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Applied and Environmental Microbiology, January 1999, p. 278-282, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
New Mobilizable Vectors Suitable for Gene Replacement in
Gram-Negative Bacteria and Their Use in Mapping of the 3' End of the
Xanthomonas campestris pv. campestris
gum Operon
Federico
Katzen,1
Anke
Becker,2
M. Verónica
Ielmini,1
Cristian G.
Oddo,1 and
Luis
Ielpi1,*
Instituto de Investigaciones
Bioquímicas Fundación Campomar, Facultad de Ciencias
Exactas y Naturales, UBA, and CONICET, 1405 Buenos Aires,
Argentina,1 and
Lehrstuhl für
Genetik, Universität Bielefeld, D-33501 Bielefeld,
Germany2
Received 20 July 1998/Accepted 29 October 1998
 |
ABSTRACT |
We describe useful vectors to select double-crossover events
directly in site-directed marker exchange mutagenesis in gram-negative bacteria. These vectors contain the gusA marker gene,
providing colorimetric screens to identify bacteria harboring those
sequences. The applicability of these vectors was shown by mapping the
3' end of the Xanthomonas campestris gum operon, involved
in biosynthesis of xanthan.
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TEXT |
Gene disruption and marker exchange
techniques are well-established tools for genetic
engineering of bacteria. Site-directed marker exchange methods
include several steps: (i) cloning of a gene region into a
suicide vector, (ii) disruption of the cloned gene by insertion of a
transposon or an interposon cassette, (iii) mobilization of the
hybrid plasmid to the recipient strain, (iv) selection of recombinant
strains, and (v) identification of double-recombinant strains. In many
cases the last step involves tedious work, since the ratio of
double-homologous-crossover to single-crossover events can be very low,
depending on the organism used. This difficulty can occasionally be
overcome by incorporating a copy of the sacB gene coding
for the levansucrase enzyme (16-18), whose expression in a sucrose-containing medium leads to a lethal phenotype in some organisms. Unfortunately, in many gram-negative bacteria the
expression of this gene in a sucrose-containing medium is not lethal or
leads to the growth of small colonies in prolonged incubations
(18). To solve this problem, we developed two plasmid vectors containing a marker gene that enables us to discern between single- and double-crossover events without any experimental step.
The gusA gene, originally identified in Escherichia
coli (11), is a widespread marker mainly used to study
plant gene expression. It encodes the 
-glucuronidase enzyme (GUS).
The advantages of GUS over other reporter systems include the
robustness of the enzyme and the simplicity of the assays. Compared to
the products of other reporter genes, GUS is a very stable gene
product. This enzyme accumulates over time, and minute quantities of
GUS activity can be accurately measured (12). A key
advantage of GUS is the generalized absence of this activity within the
taxon Eubacteria (25), with the exception of E. coli and some species of Shigella, Streptococcus, Staphylococcus,
Corynebacteria, and Clostridium. The
incorporation of the gusA gene into the mobilizable suicide vectors pK18mob and pK19mob facilitates discerning between gene replacement and plasmid integration by simply observing the color of
the colonies.
Construction of pKmobGII vectors.
A restriction map of the
resulting pKmobGII vectors and the nucleotide sequence of their
multiple cloning site (MCS) are shown in Fig.
