 2 % (w\v)
glucose for 48 h. EPS was precipitated from culture supernatants by ethanol, dried and weighed as described by Vojnov
et al. (1998). Indicator plates for protease production contained NYGB supplemented with 1 % (w\v) skimmed milk
and solidified with 2 % (w\v) agar (Tang et al., 1987). Cultures
of bacteria were washed and resuspended in water to an OD
'!!
of 0n2. Aliquots (5 µl) of these suspensions were plated and
allowed to dry before growth at 30 mC. Methods used to assay
the β-galactosidase reporter activity are given in Barber et al.
(1997). Levels of the diffusible signal molecule DSF were
assessed by a bioassay as described by Slater (1999).
DNA sequencing and analysis. The insert DNA from the
cosmid clone pIJ3020 which carries the full rpf cluster (Tang
et al., 1991) was subcloned into the pBluescript BS(k) vector
(Stratagene) using convenient restriction sites for EcoRI and
BamHI prior to sequencing. To complete the sequence of the
right border of the cluster, a cosmid clone (pIJ3092) carrying
Xcc DNA which overlapped the right-hand border of pIJ3020
was identified from a library of Xcc DNA cut with BamHI and
constructed in pLAFR3 (Staskawicz et al., 1987). A 5n0 kb
EcoRV fragment from pIJ3092, which spanned the EcoRI site
at the right-hand side of pIJ3020, was cloned in pBluescript
KS(j). A 90 bp PstI fragment spanning the EcoRI site was
subcloned into pGEM-3zf(j) (Promega) for sequencing.
Determination of the DNA sequence was done by automated
sequencing using Applied Biosystems DNA sequencers (373
and 377) in conjunction with the PRISM Ready Reaction
DyeDeoxy Terminator Cycle sequencing kit (Applied Biosystems). Double-stranded DNA templates were prepared as
recommended by the manufacturer. Plasmids were sequenced
in both directions using the forward and reverse universal
primers and by walking, using a series of synthetic primers
(Genosys) based on the determined sequence. The sequence
was analysed and compiled using the  program of Staden
(1996). The GCG suite of programs was used to analyse the
data and the predicted amino acid sequence. Homology
searches of the databases were performed using  with
 62 (Altschul et al., 1990). The  program
(Borodovsky & McIninch, 1993) and  (Bibb et al., 1984)
were used to predict potential coding regions. Protein structural predictions were made using the PredictProtein program
at EMBL (Rost, 1996).  searches against unfinished
886
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 10:23:15
Xanthomonas pathogenicity gene regulation
microbial genome sequences were performed using the websites at The Institute for Genomic Research (TIGR) at
http :\\www.tigr.org and The Sanger Centre at http :\\
www.sanger.ac.uk\.
Plant tests. For growth assays, bacterial suspensions in water
at 10& c.f.u. ml−" were introduced into the leaf lamina of
mature leaves of turnip cv. Just Right using a syringe with no
needle, and plants were maintained as described by Collinge
et al. (1987). Bacterial numbers in plants were measured as
described by Parker et al. (1993). For pathogenicity tests,
bacterial suspensions (10' c.f.u. ml−") were introduced into
the vascular system of turnip through nicks at the vein endings
and the plants were maintained in high humidity conditions as
described by Dow et al. (1990). The ability of bacterial strains
to induce the hypersensitive response was tested in pepper cv.
ECW10R as described by Newman et al. (1997).
RESULTS
Organization of genes in the right-hand section of
the Xcc DNA in pIJ3020
A physical map of the Xcc DNA in pIJ3020, with the
restriction sites for BamHI and EcoRI and the location
of the region analysed here, is shown in Fig. 1. The DNA
sequence of the right-hand section of the Xcc DNA in
pIJ3020 comprised 8165 nt. Analysis of this sequence
by the  and  programs predicted the
occurrence of eight ORFs (Fig. 1). The sizes of the
predicted gene products together with the relatedness to
sequences in the databases were revealed by 
searches (Table 1). Two of the gene products had very
high relatedness to proteins with known function : RecJ
to a single-stranded, DNA-specific exonuclease, and
GreA to a transcriptional elongation factor. The cognate
genes (greA and recJ) are predicted to be in an operon
with rpfE with the gene order greA–rpfE–recJ. As the
termination codon of greA is separated by only 3 nt
from the predicted start codon of rpfE, and the predicted
start codon of recJ overlaps the predicted stop codon of
rpfE, the three genes may be translationally coupled.
