JOURNAL OF BACTERIOLOGY, July 1996, p. 4313–4318 Vol. 178, No.

14
0021-9193/96/$04.0010
Copyright q 1996, American Society for Microbiology

Promoter Analysis of the Xanthomonas campestris pv. campestris gum

Operon Directing Biosynthesis of the Xanthan Polysaccharide
FEDERICO KATZEN,1,2 ANKE BECKER,2 ANGELES ZORREGUIETA,1
ALFRED PÜHLER,2 AND LUIS IELPI1*
Instituto de Investigaciones Bioquı́micas Fundación Campomar, Facultad de Ciencias Exactas y Naturales
and CONICET, 1405 Buenos Aires, Argentina,1 and Lehrstuhl für Genetik,
Universität Bielefeld, D-33501 Bielefeld, Germany2
Received 11 January 1996/Accepted 6 May 1996

The Xanthomonas campestris gum gene cluster is composed of 12 genes designated gumB, -C, -D, -E, -F, -G,
-H, -I, -J, -K, -L, and -M. The transcriptional organization of this gene cluster was analyzed by the construction
of gum-lacZ transcriptional fusions in association with plasmid integration mutagenesis. This analysis, coupled
with primer extension assays, indicated that the gum region was mainly expressed as an operon from a
promoter located upstream of the first gene, gumB.

Xanthomonas campestris pv. campestris is a gram-negative gum genes predicted by computer analysis, the promoterless
bacterium, originally found as the causal agent of black rot in transposon Tn3-HoHo1 (36) was used. Random insertions
crucifers. One of the products of X. campestris is an extracel-

Downloaded from jb.asm.org by on July 12, 2007

lular polysaccharide termed xanthan gum. Xanthan consists of
polymerized pentasaccharide repeating units, composed of
glucose, mannose, and glucuronic acid in a ratio of 2:2:1 (20).
The mannose residues are acetylated and pyruvylated at spe-
cific sites, but to various degrees (5, 17, 37).
Several chromosomal regions described as xpsIII, xpsIV, and
xpsVI and a 35.3-kb gene cluster are related to the first step of
the xanthan biosynthesis (15, 25). These regions comprise gene
functions involved in the biosynthesis of the sugar nucleotide
precursors. Proteins involved in subsequent steps of xanthan
biosynthesis are proposed to be encoded by the 16-kb genome
region named xpsI, or gum (6, 14, 19). Nucleotide sequence
analysis predicted the presence of 12 open reading frames
(gumB to gumM) (6 [GenBank accession number U22511]).
Genes for specific glycosyltransferases, acetyltransferases, and
a ketal pyruvate transferase and genes with unknown function
were proposed to be encoded in this region (14, 28, 41).
Control mechanisms of xanthan biosynthesis are poorly un-
derstood. However, xanthan is produced primarily in the late
logarithmic growth phase and stationary phase (14). In addi-
tion, positive regulation and negative regulation of xanthan
production have been reported (10, 39, 40). As a first step
toward understanding the regulation of xanthan biosynthesis,
we mapped the promoters mediating the expression of differ-
ent gum genes.
Cloning and transposon mutagenesis of a fragment contain-
ing the gum region. To isolate a fragment carrying the com-
plete gum gene region of X. campestris, a genomic library of the
wild-type X. campestris strain, NRRL B-1459 (22), was con-
structed with the broad-host-range cosmid vector pRK311 (12)
by cloning of total DNA partially digested with Sau3AI. This
library was mated en masse from Escherichia coli to the Gum2
X. campestris mutant 2895 (16). One of the cosmids isolated
from several mucoid exconjugants termed pIZD15-261, con-
tains a 16-kb fragment encompassing the complete gum region
shown in Fig. 1A. To assess the direction of transcription of the
* Corresponding author. Mailing address: Instituto de Investigacio-
nes Bioquı́micas Fundación Campomar, Patricias Argentinas 435, 1405
Buenos Aires, Argentina. Phone: 54(1)863-4011/19. Fax: 54(1)865-
2246. Electronic mail address: lielpi@iris.iib.uba.ar.
within the gum region in pIZD15-261 were mapped by restric-
tion analysis. b-Galactosidase (b-Gal) activities depicted by
merodiploid X. campestris strains which mapped in the same
direction of the proposed gum open reading frames displayed
b-Gal activities higher than 50 Miller units (29), while those
which mapped in the opposite orientation depicted lacZ ex-
pression that did not differ from that of the wild-type strain
(Fig. 1A). These results showed that no apparent transcription
is present in the antisense orientation of the gum genes.
Construction of a defined set of X. campestris gum mutants.
Since Northern (RNA) blot assays gave no satisfactory results,
we decided to monitor the transcription of different gum genes
employing gum-lacZ transcriptional fusion mutants. For this
purpose, a promoterless lacZ-aacC1 interposon (3) was in-
serted into fragments of the gum gene region, which were
subcloned into the mobilizable suicide vector pK18mob or
pK19mob (33). Hybrid plasmids were transferred from the
broad-host-range mobilizing strain E. coli S17-1 (35) to X.
campestris as described previously (34). Homogenotization of
lacZ-aacC1 insertions was carried out by selection for genta-
micin resistance and subsequent screening for kanamycin sen-
sitivity. All homogenotes were verified by Southern hybridiza-
tion.
Since the existence of lethal mutations within the gum gene
cluster was reported (6), in addition to the wild-type strain, in
some cases the X. campestris mutant 3192 deficient in uridine
59-diphosphoglucose pyrophosphorylase activity (15) was used
as the receptor background for homogenotization. This strain
lacks all of the xanthan biosynthetic lipid intermediates in vivo.
Therefore, it supplies a mutant background suitable for over-
coming lethal gum mutations.
Ten of 12 wild-type gum genes were finally replaced by the
gum-lacZ transcriptional fusions by two recombination events
(Fig. 1B). No homogenotized gumB or gumH mutants could be
obtained. All gumB or gumH mutants suffered chromosomal
rearrangements as judged by Southern hybridizations. X. cam-
pestris strains were grown to an optical density at 600 nm of
between 0.4 and 0.9 in tryptone-yeast extract (TY) medium
(38) and were assayed for their b-Gal activity (1). b-Gal activ-
ities of the wild-type strain and mutant 3192 were 3.1 6 1.2 and
2.2 6 0.6 Miller units, respectively. All of the homogenotized

