Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jan;81(1):351-63.
doi: 10.1128/AEM.02402-14. Epub 2014 Oct 24.

Deciphering the role of multiple betaine-carnitine-choline transporters in the Halophile Vibrio parahaemolyticus

Serge Y Ongagna-Yhombi  1 , Nathan D McDonald  1 , E Fidelma Boyd  2
Affiliations

Deciphering the role of multiple betaine-carnitine-choline transporters in the Halophile Vibrio parahaemolyticus

Serge Y Ongagna-Yhombi et al. Appl Environ Microbiol. 2015 Jan.

Abstract

Vibrio parahaemolyticus is a halophile that is the predominant cause of bacterial seafood-related gastroenteritis worldwide. To survive in the marine environment, V. parahaemolyticus must have adaptive strategies to cope with salinity changes. Six putative compatible solute (CS) transport systems were previously predicted from the genome sequence of V. parahaemolyticus RIMD2210633. In this study, we determined the role of the four putative betaine-carnitine-choline transporter (BCCT) homologues VP1456, VP1723, VP1905, and VPA0356 in the NaCl stress response. Expression analysis of the four BCCTs subjected to NaCl upshock showed that VP1456, VP1905, and VPA0356, but not VP1723, were induced. We constructed in-frame single-deletion mutant strains for all four BCCTs, all of which behaved similarly to the wild-type strain, demonstrating a redundancy of the systems. Growth analysis of a quadruple mutant and four BCCT triple mutants demonstrated the requirement for at least one BCCT for efficient CS uptake. We complemented Escherichia coli MHK13, a CS synthesis- and transporter-negative strain, with each BCCT and examined CS uptake by growth analysis and (1)H nuclear magnetic resonance (NMR) spectroscopy analyses. These data demonstrated that VP1456 had the most diverse substrate transport ability, taking up glycine betaine (GB), proline, choline, and ectoine. VP1456 was the sole ectoine transporter. In addition, the data demonstrated that VP1723 can transport GB, proline, and choline, whereas VP1905 and VPA0356 transported only GB. Overall, the data showed that the BCCTs are functional and that there is redundancy among them.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Expression analysis of the BCCT genes in V. parahaemolyticus RIMD2210633 following NaCl upshock in LB medium. Cultures were grown in LB medium–3% NaCl to log phase (A) or stationary phase (B) and then subjected to 6% and 9% NaCl upshocks. These experiments were performed in duplicate, with two biological replicates. Changes in expression levels are relative to expression levels observed in either log-phase (A) or stationary-phase (B) cultures not subjected to osmotic upshock. The data were statistically analyzed by using an unpaired Student t test with a 95% confidence interval. The P values obtained are shown as asterisks, which denote the significant differences between upshocked samples and untreated controls. The error bars indicate means ± standard errors. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
FIG 2
FIG 2
Growth analyses of V. parahaemolyticus RIMD2210633 and quadruple mutant strain BCCT1342 (ΔVP1456 ΔVP1905 ΔVPA0356 ΔVP1723). Strains were grown in M9G–1% NaCl for 5 h and then inoculated into M9G–6% NaCl medium in the absence and presence of exogenously supplied CS. Data for growth analyses carried out for 24 h are shown in the graphs. Each assay was performed in triplicate, and data are shown as pooled data from two biological replicates. The error bars indicate means ± standard errors.
FIG 3
FIG 3
Growth analyses of triple mutant strains BCCT124 and BCCT123. Strains were grown in M9G–1% NaCl for 5 h and then inoculated into M9G–6% NaCl medium in the absence and presence of exogenously supplied CS. Data for growth analyses carried out for 24 h are shown in the graphs. Each assay was performed in triplicate, and data are shown as the pooled data from two biological replicates. The error bars indicate means ± standard errors.
FIG 4
FIG 4
Growth analyses of triple mutant strains BCCT134 and BCCT234. Strains were grown in M9G–1% NaCl for 5 h and then inoculated into M9G–6% NaCl medium in the absence and presence of exogenously supplied CS. Data for growth analyses carried out for 24 h are shown in the graphs. Each assay was performed in triplicate, and data are shown as the pooled data from two biological replicates. The error bars indicate means ± standard errors.
FIG 5
FIG 5
Functional complementation of E. coli MKH13 harboring individual BCCT genes. E. coli MKH13 strains were grown in M9G–1% NaCl medium overnight and transferred into M9G–4% NaCl medium in the absence and presence of exogenously supplied CS. Data for growth analyses carried out for 24 h are shown in the graphs. For each condition tested, the sample was assayed in triplicate, with at least two biological replicates. (A) M9G–4% NaCl plus GB; (B) M9G–4% NaCl plus proline; (C) M9G–4% NaCl plus ectoine. The error bars represent means ± standard errors. ***, P ≤ 0.001.
FIG 6
FIG 6
1H NMR spectroscopy of choline and ectoine transport in complemented E. coli MKH13 strains. 1H NMR spectra were acquired with a Bruker Avance III 400-MHz NMR spectrometer. The chemical shifts (δ) are expressed in ppm. The spectral peaks corresponding to the detected compounds of interest are labeled with the initials of the compound name on the top. 1H NMR experiments were performed at least twice, with two biological replicates. E, ectoine peaks; Ch, choline peaks.
FIG 7
FIG 7
Determination of the affinity of BCCTs for GB. The affinity of each BCCT for GB was determined by growth analysis of recombinant E. coli MKH13 strains in the presence of limiting GB concentrations. Bacterial growth was monitored for 24 h, and the specific growth rates of the recombinant E. coli MKH13 strains for a given GB concentration were calculated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. DePaola A, Nordstrom JL, Bowers JC, Wells JG, Cook DW. 2003. Seasonal abundance of total and pathogenic Vibrio parahaemolyticus in Alabama oysters. Appl Environ Microbiol 69:1521–1526. doi:10.1128/AEM.69.3.1521-1526.2003. - DOI - PMC - PubMed
    1. Daniels NA, MacKinnon L, Bishop R, Altekruse S, Ray B, Hammond RM, Thompson S, Wilson S, Bean NH, Griffin PM, Slutsker L. 2000. Vibrio parahaemolyticus infections in the United States, 1973–1998. J Infect Dis 181:1661–1666. doi:10.1086/315459. - DOI - PubMed
    1. Cabanillas-Beltran H, Llausas-Magana E, Romero R, Espinoza A, Garcia-Gasca A, Nishibuchi M, Ishibashi M, Gomez-Gil B. 2006. Outbreak of gastroenteritis caused by the pandemic Vibrio parahaemolyticus O3:K6 in Mexico. FEMS Microbiol Lett 265:76–80. doi:10.1111/j.1574-6968.2006.00475.x. - DOI - PubMed
    1. Colwell RR, Kaper J, Joseph SW. 1977. Vibrio cholerae, Vibrio parahaemolyticus, and other vibrios: occurrence and distribution in Chesapeake Bay. Science 198:394–396. doi:10.1126/science.910135. - DOI - PubMed
    1. Kaneko T, Colwell RR. 1973. Ecology of Vibrio parahaemolyticus in Chesapeake Bay. J Bacteriol 113:24–32. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources