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Keywords:

  • chemotaxis;
  • cholera;
  • motility

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

The ability to move toward favorable environmental conditions, called chemotaxis, is common among motile bacteria. In particular, aerotaxis has been extensively studied in Escherichia coli and was shown to be dependent on the aer and tsr genes. Three putative aer gene homologs were identified in the Vibrio cholerae genome, designated aer-1 (VC0512), aer-2 (VCA0658), and aer-3 (VCA0988). Deletion analyses indicated that only one of them, aer-2, actively mediates an aerotaxis response, as assayed in succinate soft agar plates as well as a capillary assay. Complementation studies confirmed that Aer-2 is involved in aerotaxis in V. cholerae. In addition, overexpression of aer-2 resulted in a marked increase of the aerotactic response in soft agar plates. No observable phenotypes in V. cholerae mutants deleted in the aer-1 or aer-3 genes were detected under standard aerotaxis testing conditions. Furthermore, the V. cholerae aer-1 and aer-3 genes, even when expressed from a strong independent promoter, did not produce any observable phenotypes. As found in other bacterial species, the results presented in this study indicate the presence of a secondary aerotaxis transducer in V. cholerae.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Extensive structural and genetic analyses of the chemotaxis behavior of Escherichia coli and Salmonella enterica serovar Typhimurium have deciphered the complexity of the coordination of movement in response to environmental stimuli (reviewed in Armitage, 1999; Dahlquist, 2002; Stock et al., 2002). In E. coli, the signal for a chemical attractant or a repellent is received by one of four membrane-spanning methyl-accepting chemotaxis proteins (MCPs) that respond to a change in concentration of a limited number of periplasmic chemoeffectors. A fifth MCP, named Aer, is somewhat more unconventional, in that it responds to oxygen by monitoring the cell's intracellular energy state (Bibikov et al., 1997).

Bacteria modulate their swimming behavior in response to a variety of external stimuli (Alexandre & Zhulin, 2001). Aerotaxis is the movement of cells toward or away from oxygen, a response also termed energy taxis. This behavior guides bacteria to microenvironments with preferred oxygen concentrations, redox potential, or electron acceptors (Taylor et al., 1999). The Aer protein lacks a periplasmic-sensing domain; instead, it contains a cytoplasmic PAS domain as well as a flavin adenine dinucleotide (FAD) cofactor, which is used to monitor the redox state of the cell (Bibikov et al., 1997, 2000). Aer forms collaborative signaling complexes with other MCPs, including Tsr (Gosink et al., 2006), which acts as a secondary aerotaxis transducer (Rebbapragada et al., 1997; Edwards et al., 2006).

The genomes of E. coli and Salmonella contain only single copies of genes that play a role in the chemotaxis machinery. However, chemotaxis in several other bacteria is more complex. The genomes of a large number of bacterial species, including Vibrio cholerae, Pseudomonas aeruginosa, Rhodobacter spaeroides, Myxococcus xanthus, and Borrelia burgdorferi, encode for multiple gene paralogs of the various chemotaxis genes found in E. coli (reviewed in Szurmant & Ordal, 2004). In most cases, the detailed functions of these redundant gene paralogs have not been elucidated. In V. cholerae, only CheY-3, one of five CheY paralogs, switches flagellar rotation and only CheA-2, one of three CheA paralogs, was found to be essential for chemotaxis (Gosink et al., 2002; Hyakutake et al., 2005). Moreover, the genome sequence of V. cholerae revealed a large number of chemotaxis-related genes (Heidelberg et al., 2000). A total of 46 ORFs have been annotated as putative MCP proteins; however, there is only limited knowledge about their functions in V. cholerae. As a first step toward assigning specific functions to some of the MCPs, three putative MCPs (VC0512, VCA0658, and VCA0988) were targeted showing significant homology to the E. coli aerotaxis transducer, Aer, in V. cholerae. Of the 46 MCPs in the V. cholerae genome, only these three contain a PAS domain. Deletion constructs for each gene were created and introduced into the chromosome of V. cholerae via homologous recombination. Mutants were analyzed for aerotaxis in a variety of assays and the intact genes were provided in trans for complementation studies. This study reports the first detailed analysis of any of the many V. cholerae MCPs.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Bacterial strains and growth conditions

