Journal of Bacteriology, May 2001, p. 2715-2723, Vol. 183, No. 9
Department of Science and Mathematics,
University of Texas of the Permian Basin, Odessa, Texas
79762,1 and Section of Molecular
Genetics and Microbiology, The University of Texas at Austin, Austin,
Texas 787122
Received 22 November 2000/Accepted 20 February 2001
Plesiomonas shigelloides is a gram-negative pathogen
which can utilize heme as an iron source. In previous work, P. shigelloides genes which permitted heme iron utilization in a
laboratory strain of Escherichia coli were isolated. In the
present study, the cloned P. shigelloides sequences were
found to encode ten potential heme utilization proteins: HugA, the
putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic
binding protein; HugCD, the putative inner membrane permease; and the
proteins HugW, HugX, and HugZ. Three of the genes, hugA,
hugZ, and tonB, contain a Fur box in their putative
promoters, indicating that the genes may be iron regulated. When the
P. shigelloides genes were tested in E. coli
K-12 or in a heme iron utilization mutant of P. shigelloides, hugA, the TonB system genes, and hugW, hugX, or
hugZ were required for heme iron utilization. When the
genes were tested in a hemA entB mutant of E. coli,
hugWXZ were not required for utilization of heme as a porphyrin
source, but their absence resulted in heme toxicity when the strains
were grown in media containing heme as an iron source. hugA
could replace the Vibrio cholerae hutA in a heme iron
utilization assay, and V. cholerae hutA could complement a
P. shigelloides heme utilization mutant, suggesting that
HugA is the heme receptor. Our analyses of the TonB system of P. shigelloides indicated that it could function in tonB
mutants of both E. coli and V. cholerae and
that it was similar to the V. cholerae TonB1 system in the
amino acid sequence of the proteins and in the ability of the system to
function in high-salt medium.
Plesiomonas shigelloides
is a gram-negative bacterium associated with diarrheal disease in
humans (4). The organism has been reported to cause
several types of gastroenteritis, including acute secretory
gastroenteritis (33), an invasive shigellosis-like disease
(35), and a cholera-like illness (55).
Extraintestinal infections, such as meningitis, bacteremia
(2), and pseudoappendicitis (13), are also
associated with P. shigelloides infection.
Many bacterial pathogens have iron transport systems that play a
critical role in allowing the organism to establish an infection (for
reviews, see references 31 and 41). Several bacterial pathogens, including P. shigelloides (9),
obtain iron from heme or heme-containing compounds. Heme iron
utilization systems have been examined in many gram-negative pathogens,
including Vibrio cholerae (20, 21, 38),
Vibrio vulnificus (30), Shigella
dysenteriae (37, 60), Escherichia coli
O157:H7 (54), Serratia marcescens (16,
26, 27), yersiniae (22, 50, 51, 53),
Haemophilus (8, 11, 24, 32), neisseriae (7, 29, 52, 64), Pseudomonas (23, 28,
39), and Porphyromonas gingivalis (47).
Many heme iron utilization systems studied to date require an outer
membrane receptor which binds heme from the environment (for a review,
see reference 58). TonB, with the aid of ExbBD, is thought
to interact with the receptor to allow movement of heme into the
periplasm. A periplasmic binding protein then moves the heme across the
periplasm, and inner membrane permeases transport the heme into the cytoplasm.
A question yet to be resolved is what happens to the heme once it
enters the cytoplasm of the cell. It is clear that heme iron is
utilized as an iron source, but it is not clear how the heme is broken
down. Recently, genes have been identified in the pathogenic neisseriae
(64) and in the gram-positive pathogen Corynebacterium diphtheriae (45) that encode
heme oxygenases which may break down the heme, releasing the iron into
the cell. The heme oxygenase genes are required for heme iron
utilization in both organisms. Whether other heme iron-utilizing
bacteria use a similar mechanism to remove the iron from heme has not
been determined.
In a previous study, P. shigelloides heme iron utilization
genes were isolated (9). The goal of the present study was
to further characterize the P. shigelloides system by
examining the genes required for heme iron utilization and determining
the functions of the proteins they encode. Our results indicate that
the heme utilization system of P. shigelloides is most
similar to that of V. cholerae in terms of the amino acid
sequence and function of many of the proteins, the regulation of some
of the genes, and in the linkage of TonB system genes to the heme iron
utilization locus. In addition, we have identified a set of genes, one
or more of which is required for heme iron utilization but not
utilization of heme as a porphyrin source.
Strains.
Bacterial strains, plasmids, and their sources are
listed in Table 1. DPH-2 was a
spontaneous heme utilization mutant isolated from P. shigelloides 9 after nalidixic acid enrichment as previously described (20). DHE-1, a hemA entB mutant of
E. coli, was created by P1 transduction of a lysate from the
entB mutant E. coli AB1515.24 (48)
into the hemA mutant RK1065L.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.9.2715-2723.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of the Plesiomonas
shigelloides Genes Encoding the Heme Iron Utilization
System
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids
Media, chemicals, and enzymes.
Bacterial strains were
routinely grown at 37°C in Luria (L) broth or on L agar.
Ethylenediamine-di-(o-hydroxyphenyl acetic acid) (EDDA),
deferrated as described by Rogers (44), was added to L
broth or L agar to chelate nonheme iron. Antibiotics and supplements
were used in the following concentrations: carbenicillin, 25 µg/ml;
kanamycin, 50 µg/ml; chloramphenicol, 30 µg/ml (for E. coli) and 10 µg/ml (for P. shigelloides);
tetracycline, 12.5 µg/ml (for E. coli) and 4.2 µg/ml
(for P. shigelloides); 
-aminolevulinic acid (ALA), 80 µg/ml; and hemin, 7.6 µM.
-Galactosidase assays.
-Galactosidase assays were
performed on mid-log-phase cultures as described by Miller
(36) on E. coli DH5
that had been transformed with pHUGlac1 or pTONlac1. Overnight
L broth cultures were washed twice in M9 medium (36) and
diluted 1:25 into M9 medium containing 0.3% Casamino Acids deferrated
as described by Pugsley and Reeves (43). Low-iron medium
contained 300 µg of EDDA per ml, and high-iron medium contained 40 µM FeSO4. Independent experiments were conducted on each
strain grown under the same conditions on three separate occasions.
Growth assays. To detect the utilization of heme as an iron source, overnight cultures of E. coli 1017 containing the indicated plasmid(s) were diluted 1 to 1,000 into L broth or L broth containing 200 µg of EDDA per ml with or without hemin. Absorbance at 600 nm was measured after 15 to 18 h of growth. An absorbance of 1.4 or above was considered a positive result and 0.5 or below a negative result. In bioassays to detect utilization of heme as a porphyrin source, mid-log-phase cultures of DHE-1 were washed with saline and seeded into L agar with kanamycin at 5 × 104 cells/ml. Five microliters of 20-mg/ml ALA or 95 µM heme was spotted onto the media, and zones of growth were measured after 15 h. Bioassays to detect utilization of heme as an iron source in DHE-1 were performed in the same manner, except the cells were seeded into L agar containing ALA and EDDA (300 µg/ml). Other heme iron utilization bioassays were performed in manners similar to those of the three assays described above, with specific details being provided in the appropriate table.
Electroporation and triparental matings. The electroporation of P. shigelloides was performed as previously described (38). Triparental mating with the mobilizing strain MM294/pRK2013 also was used to transfer recombinant plasmids into P. shigelloides.
DNA sequencing and analysis. The DNA sequence of both strands of the insert in pHUG10 and other recombinant plasmids was determined with an ABI Prism 377 DNA sequencer from Applied Biosystems and was analyzed with the DNA Strider program (34). The BLAST program of the National Center for Biotechnology Information (1) was used to determine homologies of the deduced amino acid sequences, and MacVector ClustalW was used to determine protein identity and similarity.
Nucleotide sequence accession number. The nucleotide and amino acid sequences corresponding to this region can be found under GenBank/EMBL accession no. AY008342.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Nucleotide sequence analysis of P. shigelloides heme
iron utilization genes.