4313
4314 NOTES J. BACTERIOL.

Downloaded from jb.asm.org by on July 12, 2007

FIG. 1. Tn3::HoHo1 mutagenesis and mapping of promoters in the gum region. (A) Line representing cosmid pIZD15-261, along with random insertions of Tn3::
HoHo1. The lacZ genes encoded by Tn3::HoHo1 insertions indicated above the line are oriented from left to right, whereas insertions shown below the gum genes carry
lacZ oriented from right to left. Solid circles represent b-Gal activities higher than 50 Miller units; open circles represent b-Gal activities similar to the background activ-
ity of the wild type. (B) Map of relevant restriction sites and the gene structure of the gum region. The position and orientation of lacZ transcriptional fusions are
indicated by triangles. Solid triangles represent lacZ-aacC1 insertions in the X. campestris wild-type strain; open triangles represent lacZ-aacC1 insertions in X. cam-
pestris 3192. The relative activities of gum-lacZ transcriptional fusions are given in b-Gal units (Miller units) above the line. Below the line, restriction fragments used
for integration mutagenesis are presented. Potential promoters located on the fragments used for plasmid integration mutagenesis are indicated by solid boxes. The rela-
tive b-Gal activities of the gum-lacZ transcriptional fusions located downstream from the vector integration sites are listed. Relative b-Gal activities given in Miller units
(6 standard deviation) were calculated from five independent measurements. ND, not determined; NC, not considered (see text). Both A and B are in the same scale.