1. For the construction of the vectors,
the gusA gene together with the polyadenylation signal
from the nopaline synthase gene (NOS-ter) from the low-copy-number
plasmid pBI101 (GenBank accession no. U12639 [12])
were cloned between the HindIII and EcoRI
sites of pUC19, giving rise to pDNA4. The tac promoter (GenBank accession no. K01728) was released from plasmid pKK223-3 (5) by digestion with EcoRI and BamHI,
and the EcoRI end was filled in. This fragment was
cloned into pDNA4, which was previously digested with
BamHI and SmaI, originating pTACDNA4. The
final plasmids were constructed by cloning the
BamHI-SacI fragment, containing the
gusA gene from pTACDNA4, into
BstBI-digested pK18mob and pK19mob plasmids (GenBank
accession no. AF012346 [17]) upon filling in and
removing the 3' and 5' recessed ends. The additional BamHI
site regenerated at the BamHI-BstBI junction was
removed by filling in with the Klenow enzyme. These high-copy-number plasmids are 5.9 kb in length and contain the origin of replication of
pUC vectors; hence, their host range is restricted to E. coli and a few other species of the
Enterobacteriaceae family. The presence of an origin
of transfer (mobRK2) enables their transference by
conjugation into a wide range of bacteria (17). They also carry the Tn5 kanamycin resistance gene. Selection for this
antibiotic resistance does not lead to the formation of satellite
colonies and results in a high yield of DNA from standard plasmid
preparations (15). The pUC multiple cloning site located
within the gene encoding the LacZ
peptide enables the application of
these vectors as standard cloning vectors.
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FIG. 1.
(A) Physical map of pKmobGII vectors. The asterisk
symbolizes the plasmid label 18 or 19. (B) Nucleotide sequence of the
MCS. Unique restriction sites for cloning purposes are indicated. The
enzyme recognition sites and the orientation of the MCS correspond to
those in plasmids pK18mob and pK19mob. Km, kanamycin resistance gene
(black arrow); mob, origin of transfer; oriV,
origin for vegetative replication; lacZ, lac
fragment for -complementation (open arrow); ptac,
tac promoter (small gray arrow); gusA,
-glucuronidase gene (large gray arrow).
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These plasmids were successfully tested in
Xanthomonas
campestris pv. campestris FC2 (
14),
Rhizobium
meliloti 1021 (
1),
and
Agrobacterium
tumefaciens A348 (
7). For this purpose, DNA
fragments
of
X. campestris,
A. tumefaciens, and
R. meliloti were
subcloned into pK19mobGII and the resulting hybrid
plasmids were
transferred to their correspondent wild-type host
from the broad-host-range
mobilizing strain
E. coli
S17-1, as described previously (
19).
Integration of hybrid
plasmids into their correspondent host genomes
was selected by the
vector-encoded antibiotic resistance. Cells
carrying pKmobGII
sequences integrated into their chromosomes
can be easily
distinguished from those lacking these sequences
in plates
containing 50 mg of
5-bromo-4-chloro-3-indoxyl-
-
D-glucuronide
(X-gluc)/liter
or 25 mg of
5-bromo-6-chloro-3-indoxyl-
-
D-glucuronide
(magenta-gluc)/liter. Phenotypes of
X. campestris,
R. meliloti,
and
A. tumefaciens cells carrying or lacking
pKmobGII sequences
are shown in Fig.
2. GUS activities were assayed by
following
a protocol originally designed for plants (
10).
Relative units
of GUS for a strain were calculated by using the
following formula:
(picomoles of methyl-umbelliferone × reaction volume [in milliliters])/(time
[in minutes] × OD
600 of culture × cell culture [in
milliliters]),
where OD
600 is the optical density at 600 nm.
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FIG. 2.
Phenotypes of different strains grown on
X-gluc-containing medium. (A) X. campestris FC2, R. meliloti Rm1021, and A. tumefaciens A348 wild-type
cells grown for 2 days at 28°C in tryptone-yeast plates
(22) containing 50 µg of X-gluc/ml. (B) The same cells
harboring pK19mobGII sequences integrated into their genomes were grown
as described for panel A in the presence of 50 µg of kanamycin/ml.
The GUS activity of each strain is indicated in parentheses. The GUS
activities were assayed by employing five independent samples. ND, not
detected. The photographs of X. campestris cells were taken
after storing the plates for 7 days at 4°C.
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Although GUS activity that results from the expression of pKmobGII
sequences varies among different hosts (Fig.