RpfE shows amino acid sequence relatedness to hypothetical proteins predicted from genome sequencing
projects for Bordetella pertussis (Table 1) and Neisseria
gonorrhoeae ( score 8i10−)).
To the left of this predicted operon are four ORFs
(orf1–4) which are transcribed in the opposite direction.
The predicted protein products Orf1, 2 and 3 have
significant amino acid sequence similarity to each other.
An alignment of the three predicted proteins (Fig. 2)
showed that Orf1 and Orf2 have amino acid sequence
relatedness to the N-terminal and C-terminal parts of
Table 1. Genes or ORFs predicted by sequence analysis of the right-hand section of the Xcc DNA cloned in pIJ3020
.................................................................................................................................................................................................................................................................................................................
The size of the predicted gene products, the most significantly related database entries as revealed by  searches, and the
percentage similarity and identity scores are given.
Gene
Size of gene
product (aa)
Amino acid sequence relatedness
with BLAST score
Accession
no. of
homologue
Size of
homologue
(aa)
Percentage
identical (I)
and similar (S)
amino acids
No. of
positions
compared
rpfD
305
*
*
31 % I, 48 % S
235
orf1
108
*
*
61 % I, 78 % S
70
orf2
300
*
*
42 % I, 56 % S
246
orf3
454† (420)
*
*
37 % I, 55 % S
312
orf4
368
Hypothetical protein from C. crescentus
(10−"))
Hypothetical protein from C. crescentus
(10−#") ; homologous to Orf3 (9i10−#")
Hypothetical protein from C. crescentus
(10−&$) ; homologous to Orf3 (9i10−$!)
Hypothetical protein from C. crescentus
(10−%$)
Hypothetical protein from Klebsiella
pneumoniae (10−#&)
Hypothetical protein YPFG of E. coli
(10−"')
Single-stranded DNA specific exonuclease
RecJ of Erwinia chrysanthemi and E.
coli (10−"$$)
Hypothetical protein predicted from
genome sequencing of Bordetella
pertussis (5n2i10−"$)
Transcriptional elongation factor GreA
of E. coli (10−&!)
*
*
45 % I, 55 % S
172
P76559
347
29 % I, 40 % S
337
P39693
575
47 % I, 60 % S
578
*
*
33 % I, 67 % S
151
P21346
158
61 % I, 79 % S
154
recJ
582
rpfE
306
greA
158
* Data obtained from incomplete sequencing project ; accession numbers and full length of homologue not available.
† GTG start codon, all other predicted ORFs start with an ATG codon.
887
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 10:23:15
J. M. D O W a n d O T H E R S
.....................................................................................................
Fig. 2. Amino acid sequence similarities
between Orf1, 2 and 3, which are predicted
to be associated with the cytoplasmic
membrane. The sequences were aligned
using the algorithm PILEUP (Devereux et al.,
1984). Residues found in at least two of the
sequences are highlighted in bold. The
positions of the ten predicted membranespanning domains of Orf3 are indicated by
bars under the alignment and are numbered. Orf1 has three predicted membranespanning domains, equivalent in position to
domains 1–3 of Orf3. Orf2 has seven predicted membrane-spanning domains, equivalent in position to domains 4–10 of Orf3.
Orf3, respectively. There were two possible start codons
for Orf3 : a GTG, giving a product of 454 amino acids in
length, and an ATG, giving a product of 420 amino acids
in length. Optimal alignments between Orf1 and Orf3
were seen with the shorter protein product. In database
searches, Orf1, 2 and 3 were related to incomplete
hypothetical proteins predicted from the (unfinished)
genome sequence of Caulobacter crescentus (Table 1).
Orf2 and the C-terminal part of Orf3 were both highly
related to the same hypothetical protein of C. crescentus ;
Orf1 (and the N-terminal part of Orf3) was related to a
protein predicted from a different DNA sequence.