strains displayed b-Gal activities significantly higher than these
values (Fig. 1B).
The transcription of the gum genes is essentially directed by
a single promoter. To map the promoters directing gum gene
transcription, plasmids carrying different subfragments of this
region were integrated into the chromosome of X. campestris
strains carrying gum-lacZ transcriptional fusions. For this pur-
pose, subfragments of the gum region were cloned into the
mobilizable suicide vector pK18mob or pK19mob. The gum
genes encoded by the fragments cloned were transcribed in the
opposite orientation to the lacZa promoter located on the vec-
tor. Integration of these plasmids, by a single crossover event,
VOL. 178, 1996
FIG. 2. Primer extension analysis of the gumB promoter. (A) Fluorogram
obtained from a primer extension assay with a fluorescein-labelled primer cor-
responding to positions 1464 to 1445 of the gum sequence. The peak represents
the cDNA obtained after retrotranscription at 428C for 1 h. Annealing was
carried out at 428C for 2 h. The arrow indicates the direction of transcription.
The transcription start site represented by a T (11) is marked by a shaded box.
(B) The sequence of the segment upstream of gumB ranging from positions 1160
to 1196 was determined with the same primer. The fluorograms in A and B were
inverted to match the coded strand. (C) Partial sequence of the gum region
between positions 1081 and 1480. The forward arrow indicates the transcription
start site, while the backward arrow shows the primer used. The start codon for
gumB is printed in boldface. No evident Shine-Dalgarno sequence at an appro-
priate location could be found. Putative promoter regions are underlined.
caused interruption of the gum region. Transconjugants were
verified by Southern hybridization and assayed for their b-Gal
activities (Fig. 1B). The interposon placed in distinct locations
within a fragment which is transcribed as a single operon may
alter the secondary structure and half-life of the mRNA. There-
fore, the b-Gal activities shown in Fig. 1B have to be compared
within columns and not within rows. Integration of pGum01-
19AS, carrying the entire gumB coding region and 1,231 bp up-
stream of the putative gumB start codon (GenBank accession
number U22511) resulted in strains that displayed b-Gal activities
similar to those of the recipient strains without the integrated
hybrid plasmid (Fig. 1B). Integration of pGum22-18AS carrying
almost the complete gumB sequence and 28 bp upstream of its
putative start codon significantly reduced the b-Gal activities of
all recipients. Similar effects were observed for the other inte-
grated constructions shown in Fig. 1B. This proves that the frag-
ment located upstream of the putative gumB start codon is in-
volved in the transcription of the whole region.
Between positions 35 and 400 of the gum region, we found
about 70% identity to integration host factor genes from dif-
ferent microorganisms. In addition, a DNA fragment homol-
ogous to proline tRNA genes is located between positions 732
and 808. These putative genes seem to have no direct relation-
ship with xanthan biosynthesis. Therefore, transcription-pro-
moting sequences might be located within 500 bp between the
39 end of the tRNA gene and position 1308, which corresponds to
the 59 end of the fragment cloned in pGum22-18AS (Fig. 1B).
NOTES 4315
A second weak promoter may exist upstream of gumK. In-
tegration of pGum27-19AS into the chromosome reduced the
b-Gal activity of the homogenotized strain slightly more than
the preceding integrated plasmid pGum26-19AS. Compared
with the original homogenotes, integration of pGum26-19AS
reduced the b-Gal activities of the gumL-lacZ-aacC1 and the
gumM-lacZ-aacC1 mutants to 33 and 17%, respectively (Fig.
1B). In contrast, integration of pGum27-19AS, pGum17-19AS,
or pGum20-19AS diminished the b-Gal activities of the same
strains to 6 and 9%, respectively (Fig. 1B). Hence, it is possible
that a weak promoter exists upstream of gumK between posi-
tions 12246 and 12961 of the gum region, which correspond
to the 59 ends of the fragments cloned in pGum26-19AS and
pGum27-19AS, respectively (Fig. 1B). These results correlate
to the fact that the intergenic region between gumJ and gumK
is located between positions 12384 and 12764 of the gum re-
gion. b-Gal activities of the derivated Xc405J recipient strains
were not considered because an artificial transcription start site
was created upon insertion of the lacZ-aacC1 interposon (data
not shown).
Mapping of the 5* end of two gum transcripts. To identify
the transcriptional start site of both promoters mapped by
plasmid integration, primer extension experiments were car-
ried out. An automatic fluorescent primer extension assay was
Downloaded from jb.asm.org by on July 12, 2007
performed as described previously (31), with a 20-mer oligo-
nucleotide (59-CACCTTCACCAGCAACAGAT-39) comple-
mentary to the 59 terminus of the gumB gene. A single primer
extension product could be detected (Fig. 2). The first nucle-
otide to be transcribed is a thymidine located 158 nucleotides
upstream of the proposed gumB start codon. Alternatively,
32
P-radiolabelled primer extension experiments were per-
formed (43) to determine the transcription start site used by
the second promoter located upstream of gumK. In this case,
the oligonucleotide 59-CCACAGGTAACAGTCCACAC-39,
complementary to the 59 terminus of gumK, was employed.
The transcription start site corresponds to an adenosine
located 141 bp upstream of the proposed gumK start codon
(Fig. 3).
For both primer extension analyses, total RNA was pre-
pared as described previously (8), with harvest of the cells,
alternatively grown in TY or yeast-malt extract (YM) (15)
medium, at an optical density at 600 nm of between 0.4 and 0.9.
EDTA-treated X. campestris wild-type cells grown in both me-
dia are able to produce exopolysaccharide in vitro (21).
Under the rules depicted previously (42), no rho-indepen-
dent transcription terminator structure could be found at the
end of the gum sequence. We therefore suggest that the gum
region is expressed as a single operon with a size of at least 15
kb, in conjunction with a second transcript encompassing the
last three genes. Large operons related to the biosynthesis of
bacterial polysaccharides were also described in Rhizobium
meliloti as an 11.65-kb exoHKLAMONP gene cluster involved
in the biosynthesis of exopolysaccharide I (2), in Pseudomonas
aeruginosa as a 18-kb large biosynthetic gene cluster essential
for the synthesis of the alginate (7), in Pseudomonas solanacea-
rum as a 15-kb portion of the eps gene cluster related to
exopolysaccharide I biosynthesis (18), or in Erwinia amylovora
as a 16-kb transcript of the ams region required for the amy-
lovoran biosynthesis (4).
A comparison of the putative promoter regions located up-
stream of the transcription start sites with putative promoter
elements of other X. campestris genes reported previously re-
vealed some similarities (Table 1). In contrast to the high GC
content of X. campestris, putative promoter regions are AT rich
and display homology to E. coli s70 promoters (26): 3 of 6 bp
in 235 and 5 of 6 210 E. coli consensus hexamers may be
4316 NOTES J. BACTERIOL.