The bacterial strains and plasmids used in this study are listed in Table 1. Vibrio cholerae O1 classical biotype strain 0395N1 (Mekalanos et al., 1983) and El Tor biotype strain Bah-2 (Pearson et al., 1993) were grown on Luria–Bertani (LB) solidified with 1.5% agar containing 100 μg mL−1 streptomycin sulfate. Liquid cultures of V. cholerae were grown in culture tubes containing 3 mL of LB broth at 37 °C in a roller drum (New Brunswick Scientific, Edison, NJ). Escherichia coli strains RP437 (Parkinson & Houts, 1982), UU1117 (Bibikov et al., 1997), UU1250 (Bibikov et al., 2004), and SM10λpir (Miller & Mekalanos, 1988) were grown on LB plates without antibiotics. Escherichia coli TOP10 cells (Invitrogen, Carlsbad, CA) and DH5α cells were used for routine cloning and grown on LB supplemented with appropriate antibiotics. All inoculated plates were incubated at 37 °C unless otherwise noted. Liquid cultures of all E. coli strains were grown in LB broth at 37 °C in a roller drum. Antibiotics were dissolved in sterile H2O and kept as 100 mg mL−1 stock solutions at 4 °C. Antibiotics were added to LB agar cooled to 50 °C at a final concentration of 100 μg mL−1.

Table 1.   Strains, plasmids and primers used in this study
Strain or plasmidRelevant characteristicsSource or references
V. cholerae strains
 O395N1Classical biotype, wild type for chemotaxisMekalanos et al. (1983)
 Bah-2El Tor biotype, wild type for chemotaxisPearson et al. (1993)
 ΔVCAer-1Bah-2 with deletion in VC0512This study
 ΔVCAer-2O395N1 with deletion in VCA0658This study
 ΔVCAer-3O395N1 with deletion in VCA0988This study
 ΔVCAer-2/3O395N1 with deletions in VCA0658 &VCA0988This study
 ΔVCAer-1/2/3Bah-2 with deletions in all three aer homologsThis study
E. coli strains
 TOP10Host for cloning vectorInvitrogen
 SM10λpirHost for suicide cloning vectorMiller & Mekalanos (1988)
 RP437Wild type for chemotaxisParkinson & Houts (1982)
 UU1117RP437 lacking aerBibikov et al. (1997)
 UU1250RP437 lacking all MCPsBibikov et al. (2004)
Plasmids
 pWM91Suicide vectorMetcalf et al. (1996)
 pBAD24Arabinose inducible promoterGuzman et al. (1995)
 pBAD-TOPOArabinose inducible promoterInvitrogen
 pWM91-VCAer-1Suicide vector with VC0512 deletion constructThis study
 pWM91-VCAer-2Suicide vector with VCA0658 deletion constructThis study
 pWM91-VCAer-3Suicide vector with VCA0988 deletion constructThis study
 pVCAer-1Expression plasmid carrying VC0512This study
 pVCAer-2Expression plasmid carrying VCA0658This study
 pVCAer-3Expression plasmid carrying VCA0988This study
 pECAerExpression plasmid carrying E. coli aerThis study
PurposePrimerPCR product (bp)
Expression of VC0512F: 5′-TGTAAATGGAGAGCATAATG R: 5′-AGTGTTAACCTACATCTG∼1600
Expression of VCA0658F: 5′-CCCGAAATGTCAGCCTAT R: 5′-CGCGCCTATTTTTGTGC∼1600
Expression of VCA0988F: 5′-CTCTTTTATGCGCAATAACC R: 5′-GCTCTTGCGCTTTGTTTA∼1600
Expression of E. coli aerF: 5′-TCTTCTCATCCGTATGTCACCCAGC R: 5′-TTAATGCAGTACCCGTCACCG∼1600
5′ region of VC0512F: 5′-CTCGAGGGGATGGAATATAAGTAATG R: 5′-TTACATTTGATTGGTATAAATTAAG∼1000
3′ region of VC0512F: 5′-AGCTTAGTGGCAAACGCAGGTG R: 5′-TCTAGACTCAGATATTATTTCAGTATTT∼1000
5′ region of VCA0658F: 5′-GGATCCCGAAAAAGCTGAGCGTATGG R: 5′-CTCGAGGTTTGCACAAAAATAGGCG∼1000
3′ region of VCA0658F: 5′-CTCGAGGGGAGTATAGGCTGACATTT R: 5′-TCTAGAGCGATGACTTTCCCCCGT∼1000
VCA0988 plus flanking regionsF: 5′-TCTAGAGCCGAATTGCAAACGCAGA R: 5′-GAATTCCTTTGCGAGATATTGAGGTCTTGCCACT∼3500