Whether these incomplete hypothetical proteins represent different domains of the same protein will only be
revealed after assembly of the full C. crescentus sequence. Using the PredictProtein program, Orf1, 2 and 3
were suggested to be localized in the cytoplasmic
membrane. Orf3 is predicted to have ten membranespanning helices, whereas Orf2 has seven and Orf1 has
three (Fig. 2). The termination codon of orf1 overlaps
the predicted start codon of orf2. Expression studies
with Tn5lac show that orf1, 2 and 3 are only weakly
expressed (50–150 Miller units) whereas orf4 is more
highly expressed (550–1000 Miller units). Although it is
possible that orf1, 2 and 3 are transcribed as an operon,
the higher expression of orf4 suggests that it must have
its own independent promoter. Orf4 is distinct from
Orf1, 2 and 3, and has amino acid sequence relatedness
to hypothetical proteins from Klebsiella pneumoniae
and Escherichia coli (Table 1). Flanking orf1 is rpfD,
which is transcribed in the opposite direction and
encodes a protein with amino acid sequence relatedness
to a hypothetical protein from C. crescentus (Table 1)
and also, to a lesser extent, the autolysin response
regulator LytT of Bacillus subtilis ( score 10−').
Interestingly, analysis of the preliminary C. crescentus
nucleotide sequences suggests that the genes encoding
the RpfD and Orf1 homologues are also adjacent in the
chromosome and divergently transcribed. Using the
PredictProtein program, RpfD, Orf4 and RpfE were
suggested to be globular soluble proteins.
Analysis of effects of transposon insertion on the
production of extracellular enzymes and EPS
We have previously reported the mutagenesis of pIJ3020
with Tn5 and Tn5lac and the effects that some of these
insertions have on production of extracellular enzymes,
EPS and pathogenicity (Tang et al., 1991). The positions
of transposon insertion in the right-hand section of the
Xcc DNA in pIJ3020 are shown in Fig. 1. The effect of
these transposon insertions on the production of certain
888
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 10:23:15
Xanthomonas pathogenicity gene regulation
Table 2. Levels of extracellular enzymes and EPS in
strains of Xcc
.................................................................................................................................................
Activities of PGL and endoglucanase and the levels of EPS are
expressed (ml culture)−". Units and growth conditions are given
in Methods. Data given are the means p of three
independent determinations.
Strain
8004 (wild-type)
8478 (rpfE)
8535 (orf4)
8004
Endoglucanase
(U ml−1)
PGL
(mU ml−1)
EPS
(mg ml−1)
202p30
66p10
78p10
10p2
31p7
10p2
2n16p0n05
0n98p0n02
0n39p0n02
8535
8478
.................................................................................................................................................
Fig. 3. Protease production by strains of Xcc as revealed by
zones of clearing around colonies on skimmed milk indicator
plates. The strains were 8004 (wild-type), 8478 (rpfE) and 8535
(orf4).
extracellular enzymes and EPS was assessed. Tn5
insertions within orf1, orf2 and recJ and Tn5lac
insertions within recJ and orf3 had no effect on the
production of extracellular enzymes or EPS. Tn5lac
insertion into rpfD had only minor effects (Tang et al.,
1991 ; and data not shown). In contrast, mutation of orf4
and rpfE with Tn5lac and Tn5, respectively, gave
significant alterations in the levels of pathogenicity
factors. Mutation of orf4 led to a reduction in endoglucanase and protease levels and in EPS (Table 2 and
Fig. 3), although the levels of PGL were unaltered.
Mutation of rpfE had more complex effects than
originally reported by Tang et al. (1991) leading to a
reduction in levels of endoglucanase, protease and EPS
but an increase in PGL activity (Table 2 and Fig. 3). The
effect of transposon insertion in rpfE on production of
extracellular enzymes cannot be due to polar effects on
the expression of recJ (which is downstream) since
insertions in recJ alone had no effect. Although there
were alterations in the levels of extracellular enzymes in
rpfE and orf4 mutants, both of these strains produced
levels of the diffusible signal factor DSF comparable to
that of the wild-type. None of the mutations had an
effect on the growth rate in nutrient medium.