Downloaded from jb.asm.org by on July 12, 2007

FIG. 3. Primer extension analysis of the gumK promoter. (A) Primer extension assay with a radiolabelled primer corresponding to positions 12703 to 12684 of the
gum sequence. Lanes: 1, assay with 100,000 cpm of radiolabelled primer and 50 mg of total RNA; 2, assay with 100,000 cpm of radiolabelled primer without RNA; 3,
assay with 50,000 cpm of radiolabelled primer and 50 mg of total RNA. In all cases, the annealing step was performed at 508C for 2 h, and the extension reactions were
carried out at 458C for 1 h. A, C, G, and T correspond to the parallel sequence ladder created with the same primer. The sequence depicted is the reverse complement
of the one in panel B. (B) Partial sequence of the gum region between positions 12541 and 12780. The forward arrow indicates the transcription start site, while the
backward arrow shows the primer used. The start codon for gumK and its corresponding. Shine-Dalgarno sequence are printed in boldface. Putative promoter regions
are underlined.

conserved in X. campestris. Compared with E. coli promoters,
X. campestris displays larger spacers between the putative left
and right promoter regions (Table 1): the spacer size in X.
campestris ranges from 17 to 43 bp, while the functional spacer
length in E. coli may vary from 15 to 19 bp (26). Whether the
proposed sequence motifs might function as promoter ele-
ments in the X. campestris gum region deserves further exper-
iments.
No promoter activity was found upstream of gumD. It was
suggested that a promoter element exists upstream of gumD (6,
32). Plasmid integration mutagenesis showed no evidence for
the existence of a promoter in that area (Fig. 1B). Neverthe-
less, primer extension analysis was performed to identify a
putative transcription initiation in this region, but no product
could be obtained (data not shown). In addition, we subcloned
250 bp adjacent to the 59 terminus of gumD into a broad-host-
range promoter probe vector, and its expression level in X.
campestris did not differ from that in control experiments (data
not shown). Although this fragment presents homology to s70
promoters (26), we suggest that this promoter-like sequence is
not functional in X. campestris under our experimental condi-
tions.

TABLE 1. Compilation of promoter region sequences of X. campestris

X. campestris Transcription
Gene Promoter sequence motifa Source or reference
pathovar startb

gumB campestris TTGTGC [43] GATTCA [19]T This work

gumJ-lacZ campestris TTGTTG [17] GATCAA [19]A This work
gumK campestris GTGGCA [22] GATACA [14]A This work
Protease campestris TTGTCC [37] GATAAC ND 27
xpsE campestris TTGTGG [26] GATCCA ND 13
fruA campestris CTGGCA [18] TCGAAT ND 9
rpfC campestris CTGTCA [29] TCTGAT ND 40
avrb6 malvacearum GTGTAC [20] TATGTA ND 11
avrBs3 vesicatoria GTGTAC [20] TATGTA [8]A 24
Putative poannua TTGTCA [21] TAGAAT ND GenBank accession no. U09965
opsX citrumelo TTGGGT [17] TGAAAT [7]G 23
hemL phaseoli TTCCAA [21] GATCAT ND 30

Consensus X. campestris TTGTNN [ND] T A ND This work

G ATNAT
Consensus E. coli TTGACA [15–19] TATAAT ND 26
a
Numbers in brackets represent base pairs between left and right promoter regions. Letters in boldface are part of the consensus sequence.
b
Numbers in brackets represent base pairs between the right region and the transcription start site when determined. ND, not determined.
VOL. 178, 1996
Conclusions. In this work, two promoters, including their
respective transcription start sites, were identified in X. cam-
pestris through application of gum-lacZ transcriptional fusions
combined with plasmid integration and primer extension as-
says. Nevertheless, the possibility of the existence of other
secondary promoters, silent under our growth conditions, can-
not be ruled out. The identification of promoter regions in-
volved in xanthan production sets the basis for further studies
of gum gene regulation. Furthermore, modifying the described
promoter regions or changing them by using others with dif-
ferent host specificity may allow the expression of these genes
in heterologous systems. In addition, gum-lacZ transcriptional
fusions are a useful tool with which to study gum gene expres-
sion under different environmental conditions.
We thank Nancy E. Harding (Kelco, a unit of Monsanto Co.) for
kindly providing X. campestris 2895 and 3192. We also thank Helge
Küster for critically reviewing the manuscript.
F.K. acknowledges Ph.D. grants from Universidad de Buenos Aires,
Deutscher Akademischer Austauschdienst, and the Deutsche For-
schungsgemeinschaft (Sonderforschungbereich 223). This work was
supported partly by grant Ex-240 from Universidad de Buenos Aires to
L.I. A.Z. and L.I. are members of Carrera del Investigador
(CONICET, Buenos Aires, Argentina).
REFERENCES
1. Aguilar, O. M., D. Kapp, and A. Pühler. 1985. Characterization of a Rhizo-
bium meliloti fixation gene (fixF) located near the common nodulation re-
gion. J. Bacteriol. 164:245–254.
2. Becker, A., A. Kleickmann, M. Keller, W. Arnold, and A. Pühler. 1993.
Identification and analysis of the Rhizobium meliloti exoAMONP genes in-
volved in exopolysaccharide biosynthesis and mapping of promoters located
on the exoHKLAMONP fragment. Mol. Gen. Genet. 241:367–379.
3. Becker, A., M. Schmidt, W. Jäger, and A. Pühler. 1995. New gentamicin-
resistance and lacZ promoter-probe cassettes suitable for insertion mutagen-
esis and generation of transcriptional fusions. Gene 162:37–39.
4. Bugert, P., and K. Gelder. 1995. Molecular analysis of the ams operon
required for exopolysaccharide synthesis of Erwinia amylovora. Mol. Micro-
biol. 15:917–933.
5. Cadmus, M., S. Rogovin, K. Burton, J. Pittsley, C. Knutson, and A. Jeanes.
1976. Colonial variation in Xanthomonas campestris NRRL B-1459 and char-
acterization of the polysaccharide from a variant strain. Can. J. Microbiol.
22:942–948.
6. Capage, M. A., D. H. Doherty, M. R. Betlach, and R. W. Vanderslice. March
1987. Recombinant-DNA mediated production of xanthan gum. Interna-
tional patent WO87/05938.
7. Chitnis, C., and D. Ohman. 1993. Genetic analysis of the alginate biosyn-
thetic gene cluster of Pseudomonas aeruginosa shows evidence of an operonic
structure. Mol. Microbiol. 8:583–590.
8. Chomczynski, P., and N. Sacchi. 1987. Single step method of RNA isolation
by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Bio-
chem. 162:156–159.
9. de Crécy-Lagard, V., O. Bouvet, P. Lejeune, and A. Danchin. 1991. Fructose
catabolism in Xanthomonas campestris pv. campestris. Sequence of the PTS
operon, characterization of the fructose-specific enzymes. J. Biol. Chem.
266:18154–18161.
10. de Crécy-Lagard, V., P. Glaser, P. Lejeune, O. Sismeiro, C. E. Barber, M. J.