Construction of V. cholerae Aer deletion mutants

The primers used for PCR are listed in Table 1. Deletions in the V. cholerae aer-1 (VC0512), aer-2 (VCA0658), and aer-3 (VCA0988) genes were made by PCR amplifying two 1000-bp fragments of the up- and downstream regions of the genes, respectively. These fragments were cloned into pUC19, spliced together using restriction sites engineered into the primers, and then subcloned into pWM91 (Metcalf et al., 1996) using standard procedures (Sambrook et al., 1989). For VCA0988, a large PCR product was generated and internal restriction sites were utilized to delete the majority of the ORF. The deletion constructs were then cloned into pWM91 (Metcalf et al., 1996). A protocol for introducing cloned mutations into the chromosome of V. cholerae by homologous recombination using a suicide vector system, followed by sucrose selection was used (Donnenberg & Kaper, 1991). For complementation studies, PCR was used to amplify the V. cholerae ORFs VC0512, VCA0658, and VCA0988 as well as the E. coli aer gene. The PCR products were cloned under the control of the arabinose-inducible promoter (pBAD) and introduced into V. cholerae via electroporation.

Soft agar swarm plates

Minimal media soft agar swarm plates were used to assess aerotactic behavior (Ames & Parkinson, 1988; Bibikov et al., 1997). Succinate was used as the sole carbon source at a final concentration of 50 mM. Plates were made fresh for each experiment and used on the same day. Sterile toothpicks were used to inoculate swarm plates by touching a bacterial colony on an LB plate and stabbing the toothpick to the bottom of the swarm plate. The plates were incubated at 30 °C for 24 h. Plates were photographed using a UVP BioDocIt imaging station. Swarm diameters were measured using imagej analysis software (http://rsb.info.nih.gov/ij/). Statistical analysis was performed using microsoft excel software. To assess swarming behavior under anaerobic conditions, KNO3 was used as an alternative electron acceptor at a final concentration of 50 mM in the swarm plates. Plates were inoculated as described above and placed in an anaerobic chamber together with a gas pack and incubated at 30 °C for 4 days. For complementation assays, l-arabinose was added to the medium at a final concentration of 0.05% for promoter induction, as well as 100 μg mL−1 of ampicillin to maintain the plasmids in the host strains.

Capillary assays

Capillary assays were performed essentially as described (Wong et al., 1995). In brief, a flat capillary (Vitro Dynamics Inc., Rockaway, NJ) was placed in a cell suspension (mid-log phase) and the liquid was allowed to rise to within 1 cm of the end of the capillary, leaving an air bubble at the end. Cells for this assay were grown in the same medium that was used for the swarm plates, except that no agar was added. The ends of the capillary were sealed using melted candle wax to avoid evaporation of the liquid. Capillaries were mounted on a microscope slide to allow for easier handling and observation under a microscope and cells were checked for motility within 5 min using an inverted microscope. Capillaries were left at room temperature overnight and observed the following day by light microscopy.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Sequence analysis of V. cholerae MCP homologs