The genetic organization of the P. shigelloides heme iron utilization locus was determined by DNA
sequence analysis of pHUG10, a plasmid which enables E. coli
1017 to utilize heme as an iron source, and of pHUG16, which contains
hugBCD. Our analyses indicated the presence of 10 open
reading frames (ORFs) which spanned approximately 10,000 nucleotides.
Figure 1A shows the genetic organization
of the region. The calculated molecular weights and pIs for these proteins and the percent identity and similarity of the predicted proteins to selected proteins in other organisms are shown in Table
2.
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Predicted promoters for heme utilization genes.
Each of the
genes in the P. shigelloides heme utilization locus except
hugW, exbD, and hugBCD appears to contain its own
promoter (Fig. 1A). The predicted promoter for hugZ contains
a potential Fur box upstream of the putative 
35 region (Fig.
2A). The putative promoters for
hugAW and tonB are divergent overlapping
promoters and also contain a sequence that resembles a Fur box (Fig.
2B). The putative Fur box, which overlaps the 10 region of each
promoter, shares 12 of 19 nucleotides with the consensus sequence of
the E. coli Fur box (10) and 13 of 19 nucleotides with the V. cholerae viuA downstream Fur box
(5) (data not shown). To assess regulation of the
predicted hugAW-tonB promoters, the hugA-tonB
intergenic region was cloned in both orientations upstream of the
promoterless lacZ gene in pQF50 to create
pHUGlac1 and pTONlac1. -Galactosidase assays
were performed on E. coli DH5 containing the indicated plasmids following growth in high- or low-iron medium. When either transformed strain was grown in high-iron medium, the level of -galactosidase activity was approximately 30 U. When the strains were grown under low-iron conditions, activity increased approximately 19-fold for both strains (577 in DH5/pHUGlac1 and 558 in
DH5/pTONlac1). These data indicate that promoters in the
hugA-tonB intergenic region are iron regulated.
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Reconstitution of the P. shigelloides heme iron
utilization system in E. coli 1017.
To determine which
genes are necessary for heme iron utilization, the Ent
E. coli strain 1017 containing subclones of pHUG10 was
tested for growth in L broth containing heme as the sole iron source (Fig. 1A). E. coli 1017/pHUG10 grew well in this medium,
indicating that hugBCD are not required for heme iron
utilization. E. coli 1017/pHUG4 also grew in media with heme
as the iron source, indicating that sequences downstream of
hugZ are not required for heme iron utilization. E. coli 1017/pHUG3, containing only hugA, or 1017/pHUG7, containing hugAWXZ, did not utilize heme as an iron source,
indicating that the P. shigelloides TonB system genes are
required. This was confirmed when 1017 containing both pHUG7 and pHUG2
was able to utilize heme. Heme iron utilization did not occur when
1017/pHUG7 was transformed with pHUG2.1, which contains a kanamycin
cassette that disrupts tonB, or when the exbBD
genes from pHUG2 were removed (pHUG1). These data indicate that
P. shigelloides TonB requires ExbBD to function and that
E. coli ExbBD cannot substitute for the P. shigelloides counterparts. To determine if the genes downstream of
hugA are required for heme iron utilization, E. coli 1017 transformed with pHUG8, which contains hugA,
tonB, and exbBD but is missing hugWXZ, was
tested, and the strain failed to utilize heme.
Genes required for utilization of heme as a porphyrin source. We wanted to determine if the genes needed for utilization of heme as an iron source are also required for utilization of heme that is incorporated in intact form into heme proteins. Bioassays were conducted with DHE-1, a hemA entB mutant of E. coli, which cannot synthesize heme in the absence of the precursor ALA and cannot transport heme to meet the porphyrin requirement. In the assay, the strains were seeded into L agar, and ALA or heme was spotted onto the plates.