Daniels, and A. Danchin. 1990. A Xanthomonas campestris pv. campestris
protein similar to catabolite activation factor is involved in regulation of
phytopathogenicity. J. Bacteriol. 172:5877–5883.
11. De Feyter, R., Y. Yang, and D. Gabriel. 1993. Gene-for-genes interactions
between cotton R genes and Xanthomonas campestris pv. malvacearum avr
genes. Mol. Plant-Microbe Interact. 6:225–237.
12. Ditta, G., T. Schmidhauser, E. Yakobson, P. Lu, X. Liang, D. Finlay, D.
Guiney, and D. R. Helinski. 1985. Plasmid related to the broad host range
vector, pRK290, useful for gene cloning and for monitoring gene expression.
Plasmid 13:149–153.
13. Dums, F., J. Dow, and M. J. Daniels. 1991. Structural characterization of
protein secretion genes of the bacterial phytopathogen Xanthomonas cam-
pestris pathovar campestris: relatedness to secretion systems of other Gram-
negative bacteria. Mol. Gen. Genet. 229:357–364.
14. Harding, N. E., J. M. Cleary, and L. Ielpi. 1995. Genetics and biochemistry
of xanthan gum production by Xanthomonas campestris, p. 495–514. In Y. H.
Hui and G. Khachatourians (ed.), Food biotechnology microorganisms.
VCH Publishers, Inc., New York.
15. Harding, N. E., S. Raffo, A. Raimondi, J. M. Cleary, and L. Ielpi. 1993.
NOTES 4317
Identification, genetic and biochemical analysis of genes involved in syn-
thesis of sugar nucleotide precursors of xanthan gum. J. Gen. Microbiol.
139:447–457.
16. Harding, N. E., J. M. Cleary, D. K. Cabañas, I. G. Rosen, and K. S. Kang.
1987. Genetic and physical analysis of genes essential for xanthan gum
biosynthesis in Xanthomonas campestris. J. Bacteriol. 169:2854–2861.
17. Hassler, R. A., and D. Doherty. 1990. Genetic engineering of polysaccharide
structure: production of variants of xanthan gum in Xanthomonas campestris.
Biotechnol. Prog. 6:182–187.
18. Huang, J., and M. Schell. 1995. Molecular characterization of the eps gene
cluster of Pseudomonas solanacearum and its transcriptional regulation at a
single promoter. Mol. Microbiol. 16:977–989.
19. Ielpi, L., R. O. Couso, and M. A. Dankert. 1993. Sequential assembly and
polymerization of the polyprenol-linked pentasaccharide repeating unit of
the xanthan polysaccharide in Xanthomonas campestris. J. Bacteriol. 175:
2490–2500.
20. Jansson, P. E., L. Keene, and B. Lindberg. 1975. Structure of the extracel-
lular polysaccharide from Xanthomonas campestris. Carbohydr. Res. 45:275–
282.
21. Katzen, F., and L. Ielpi. Unpublished data.
22. Kidby, D., P. Sandford, A. Herman, and M. Cadmus. 1977. Maintenance
procedures for the curtailment of genetic instability: Xanthomonas campes-
tris NRRL B-1459. Appl. Environ. Microbiol. 33:840–845.
23. Kingsley, M., D. W. Gabriel, G. C. Marlow, and P. D. Roberts. 1993. The
opsX locus of Xanthomonas campestris affects host range and biosynthesis of
lipopolysaccharide and extracellular polysaccharide. J. Bacteriol. 175:5839–
5850.
24. Knoop, V., B. Staskawicz, and U. Bonas. 1991. Expression of the avirulence
gene avrBs3 from Xanthomonas campestris pv. vesicatoria is not under the
control of hrp genes and is independent of plant factors. J. Bacteriol. 173:
Downloaded from jb.asm.org by on July 12, 2007
7142–7150.
25. Köplin, R., W. Arnold, B. Hötte, R. Simon, G. Wang, and A. Pühler. 1992.
Genetics of xanthan production in Xanthomonas campestris: the xanA and
xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis. J.
Bacteriol. 174:191–199.
26. Lisser, S., and H. Margalit. 1993. Compilation of E. coli mRNA promoter
sequences. Nucleic Acids Res. 21:1507–1516.
27. Liu, Y., J. Tang, B. Clarke, J. Dow, and M. J. Daniels. 1990. A multipurpose
broad host range cloning vector and its use to characterize an extracellular