Analysis of the published genome sequence of V. cholerae (Heidelberg et al., 2000) (http://www.tigr.org) revealed the presence of a multitude of putative genes encoding proteins with possible sensory signal transducer domains that show high similarity to the E. coli MCP transducers. Overall, 46 V. cholerae ORFs can be classified as encoding putative MCPs (Heidelberg et al., 2000; Boin et al., 2004). This identification was made on the basis of the highly conserved domain (HCD) of chemotaxis transducer genes (Bourret et al., 1991). In particular, three putative V. cholerae MCPs (VC0512, VCA0658, and VCA0988) were identified as being homologs of the aer genes found in several Gram-negative bacterial species. The amino-terminal amino acid sequences of each of the V. cholerae aer homologs predict a PAS domain, common in a large family of proteins that sense oxygen, redox potential, or light (Taylor & Zhulin, 1999). In combination with other conserved residues, these proteins can be placed in the PAS-Aer subfamily (Repik et al., 2000). Furthermore, their respective two transmembrane regions, found in conventional MCPs, are positioned closely together, a feature found in other Aer proteins (Nichols & Harwood, 2000; Hong et al., 2004). This places their sensing domains inside the cytoplasm while anchoring the proteins in the cell membrane. The HAMP domain of the E. coli Aer protein, a feature found to be important to aerotactic signaling, has no apparent amino acid homology to the putative V. cholerae Aer proteins (Fig. 1). The amino acid alignment of these putative V. cholerae proteins to the E. coli Aer protein is shown in Fig. 1. Interestingly, VC0512 is located on chromosome I and is part of a 26.9 kb genomic island in the sequenced V. cholerae El Tor biotype strain, but was found to be absent in the classical biotype V. cholerae strain O395N1 (O'Shea et al., 2004). In contrast, VCA0658 and VCA0988 are located on chromosome II in the V. cholerae genome and are common to both biotypes of V. cholerae.

image

Figure 1.  Amino acid sequence alignment of the predicted proteins of the three putative Vibrio cholerae aer genes to the Escherichia coli Aer proteins. The predicted HCD and possible PAS domains are marked by a heavy line or a double line, respectively. The HAMP domain in E. coli Aer is underlined with a dotted line. Globally conserved residues are shaded black, whereas identical residues are shaded light gray, and similar residues are shaded dark gray.

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Characterization of V. cholerae aer deletion mutants

Vibrio cholerae strains carrying deletions in each of the putative aer genes as well as in several combinations were constructed (Table 1). The aer-2 and aer-3 deletions were introduced into the classical biotype V. cholerae strain O395N1, resulting in the strains ΔVCAer-2 and ΔVCAer-3, respectively, as classical biotype strains allow for easier analysis of virulence gene expression in vitro than El Tor biotype strains of V. cholerae. A V. cholerae O395N1 double mutant carrying deletions in aer-2 as well as aer-3 (ΔVCAer-2/3) was also constructed. The aer-1 gene was deleted in the V. cholerae El Tor biotype strain Bah-2, resulting in strain ΔVCAer-1. In addition, a Bah-2 mutant strain deleted in all three putative aer genes (ΔVCAer-1/2/3) was constructed.

Several methods were used to assess whether the three constructed mutant strains showed a defect in aerotaxis, including soft agar swarm plates (Fig. 2). Interestingly, only ΔVCAer-2 showed a reduced swarm circle diameter (P≤0.05) compared with its parent strain after a 24-h incubation at 30 °C. No significant difference between the VCAer-3 mutant and the parent strain was observed (P=0.08). In the El Tor biotype, deletion of aer-1 had no effect on the swarm circle diameter (P=0.25). Deletion of all three genes in the El Tor biotype showed results similar to the classical biotype double mutant (data not shown). To ensure that the smaller swarm circle diameter for strain ΔVCAer-2 was not due to a growth defect, growth rates of the parent and ΔVCAer-2 strains in liquid succinate minimal medium were compared and were found to be essentially identical (data not shown).

image

Figure 2.  Swarm patterns of the Vibrio cholerae parental and various putative aer deletion strains in succinate soft agar plates. El Tor biotype V. cholerae strain Bah-2 and its aer-1 (ΔVCAer-1) deletion derivative (a). Classical biotype V. cholerae strain O395N1 and its aer-2 (ΔVCAer-2) and aer-3 (ΔVCAer-3) single mutant derivatives as well as its aer-2/aer-3 (ΔVCAer-2/3) double mutant derivative (b). The lower panels show the corresponding histograms comparing averages of swarm circle diameters. Plates were incubated at 30°C for 24 h.