2), a correlation
between
GUS activity and cell color development was not observed.
Whereas blue
colonies of
A. tumefaciens and
R. meliloti
strains
could be observed 2 days after plating,
X. campestris cells harboring
a copy of pKmobGII needed to be
stored at 4°C for a week to become
green (Fig.
2). This might
be due to differences in the cell wall
composition that could make
X. campestris cells more impermeable
to chromogenic
substrates.
The only requisite for the usefulness of these vectors is that
the synthesized GUS protein should become accessible to its
substrate (X-gluc). There is no need for any specific sugar
consumption
by the cell for color development. The catalysis of X-gluc
might
proceed either in the cytoplasm, if the substrate gains access
to
it, or outside the cell, if the enzyme is released by spontaneous
lysis. To test whether the presence of the
glucuronide-specific
permease gene
gusB (
11)
facilitated color development, this
gene was cloned into the
broad-host-range vector pRK293 and introduced
into a
X. campestris mutant with pKmobGII sequences integrated
into
its chromosome. The presence of the
gusB-containing
plasmid
did not shorten the time in which cells developed a green
color.
Mapping of the 3' end of the gum operon in X. campestris.
The plant-pathogenic bacterium X. campestris produces an acidic exopolysaccharide termed xanthan
gum, which is composed of D-glucosyl,
D-mannosyl, and D-glucuronyl acid residues in a
molar ratio of 2:2:1 and variable proportions of
O-acetyl and pyruvyl residues (9, 21).
Because of its physical properties, xanthan is widely used as a
thickener or viscosifier in both the food and nonfood industries
(reviewed in reference 3). The biosynthesis of
xanthan mainly consists of a stepwise assembly and decoration of
pentasaccharide subunits attached to a polyprenol phosphate carrier,
which are subsequently polymerized and exported (8). The
enzymes required for these processes are encoded by a 16-kb genome
region named xpsI, or gum (14). This
region is composed of 12 genes designated gumB to
gumM (GenBank accession no. U22511). Transcription of the
X. campestris gum gene cluster was proved to be directed by
a promoter located upstream of the first gene, gumB
(13), but the 3' end of the operon has not been mapped.
In order to assess whether this operon continues downstream of
gumM, we sequenced a 3.6-kb DNA fragment adjacent to the
gum region contained in the plasmid pJC372 (J. M. Cleary, Kelco Biopolymers
Group of Monsanto Company) (Fig.
3A). This plasmid was obtained
from a
X. campestris genomic library constructed by
cloning total
DNA digested with
SacI into pJC106, a
derivative of the broad-host-range
vector RSF1010. DNA sequences were
obtained from overlapping nested
deletion clones generated by
exonuclease III digestion and processed
by using an ALFexpress DNA
sequencer (Amersham Pharmacia Biotech,
Uppsala, Sweden) according to
the manufacturer's instructions.
The nucleotide and amino acid
sequences were analyzed by using
the MacVector sequence analysis
software (Oxford Molecular Ltd.,
United Kingdom). The amino acid
sequences deduced from the nucleotide
sequence were compared to the
GenBank database with the gapped
BLAST algorithm (
2). A
coding region analysis, using a
X. campestris codon usage
table, suggested the presence of three open reading
frames, which were
named, from 5' to 3', ORF14, ORF15, and ORF16
(Fig.
3B). While a
comparison of the deduced amino acid sequences
of ORF14 and ORF16
revealed no homologies in the GenBank database,
the deduced sequence of
ORF15 was found to be homologous to those
of a large family of
3-ketoacyl-acyl carrier proteins involved
in the biosynthetic pathway
of fatty acids (
24).
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FIG. 3.
Mapping of the 3' end of the gum operon. (A)
Physical map of the plasmid pJC372. DNA sequences that overlap the
gum region (GenBank accession no. U22511) are indicated by a
solid line. The dotted line represents the nucleotide sequence of a
3.6-kb DNA fragment determined in this work (GenBank accession no.