Mutation of rpfE, orf3 and orf4 has no detectable
effect on the bacterial interaction with plants
The wild-type strain 8004 and the rpfE, orf3 and orf4
mutants were inoculated into the leaf lamina of turnip
plants at 10& c.f.u. ml−" and bacterial growth was
assessed over a period of 5 d. No significant difference in
the growth rate or final populations was observed (data
not shown). Pathogenicity tests in which bacteria were
introduced into the vascular system of the plant through
cuts at the leaf vein endings showed no loss of virulence
associated with mutation of rpfE, orf3 or orf4. In
addition, rpfE, orf3 and orf4 mutants, as well as the
wild-type, were all able to induce a hypersensitive
response in the non-host plant pepper (Capsicum
annuum) cv. ECW10R.
DISCUSSION
Our analysis of the right-hand section of the 21n9 kb Xcc
DNA fragment cloned in pIJ3020 has identified two
novel genes, orf4 and rpfE, which appear to regulate the
production of extracellular enzymes and EPS. The
mechanism by which the gene products effect changes in
the level of these pathogenicity factors is obscure. The
effects of mutation of orf4 and rpfE are considerably
different from those seen after mutation of other rpf
genes. Mutation of rpfE led to a less pronounced
reduction of protease, endoglucanase and EPS production than those seen after mutation of rpfC, rpfG,
rpfH, rpfB and rpfF. More significantly, mutation of
rpfE led to an elevated level of PGL, an effect that is not
seen as a consequence of mutation of any other gene
within the rpf cluster. Mutations in an unlinked gene,
rpfN, do lead to an increase in PGL, but the levels of
other enzymes also increase co-ordinately (Tang et al.,
1990). Divergent effects on the levels of different
extracellular enzymes are seen after mutation of clp, a
gene encoding a catabolite-activation-factor-like protein
in X. campestris (De Cre' cy-Lagard et al., 1990). A clp
mutant has elevated levels of protease but reduced levels
of endoglucanase and PGL. Mutation of orf4 had no
effect on the production of PGL although levels of EPS
were fivefold lower. These less pronounced effects on
enzymes and EPS compared to other rpf mutants may
explain the lack of a detectable effect of mutation of
either rpfE or orf4 on pathogenesis or on growth in
plants. It is possible that regulation of extracellular
enzyme and EPS production by orf4 and rpfE is
important in other phases of the disease cycle of Xcc or
may be more pronounced under other culture conditions.
The two genes whose functions could be unequivocally
assigned were recJ and greA. No detectable change in
phenotype was associated with mutation of recJ, a
situation which is found in other bacteria where RecJ is
thought to have functions which are redundant with
other single-stranded DNA exonucleases (Viswanathan
& Lovett, 1998 ; Harris et al., 1998). We did not obtain
any transposon insertions in greA. The significance (if
any) of the organization of greA within an operon
containing recJ and rpfE is not known. greA is known to
occur in different (genomic) contexts in different bacteria. In Pseudomonas aeruginosa, greA is the most
downstream gene in an operon encoding enzymes
involved in pyrimidine biosynthesis (Lu et al., 1997)
whereas in Rickettsia prowazekii it is the most upstream
889
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 10:23:15
J. M. D O W a n d O T H E R S
gene in the major macromolecular synthesis operon
(Shaw et al., 1997), which includes the gene encoding the
major sigma factor RpoD.
Mutation of rpfE and orf4 had no effect on the
production of the diffusible signal molecule DSF. DSF
may act as a stress- or starvation-induced signal for the
synthesis of all extracellular enzymes and EPS (Barber et
al., 1997). Its synthesis requires RpfB and RpfF (Barber
et al., 1997) and its perception may involve the twocomponent regulatory system encoded by rpfG\H\C
(Slater, 1999). The phenotypes of rpfE and orf4 mutants
suggest that RpfE and Orf4 are not essential components
within the DSF regulatory system. Similar considerations apply to RpfA, which is implicated in iron
homeostasis but not in DSF-dependent regulation
(Wilson et al., 1998).The organization of the genes
within the region of the Xcc chromosome cloned in
pIJ3020 reflects the functional connection to the DSFdependent regulation of extracellular enzyme and EPS
synthesis ; the genes rpfB, rpfF, rpfC, rpfH and rpfG are
present in two convergently transcribed operons which
are contiguous (Slater, 1999). This group of genes is
flanked on the left by rpfA (Wilson et al., 1998).