protease gene of Xanthomonas campestris pathovar campestris. Mol. Gen.
Genet. 220:433–440.
28. Marzocca, M. P., N. E. Harding, E. A. Petroni, J. M. Cleary, and L. Ielpi.
1991. Location and cloning of the ketal pyruvate transferase gene of Xan-
thomonas campestris. J. Bacteriol. 173:7519–7524.
29. Miller, J. 1992. A short course in bacterial genetics. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.
30. Murakami, K., S. Korbsrisate, N. Asahara, Y. Hashimoto, and Y. Murooka.
1993. Cloning and characterization of the glutamate 1-semialdehyde amin-
omutase gene from Xanthomonas campestris pv. phaseoli. Appl. Microbiol.
Biotechnol. 38:502–506.
31. Myöhänen, S., and J. Wahlfors. 1993. Automated fluorescent primer exten-
sion. BioTechniques 14:16–17.
32. Pollock, T. J., L. Thorne, M. Yamazaki, M. Mikolajczak, and R. W. Armen-
trout. 1994. Mechanism of bacitracin resistance in gram-negative bacteria
that synthesize exopolysaccharides. J. Bacteriol. 176:6229–6237.
33. Schäfer, A., A. Tauch, W. Jäger, J. Kalinowski, G. Thierbach, and A.
Pühler. 1994. Small mobilizable multi-purpose cloning vectors derived
from the Escherichia coli plasmids pK18 and pK19: selection of defined
deletions in the chromosome of Corynebacterium glutamicum. Gene 145:
69–73.
34. Simon, R. 1984. High frequency mobilization of Gram-negative bacterial
replicons by the in vitro constructed Tn5-Mob transposon. Mol. Gen. Genet.
196:413–420.
35. Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilization
system for in vivo genetic engineering: transposon mutagenesis in Gram-
negative bacteria. Bio/Technology 1:784–791.
36. Stachel, S., G. An, C. Flores, and E. Nester. 1985. A Tn3 lacZ transposon for
the random generation of b-galactosidase gene fusions: application to the
analysis of gene expression in Agrobacterium. EMBO J. 4:891–898.
37. Stankowski, J., B. Mueller, and S. Zeller. 1993. Location of a second O-
acetylgroup in xanthan gum by the reductive-cleavage method. Carbohydr.
Res. 241:321–326.
38. Steinmann, D., H. Wiggerich, K. Bärbel, U. Schramm, and A. Pühler. 1993.
Saturation mutagenesis in Escherichia coli of a cloned Xanthomonas cam-
pestris DNA fragment with the lux transposon Tn4431 using the delivery
plasmid pDS1, thermosensitive in replication. Appl. Microbiol. Biotechnol.
40:356–369.
39. Tang, J., C. Gough, and M. J. Daniels. 1990. Cloning of genes involved in
negative regulation of production of extracellular enzymes and polysaccha-
ride of Xanthomonas campestris pathovar campestris. Mol. Gen. Genet. 222:
157–160.
40. Tang, J., Y. Liu, C. Barber, J. Dow, J. Wooton, and M. J. Daniels. 1991.
4318 NOTES
Genetic and molecular analysis of a cluster of rpf genes involved in positive
regulation of synthesis of extracellular enzymes and polysaccharide in Xan-
thomonas campestris pathovar campestris. Mol. Gen. Genet. 226:409–417.
41. Vanderslice, R. W., D. H. Doherty, M. A. Capage, M. R. Betlach, R. A. Has-
sler, N. M. Henderson, J. Ryan-Graniero, and M. Tecklenburg. 1989. Ge-
netic engineering of polysaccharide structure in Xanthomonas campestris, p.
145–156. In V. Crescenzi, I. C. M. Dea, S. Paoletti, S. S. Stivala, and I. W.
J. BACTERIOL.
Sutherland (ed.), Biomedical and biotechnological advances in industrial
polysaccharides. Gordon and Breach, New York.
42. von Heijne, G. 1987. Sequence analysis in molecular biology. Treasure trove
or trivial pursuit. Academic Press, Inc., London.
43. Waldbeser, L., M. E. Tolmasky, L. Actis, and J. Crossa. 1993. Mechanisms
for negative regulation by iron of the fatA outer membrane protein gene
expression in Vibrio anguillarum 775. J. Biol. Chem. 268:10433–10439.
Downloaded from jb.asm.org by on July 12, 2007