U70053). The relevant restriction sites are indicated. (B) Genetic map
of the cosmid pJC372. The position and orientation of lacZ
transcriptional fusions are indicated by solid flags. The relative
activities (± standard deviations) of lacZ transcriptional
fusions are given in -Gal units (Miller units) above the line. Below
the line, the restriction fragments used for integration mutagenesis
are presented. A potential promoter is indicated by a solid box. The
relative -Gal activities (± standard deviations) of the
lacZ transcriptional fusions located downstream from the
vector integration sites are listed; the activities were calculated
from five independent measurements.
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To test whether ORF14 is cotranscribed with
gumM, we
employed a method consisting of the application of
lacZ
transcriptional
fusions combined with plasmid integration
(
13). A promoterless
lacZ-aacC1 interposon
(
4) was inserted into the unique
BamHI
site of
the
SmaI-
ClaI fragment (nucleotides 14056 [GenBank accession
no.
U22511] and 2071 [GenBank accession no.
U70053], respectively),
which was previously subcloned into
pK19mobGII. The hybrid plasmid
was transferred from the
broad-host-range mobilizing strain
E. coli S17-1
(
21) to wild-type
X. campestris FC2
(
14), as described
previously (
19).
Exconjugants were selected in agar medium containing
gentamicin,
rifampin, and X-gluc. Yellow colonies appeared with
a frequency of
10
4 and were sensitive to kanamycin. Correct gene
replacement was
verified by Southern hybridization. The strain mutated
in ORF14
(XcORF14) produced normal amounts of xanthan, as judged by
precipitation
of the polymer from the broth as previously
described (
6) (not
shown). Also, the biochemical
characterization of their lipid-linked
intermediates, following
established protocols (
14), revealed
that XcORF14 is capable
of both synthesizing and decorating the
xanthan lipid-linked repeating
unit (not shown). A second mutant
in ORF14 was built up by plasmid
integration. An internal fragment
of ORF14 (nucleotides 763 to 1170;
GenBank accession no.
U70053)
was cloned into the mobilizable suicide
vector pK18mob. The resulting
plasmid was transferred to
X. campestris FC2, and the integration
of the hybrid plasmid
into the
X. campestris genome was selected
by the
vector-encoded antibiotic resistance and verified by Southern
hybridization. The phenotype of this mutant was identical to that
of
XcORF14, reinforcing the hypothesis that ORF14 might not be
related to
xanthan biosynthesis. Further studies are required
to determine the
function of the ORF14 gene
product.
To investigate whether ORF14 belongs to the
gum operon,
subfragments of the
gum region were cloned into pK18mob or
pK19mob
cloning vectors. The
gum genes encoded by
the cloned fragments
were transcribed in the orientation opposite
to that of the
lacZ promoter located on the vector. The
hybrid plasmids were transferred
to XcORF14. Integration of these
plasmids, by a single-crossover
event, caused interruption of the
gum region. Transconjugants
were verified by Southern
hybridization and assayed for their
-galactosidase (
-Gal)
activities (Fig.
3B). Integration of pGum01-19AS
resulted in strains
that displayed
-Gal activities similar to
those of the recipient
strains without the integrated plasmid
(
13) (Fig.
3B).
Whereas the integration of pGum20-19AS reduced
the
-Gal activity
mediated by the
gumM transcriptional fusion
to 10%
(
13) (Fig.