Transposon insertions in genes immediately to the left of
rpfA have no effect on the synthesis of extracellular
enzymes or EPS (T. J. G. Wilson & M. J. Daniels,
unpublished). The other rpf genes (rpfD, rpfE and orf4)
are to the right of rpfB–G and are grouped along with
genes of diverse function including those with broad
regulatory functions such as prfB, lysU and greA, and
those with, as yet, no defined function (orf1, 2 and 3).
The significance of the clustering of so many genes with
regulatory functions within the region of the Xcc
chromosome cloned as pIJ3020 is not understood.
In conclusion, our analysis has identified two genes (orf4
and rpfE) with roles in the regulation of pathogenicity
factors in Xcc ; we propose the gene designation rpfI for
orf4. Although mutation of rpfE and orf4 had no
detectable effect on the interaction of Xcc with plants,
the role in pathogenesis of the related genes that occur in
animal pathogens certainly warrants investigation.
ACKNOWLEDGEMENTS
The Sainsbury Laboratory is supported by a grant from the
Gatsby Charitable Foundation. This work was carried out
according to the provisions of the Ministry of Agriculture,
Fisheries and Food, licence PHF 1185\8(48) issued under the
Plant Health (Great Britain) Order 1987 (Statutory Instrument
1758).
REFERENCES
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. P.
(1990). Basic local alignment search tool. J Mol Biol 215, 403–410.
Barber, C. E., Tang, J.-L., Feng, J.-X., Pan, M.-Q., Wilson, T. J. G.,
Slater, H., Dow, J. M., Williams, P. & Daniels, M. J. (1997). A novel
regulatory system required for pathogenicity of Xanthomonas
campestris is mediated by a small diffusible signal molecule. Mol
Microbiol 24, 555–566.
Bibb, M. J., Findlay, P. R. & Johnson, M. W. (1984). The relationship between base composition and codon usage in bacterial
genes and its use for the simple and reliable identification of
protein-coding sequences. Gene 30, 157–166.
Borodovsky, M. & McIninch, J. D. (1993).  : parallel gene
recognition for both DNA strands. Comput Chem 17, 123–133.
Collinge, D. B., Milligan, D. E., Dow, J. M., Scofield, G. & Daniels,
M. J. (1987). Gene expression in Brassica campestris showing a
hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians. Plant Mol Biol 8, 405–414.
Daniels, M. J., Barber, C. E., Turner, P. C., Cleary, W. G. & Sawczyc,
M. K. (1984). Isolation of mutants of Xanthomonas campestris
pathovar campestris showing altered pathogenicity. J Gen
Microbiol 130, 2447–2455.
De Cre' cy-Lagard, V., Glaser, P., Lejeune, P., Sismeiro, O., Barber,
C. E., Daniels, M. J. & Danchin, A. (1990). A Xanthomonas
campestris pv. campestris protein similar to catabolite activation
factor is involved in regulation of phytopathogenicity. J Bacteriol
172, 5877–5883.
Devereux, V., Haeberli, P. & Smithies, O. (1984). A comprehensive
set of sequence analysis programs for the VAX. Nucleic Acids Res
12, 387–395.
Dow, J. M. & Daniels, M. J. (1994). Pathogenicity determinants
and global regulation of pathogenicity in Xanthomonas campestris pv. campestris. In Molecular and Cellular Mechanisms in
Bacterial Pathogenesis of Plants and Animals, pp. 29–41. Edited
by J. L. Dangl. Berlin : Springer.
Dow, J. M., Scofield, G., Trafford, K., Turner, P. C. & Daniels,
M. J. (1987). A gene cluster in Xanthomonas campestris pv.
campestris controls the excretion of polygalacturonate lyase and
other enzymes. Physiol Mol Plant Pathol 31, 261–271.
Dow, J. M., Clarke, B. R., Milligan, D. E., Tang, J. L. & Daniels,
M. J. (1990). Extracellular proteases from Xanthomonas cam-
pestris pv. campestris, the black rot pathogen. Appl Environ
Microbiol 56, 2994–2998.
Gough, C. L., Dow, J. M., Barber, C. E. & Daniels, M. J. (1988).
Cloning of two endoglucanase genes from Xanthomonas campestris pv. campestris : role of the major enzyme in pathogenesis.
Mol Plant–Microbe Interact 1, 275–281.