DHE-1/pHUG10 grew around the heme spot, indicating that pHUG10 contained the necessary genes for heme to be utilized as a porphyrin source (Fig. 1A). DHE-1 containing pHUG8, which has only hugA, tonB, and exbBD, also could utilize heme as a porphyrin source, suggesting that hugWXZ are not required for utilization of heme as a porphyrin source. The strain transformed with pHUG3.1, which carries hugA, could not utilize heme as a porphyrin source. These data indicate that the heme receptor gene and the TonB system genes are required for the utilization of heme as a porphyrin source.hugW, -X, or -Z is needed to prevent heme toxicity. We also seeded the strains into iron-restricted medium containing ALA and supplied heme as an iron source. As expected, E. coli DHE-1/pHUG10 grew well in the assay, exhibiting a zone of growth of approximately 12 mm. In contrast, DHE-1/pHUG8 grew poorly and appeared to be inhibited by the higher concentration of heme present in the center of the spot. The strain exhibited a 14-mm zone of inhibition surrounded by a very faint 3-mm zone of growth. This suggested that the high level of heme was toxic to the cells.
We also determined the effect of several different concentrations of heme on the growth of DHE-1/pHUG8 and found that at higher heme concentrations, the zone of inhibition increased in diameter (data not shown). Because it is possible that growing the strain in media containing both ALA and heme resulted in the accumulation of toxic intermediates in the heme biosynthetic pathway that would not be present when either heme or ALA were present alone, the heme iron assay was performed with no ALA in the L agar. Growth around the heme spots was essentially the same as that observed when ALA was supplied in the L agar (data not shown). Growth inhibition in the presence of heme was also observed in a HemA+ strain (E. coli 1017), indicating that the inhibition is unlikely to be associated with the hemA mutation. These data suggest that hugW, -X, or -Z is necessary to prevent heme toxicity when heme is supplied as an iron source. It is not clear why heme supplied as an iron source, but not as a porphyrin source, is toxic to the cells. This could be due to the fact that when heme was supplied as an iron source, the cells are grown under low-iron conditions, which increases the expression of heme utilization proteins and which may allow higher levels of heme to enter the cytoplasm.Determination that HugA is a heme receptor.
Our sequencing
data indicated that HugA shares homology with a number of heme
receptors, including V. cholerae HutA. Thus, we determined
if HugA could function as a heme receptor by testing its ability to
substitute for V. cholerae HutA. The V. cholerae heme utilization system can be reconstituted in E. coli 1017 by transforming the strain with recombinant plasmids pHUT3, which carries hutA, and pHUT10, which carries tonB1,
exbB1D1, hutBCD, and hutWXZ. When E. coli
1017/pHUT3/pHUT10 was tested for growth in L broth with EDDA and heme,
the strain utilized heme as an iron source, whereas E. coli
1017/pHUG10 could not, confirming previously published results
(20) (Table 3). To determine
if HugA can substitute for HutA, we moved pHUG3, which carries the P. shigelloides gene hugA, into 1017/pHUT10 to
create a chimeric heme iron utilization system. This strain grew as
well as 1017/pHUT3/pHUT10 in L broth with EDDA and heme, indicating
that hugA encodes a protein that can replace the function of
the V. cholerae HutA.
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Complementation of tonB mutants with P. shigelloides TonB system genes.
Because P. shigelloides TonB and ExbBD resemble proteins found in TonB
systems from other organisms, we wanted to determine if these proteins
function as a TonB system. We moved the P. shigelloides TonB
system on pHUG2 or E. coli tonB on pETONBX into KP1032, an E. coli tonB mutant, and performed complementation assays on
the strains. The strains were grown in low-iron medium, which tests for
the ability to acquire iron via the siderophore enterobactin, a
TonB-dependent process. E. coli KP1032/pHUG2 grew as well
in low-iron medium as the positive control,
KP1032/pETONBX (Table 5),
indicating that the P. shigelloides TonB system complements an E. coli tonB mutant. However, when the P. shigelloides tonB gene without exbBD was provided on
pHUG1, the strain failed to grow. The fact that the P. shigelloides TonB by itself could not restore function but the
entire TonB system could suggests that P. shigelloides TonB
cannot function with the E. coli ExbBD proteins but that it
can function with the enterobactin receptor in E. coli.