3B), the
-Gal activity of the XcORF14 strain
was not significantly affected by the integration of pGum20-19AS
or
pGum70-18AS. However, the integration of pGum71-18AS reduced
the
-Gal activity of XcORF14 more than 50%. These results suggest
that
transcription of the
gum operon may terminate within a
region
of 589 bp between the stop codon of
gumM and the
start codon of
ORF14. No clear transcriptional terminators were located
in this
fragment, although a short open reading frame, not described in
the original
gum sequence, was found. This open reading
frame,
whose deduced amino acid sequence displayed no homology to those
of proteins of known function, was termed ORF13, and it partially
overlaps sequences with weak promoter activity located immediately
upstream of ORF14, responsible for the reduced
-Gal activity
due to
integration of pGum71-18AS. These sequences display no
clear homology
to the proposed
X. campestris consensus promoter
sequences (
13) or to
E. coli 
70 promoters.
To determine whether ORF13 and
gumM belong to
the same transcriptional unit, reverse transcriptase (RT) PCR
experiments were
performed as described previously (
23),
applying a single amplification
round. Whole-cell RNA was isolated from
strain FC2 as described
previously (
13), with a further
treatment with RNase-free DNase
I (GIBCO BRL, Gaithersburg, Md.). The
nucleotide sequences of
the primers used were as follows: F1
(5'-CCGACCTGATTCCGTACCTTTG-3'),
B1
(5'-GAGAGAAAATCCAGCAAGGCG-3'), F2
(5'-CGCCAGGTGCTGGATGACAG-3'),
and B2
(5'-CGAAACCGTGCAGCCCAC-3'). A diagram showing the position
and orientation of each primer is presented in Fig.
4A. The right
primer of each
correspondent pair was employed for reverse transcription.
The results
are shown in Fig.
4B. While internal sequences of
gumM and
ORF13 could be RT-PCR amplified (Fig.
4B, lanes 2 and
5, respectively),
the expected 893-bp RT-PCR product encompassing
both sequences could
not be obtained (Fig.
4B, lane 8). These
results indicate that ORF13 is
transcribed separately from
gumM.
Therefore, the
gum transcript terminates within 418 bp downstream
of
gumM. These results, in conjunction with previous data
(
13),
show that the
gum gene cluster is expressed
as a 12-cistron operon,
from
gumB to
gumM.
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FIG. 4.
RT-PCR analysis of the 3' end of the gum
operon. (A) Scheme showing the position and orientation of the primers
(black arrows) in respect to gumM and ORF13. (B) Ethidium
bromide-stained agarose gel (1.8%) of whole-cell RNA-based RT-PCR
(lanes 2, 3, 5, 6, 8, and 9) and genome-based PCR (lanes 4, 7, and 10)
products amplified with the following primers: F1 and B1 (lanes 2 to
4), F2 and B2 (lanes 5 to 7), and F1 and B2 (lanes 8 to 10). Controls
without RT added were included (lanes 3, 6, and 9). DNA size standards
(in base pairs) are indicated (lane 1). +, present;  , absent.
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Nucleotide sequence accession number.
The GenBank
accession number of the DNA fragment determined in this work is
U70053.
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ACKNOWLEDGMENTS |
We are very grateful to Jürgen Brosius, Andreas
Schäfer, Joseph M. Cleary, and Mario Aguilar for providing
pKK223-3, the pKmob vectors, pJC372, and the strain Rm1021,
respectively. We thank Pablo Cerdán for assistance in GUS assays
and A. J. Parodi for critically reading the manuscript.
Photographs were kindly taken by Eduardo J. Rattner.
F.K. acknowledges Ph.D. grants from Universidad de Buenos Aires. Work
at the L.I. laboratory was partly supported by grants from Universidad
de Buenos Aires and from CONICET. L.I. is a member of Carrera del
Investigador (CONICET, Buenos Aires, Argentina).
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FOOTNOTES |
*
Corresponding author. Mailing address: Instituto
de Investigaciones Bioquímicas Fundación
Campomar, Patricias Argentinas 435, 1405 Buenos Aires, Argentina.
Phone: 54(1) 863-4011/19. Fax: 54(1) 865-2246. E-mail:
LIELPI{at}iib.uba.ar.
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Applied and Environmental Microbiology, January 1999, p. 278-282, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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