Harris, R. S., Ross, K. J., Lombardo, M. J. & Rosenberg, S. M.
(1998). Mismatch repair in Escherichia coli cells lacking single-
strand exonucleases ExoI, ExoVII, and RecJ. J Bacteriol 180,
989–993.
Lu, C. D., Kwon, D. H. & Abdelal, A. T. (1997). Identification of
greA encoding a transcriptional elongation factor as a member of
the carA-orf-carB-greA operon in Pseudomonas aeruginosa
PAO1. J Bacteriol 179, 3043–3046.
Newman, M.-A., Daniels, M. J. & Dow, J. M. (1997). The activity
of lipid A and core components of bacterial lipopolysaccharides
in the prevention of the hypersensitive response in pepper. Mol
Plant–Microbe Interact 10, 926–928.
Onsando, J. M. (1992). Black rot of crucifers. In Plant Diseases of
International Importance. II : Diseases of Vegetable and Oil Seed
Crops, pp. 243–252. Edited by H. S. Chaube, J. Kumar, A. N.
Mukhopadhyay & U. S. Singh. Englewood Cliffs, NJ : Prentice
Hall.
Parker, J. E., Barber, C. E., Fan, M.-J. & Daniels, M. J. (1993).
Interaction of Xanthomonas campestris with Arabidopsis thaliana : characterization of a gene from X.c. pv. raphani that
confers avirulence to most A. thaliana accessions. Mol Plant–
Microbe Interact 6, 216–224.
Rost, B. (1996). PHD : predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol 266,
525–539.
890
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 10:23:15
Xanthomonas pathogenicity gene regulation
Shaw, E. I., Mark, G. L., Winkler, H. H. & Wood, D. O. (1997).
Transcriptional characterization of the Rickettsia prowazekii
major macromolecular synthesis operon. J Bacteriol 179,
6448–6452.
Slater, H. (1999). The regulation of pathogenicity factor
production in Xanthomonas campestris by a small diffusible
molecule. PhD thesis, University of East Anglia.
Staden, R. (1996). The Staden sequence-analysis package. Mol
Biotechnol 5, 233–241.
Staskawicz, B. J., Dahlbeck, D., Keen, N. T. & Napoli, C. (1987).
Molecular characterization of cloned avirulence genes from race
0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol
169, 5789–5794.
Tang, J.-L., Gough, C. L., Barber, C. E., Dow, J. M. & Daniels,
M. J. (1987). Molecular cloning of protease gene(s) from Xantho-
monas campestris pv. campestris : expression in Escherichia
coli and role in pathogenicity. Mol Gen Genet 210, 443–448.
Tang, J.-L., Gough, C. L. & Daniels, M. J. (1990). Cloning of genes
involved in negative regulation of production of extracellular
enzymes and polysaccharide of Xanthomonas campestris
pathovar campestris. Mol Gen Genet 222, 157–160.
Tang, J.-L., Liu, Y.-N., Barber, C. E., Dow, J. M., Wootton, J. C. &
Daniels, M. J. (1991). Genetic and molecular analysis of a cluster
of rpf genes involved in positive regulation of synthesis of
extracellular enzymes and polysaccharide in Xanthomonas campestris pathovar campestris. Mol Gen Genet 226, 409–417.
Viswanathan, M. & Lovett, S. T. (1998). Single-strand DNAspecific exonucleases in Escherichia coli : roles in repair and
mutation avoidance. Genetics 149, 7–16.
Vojnov, A. A., Zorreguieta, A., Dow, J. M., Daniels, M. J. &
Dankert, M. A. (1998). Evidence for a role for the gumB and
gumC gene products in the formation of xanthan from its
pentasaccharide repeating unit by Xanthomonas campestris.
Microbiology 144, 1487–1493.
Wilson, T. J. G., Bertrand, N., Tang, J.-L., Feng, J.-X., Pan, M.-Q.,
Barber, C. E., Dow, J. M. & Daniels, M. J. (1998). The rpfA gene of
Xanthomonas campestris pathovar campestris which is involved
in regulation of pathogenicity factor production encodes an
aconitase. Mol Microbiol 28, 961–970.
.................................................................................................................................................
Received 15 September 1999 ; revised 11 November 1999 ; accepted
21 December 1999.
891
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 10:23:15