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strain of V. cholerae. DHH-11/pHUG2 could
acquire iron from heme and the siderophore vibriobactin, which are
TonB-dependent processes, whereas the untransformed strain could not
(data not shown).
Salt sensitivity of P. shigelloides TonB.
Seliger
et al. (46) reported that high levels of salt diminish the
function of V. cholerae TonB2 but have no impact on TonB1 function. This is thought to occur because TonB1 is longer than TonB2
(244 amino acids versus 206 amino acids). Thus, TonB1 can extend across
the periplasm and interact with the receptor under high-salt conditions
when the periplasm may become larger due to possible shrinkage of the
cytoplasm. To determine if P. shigelloides TonB, which is
288 amino acids in length, can also function under high-salt
conditions, we moved plasmids containing various TonB systems into
E. coli 1017/pHUG7 (contains hugAWXZ). The
following TonB systems were moved into the strain: the P. shigelloides TonB system on pHUG2, the V. cholerae
TonB1 system on pTEE1, and the V. cholerae TonB2 system on
pCos3. All the strains grew well in high-salt L broth, which showed
that high levels of salt in iron-rich media did not inhibit growth
(Table 6). The strains also grew in
low-salt medium with heme as the iron source, which indicated that each
of the three TonB proteins could interact with P. shigelloides HugA to allow heme to enter the cell (Table 6). When
the strains were tested for their ability to utilize heme as an iron
source in high-salt media, substantial growth was observed with
E. coli 1017/pHUG7 containing the P. shigelloides
TonB system or the V. cholerae TonB1 system but not in the
strain containing the V. cholerae TonB2 system. These data
suggest that P. shigelloides TonB is like V. cholerae TonB1 in its ability to function under high-salt
conditions.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Our analyses of the P. shigelloides heme iron utilization system indicates it contains an outer membrane receptor, HugA, which shares significant homology to other heme iron receptors. HugA also contains several motifs that are present in most heme receptors but are absent in receptors for other ligands. Our data indicate that HugA functions as a heme receptor in that it is interchangeable with the V. cholerae heme receptor in both a hugA mutant of P. shigelloides and in an E. coli strain that contains a hybrid heme iron utilization system consisting of hugA and V. cholerae tonB1, exbB1D1, hutBCD, and hutWXZ. The ability of these two heme receptors to function interchangeably may be due to the similar amino acid sequences of their TonB boxes (NEVLVTA in HugA and DEVVVST in HutA). The TonB box is the region of an outer membrane receptor that is thought to physically interact with the TonB protein. Other regions that the two proteins share in common also may play a role in allowing HugA and HutA to be interchangeable, as the two proteins share significant homology overall (Table 2).
P. shigelloides also has a TonB system which is required for heme iron utilization. The TonB system genes are arranged in the same order as those in the V. cholerae heme iron utilization system (tonB, exbB, exbD) and are linked to other genes specifically required for heme iron utilization. Linkage of a ligand transport system to tonB is unusual. P. shigelloides, V. cholerae (38), and possibly Vibrio parahaemolyticus (40) are the only organisms characterized to date which share this feature. The fact that these three organisms have TonB system genes linked to other heme iron utilization genes suggests that the genes were acquired simultaneously by horizontal gene transfer, and that at least initially these TonB systems were specific for heme iron utilization.
The P. shigelloides TonB and ExbBD sequences most closely resemble their counterparts in the V. cholerae TonB1 system. P. shigelloides TonB also is similar in size to V. cholerae TonB1 and like TonB1 can function in high-salt conditions. However, the P. shigelloides TonB system differs from the V. cholerae TonB1 system in that it can complement an E. coli TonB mutant, whereas the V. cholerae TonB1 system cannot (38). V. cholerae has a second TonB system (38) which, like the P. shigelloides system, complements an E. coli tonB mutant. Thus, it appears that the P. shigelloides TonB system shares features with both TonB systems in V. cholerae.