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Only the mutant strain carrying a deletion in one of the three identified aer homologs, ΔVCAer-2, showed a reduced swarm circle diameter compared with the parent strain. Loss of the other two V. cholerae aer gene paralogs did not result in any observable phenotypes and these genes might not be expressed under the conditions tested, or might encode for proteins that are not functional, or might be involved in other cellular functions. As noted before, V. cholerae has multiple paralogs of many chemotaxis-related genes in its genome (Boin et al., 2004) and in several cases, only one of them is actively involved in the traditional chemotactic phenotype (Gosink et al., 2002; Hyakutake et al., 2005).

Complementation of the V. choleraeΔVCAer-2 mutant

In order to ensure that the observed phenotype of ΔVCAer-2 was due to the deletion of the gene, plasmids carrying various aer genes were constructed and introduced into ΔVCAer-2. As expected, pVCAer-2 fully complemented the mutant phenotypes of ΔVCAer-2 only in the presence, but not in the absence, of arabinose. Under the arabinose concentrations used, pVCAer-2 not only complemented the swarm circle diameter of the ΔVCAer-2 mutant strain to normal levels but also increased the swarm circle diameter beyond that of the parent carrying the empty plasmid (Fig. 3). These results are comparable with findings in E. coli and P. aeruginosa where gene expression levels also corresponded to the aerotactic responses (Bibikov et al., 1997; Hong et al., 2004). In contrast, neither pVCAer-1 nor pVCAer-3 had any effect on the swarm circle diameter of ΔVCAer-2 (Fig. 3), giving further indication that they do not play a role in aerotaxis, at least not under the conditions tested in this study. In addition, the expression of the E. coli aer gene in ΔVCAer-2 seemed to increase the swarm circle diameter slightly (Fig. 3). As expected, pECAer completely restored the loss of aerotaxis behavior in succinate swarm plates in E. coli strain UU1117, an aer deletion strain (Bibikov et al., 1997) (data not shown). However, introduction of the V. cholerae aer-2 gene into UU1117 and UU1250 (E. coli strain lacking all MCPs, Bibikov et al., 2004) did not restore aerotaxis in either strain (data not shown). This is in contrast to previous studies of chemotaxis in V. cholerae that showed the V. cholerae CheA-2 and CheY-3 proteins complementing their E. coli homologs (Gosink et al., 2002; Hyakutake et al., 2005).

image

Figure 3.  Complementation of swarm plate phenotypes in succinate soft agar plates. Swarming abilities in the presence of arabinose of the Vibrio cholerae O395N1 carrying pBAD-24 as well as of the V. cholerae aer-2 deletion strain (VCAer-2) complemented by plasmids carrying the V. cholerae aer-1 (pVCAer-1), aer-2 (pVCAer-2), or aer-3 (pVCAer-3) gene or the Escherichia coli aer gene. pBAD-24 is the parent plasmid vector and contains no aer gene. Plates were incubated at 30°C for 24 h.

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Anaerobic conditions

To test whether the outward movement of V. cholerae cells in semi-solid succinate agar plates is due to the cells responding to an oxygen gradient, anaerobic conditions were used to eliminate the oxygen stimulus. To allow the cells to perform anaerobic respiration, KNO3 was added to the medium as an alternative electron acceptor. Owing to the slow growth under anaerobic conditions in minimal medium, the incubation time of the plates was increased to 96 h. Although the outward movement from the initial inoculation site in the absence of oxygen was severely limited in all strains, no notable differences in swarm circle sizes were observed between the parent and mutant strains (Fig. 4a). To ensure that the addition of KNO3 to the medium was not causing this limited outward movement, similar plates were also tested under normal aerobic conditions. Figure 4b shows that the bacterial swarm circles formed in the KNO3-supplemented plates were essentially the same as in regular succinate plates. To check that the swarming ability of the bacteria under anaerobic conditions is not generally impaired, succinate plates supplemented with the amino acids histidine, leucine, threonine, and methionine [known to be chemoattractants for V. cholerae (Freter & O'Brien, 1981)] were used under anaerobic conditions. All tested strains formed chemotactic rings in the swarm plates, and again no dramatic differences between the swarm circles of the different strains were observed (Fig. 4c). To ensure that the chemotactic signal received from the added amino acids did not override any aerotactic response, the amino acid-containing plates were also incubated aerobically (Fig. 4d). Similar to regular succinate plates, only the strains carrying a deletion in VCAer-2 showed a phenotype.