O'Malley et al. (40) suggested that V. parahaemolyticus and Vibrio alginolyticus have two tonB genes similar to those identified in V. cholerae, and two tonB genes have been identified in Pseudomonas aeruginosa (63). It is not clear if P. shigelloides also has two different tonB genes. No signal was detected in Southern blots of P. shigelloides chromosomal DNA probed with V. cholerae tonB2. This suggests that P. shigelloides does not have a tonB2 homologue but does not rule out the possibility that a second tonB is present that cannot be detected with the V. cholerae tonB2 probe at low stringency.
Many heme iron utilization systems characterized to date have genes encoding periplasmic and inner membrane permeases, which move the heme across the periplasm and into the cytoplasm. In V. cholerae these genes (hutBCD) are located downstream of exbD1. P. shigelloides also has homologues to these genes (hugBCD) which are located downstream of exbD. However, hugBCD are not needed to reconstitute the P. shigelloides heme iron utilization system in E. coli 1017, whereas V. cholerae hutBCD are needed to reconstitute the V. cholerae system in the same E. coli strain (38).
The role of the proteins encoded by P. shigelloides hugWXZ is not clear. Several bacterial heme iron utilization systems contain homologues of one or more of these proteins (Table 2), but functions have yet to be assigned to these proteins. Our results indicate that these three genes are not required for utilization of heme as a porphyrin source but are needed when heme is used as an iron source. When the P. shigelloides heme utilization system is reconstituted in E. coli in the absence of hugWXZ, heme supplied as an iron source is toxic to the cells. Stojiljkovic and Hantke (51) observed a similar result when they reconstituted the Y. enterocolitica heme iron utilization system in E. coli. In those experiments, hemS was required for utilization of heme as an iron source but not for utilization of heme as a porphyrin source, and heme toxicity was observed in the absence of hemS. The researchers hypothesized, but have not confirmed, that HemS breaks down the heme and releases iron into the cytoplasm. One or more of the proteins encoded by hugWXZ could serve a similar function in P. shigelloides, although none were found to share homology with HemS. It is also possible that these proteins may not be involved in breaking down the heme but rather in controlling the level of heme that enters the cytoplasm of the cell. The proteins could also be involved in heme storage, and their absence could result in accumulation of toxic levels of heme in the cytoplasm.
While P. shigelloides and V. cholerae have heme iron utilization systems that are similar to one another, the two organisms appear to be very different in the number of strategies each possesses for acquiring iron in the host. V. cholerae has several methods of acquiring iron from the host. V. cholerae utilizes the siderophores vibriobactin (17), ferrichrome (17), schizokinen (46), and enterobactin (61). P. shigelloides may not exhibit the same versatility. Daskaleros et al. (9) demonstrated that P. shigelloides does not synthesize a siderophore and that it cannot utilize vibriobactin or enterobactin. We conducted additional tests and found that it could not utilize ferrichrome or schizokinen or be crossfed by V. parahaemolyticus, which produces vibrioferrin, or by V. alginolyticus, which produces a yet-to-be-characterized siderophore (data not shown). Thus, the organism can use heme but not siderophores as an iron source. One could speculate that the absence of siderophore uptake systems in P. shigelloides may have a negative impact on its ability to survive in the environment and to infect potential hosts. This may explain why P. shigelloides appears to be less successful than V. cholerae in causing disease in a large number of humans.
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ACKNOWLEDGMENTS |
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This study was supported by Grant Development funds from the University of Texas of the Permian Basin and Alliance for Minority Participation funds from the University of Texas System.
We thank Shelley Payne for her guidance and critical reading of the manuscript, Donald Allen and Douglas Hale for their strong support, and Alexandra Mey for kindly providing recombinant plasmids.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Science and Mathematics, University of Texas of the Permian Basin, 4901 E. University Blvd., Odessa, TX 79762-0001. Phone: (915) 552-2270. Fax: (915) 552-2374. E-mail: henderson_d{at}utpb.edu.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, May 2001, p. 2715-2723, Vol. 183, No. 9
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.9.2715-2723.2001
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