image

Figure 4.  Swarm patterns in the presence of an alternative electron acceptor. Swarming abilities in succinate soft agar plates in the presence of KNO3, as an alternative electron acceptor, of the Vibrio cholerae strain O395N1 and its aer-2 (ΔVCAer-2) and aer-3 (ΔVCAer-3) single mutant derivatives as well as its aer-2/aer-3 (ΔVCAer-2/3) double mutant derivative. Succinate soft agar plates containing KNO3 were incubated in an anaerobic chamber for 96 h (a) or aerobically for 24 h (b) at 30°C. Succinate soft agar plates containing KNO3 and 0.1 mM of the amino acids histidine, leucine, threonine, and methionine were incubated in an anaerobic chamber for 96 h (c) or aerobically for 24 h (d).

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These results served to show that succinate itself is not a strong chemoattractant for V. cholerae, as chemotactic outward movement should have occurred in response to a chemoattractant. Control experiments showed that, even under anerobic conditions, the bacteria are fully capable of performing chemotaxis in the presence of an appropriate attractant. Under the current model of how Aer functions in E. coli, redox changes in the cell are the signal sensed by Aer. Possible ways by which this is accomplished are direct reduction of Aer by a member of the respiratory complex, a cytosolic electron donor, or a diffusible redox component (Edwards et al., 2006). Because redox changes should still occur even under anaerobic respiration, the lack of a strong outward movement in soft agar plates under anaerobic conditions by both the parent as well as the ΔVCAer-2 strain indicates a possible mechanistic difference between the V. cholerae and the E. coli aerotaxis transducers in how the aerotactic signal is transmitted.

Capillary assay

To further establish the V. cholerae aer-2 gene as an aerotaxis transducer, the different V. cholerae aer deletion strains were analyzed in capillary assays (Wong et al., 1995). Whereas the parental cells clustered close to the meniscus of the liquid–air interface, the ΔVCAer-2 cells were more diffuse and further back (Fig. 5). Interestingly, it seemed that the mutant cells can still accumulate in an oxygen/air gradient, but at a lower concentration than the parent strain, indicating that V. cholerae posseses a secondary aerotaxis transducer. Actively swimming bacterial cells were observed in the dense zones of bacteria in both strains (data not shown). As expected, the ΔVCAer-1 and ΔVCAer-3 strains did not show an altered response in the capillary assay compared with their parental strains (data not shown).

image

Figure 5.  Capillary assay. The aerotactic behavior of the Vibrio cholerae strain O395N1 and its aer-2 (ΔVCAer-2) derivative. The dark line on the left indicates the meniscus of the liquid–air interface.

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Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

The fact that the aerotactic response of the V. cholerae mutant lacking all identified aer genes is not completely abolished in soft agar plates suggests the presence of a second aerotaxis transducer, as has been observed in E. coli (Rebbapragada et al., 1997), P. aeruginosa (Hong et al., 2004), and Pseudomonas putida (Nichols & Harwood, 2000). Moreover, in an aerotaxis capillary assay the VCAer-2 mutant cells clearly have an altered response to air, implying that other transducers contribute to the residual oxygen/air response in the V. cholerae. Although the role of chemotaxis and particularly aerotaxis in the biology of V. cholerae, including its environmental and infectious life stages, remains to be fully understood, this study provides a solid foundation for future studies into the functions of the multiple chemosensors found in this organism. The enormous complexity of the potential signals perceived, including oxygen, makes V. cholerae a particularly interesting model organism to study chemotactic behavior.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

The authors thank Sandy Parkinson for many critical comments and for providing E. coli strains. The authors would like to thank Khoosheh Gosink for her initial work on this project. This study was supported by the Ellison Medical Foundation and NIH Grant AI-063120-01A2.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
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