Skip to main content
SpringerLink
Log in

Genetic analysis of phosphomannomutase/phosphoglucomutase from Vibrio furnissii and characterization of its role in virulence

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

The pmm gene from Vibrio furnissii, which encodes phosphomannomutase (PMM), was cloned and sequenced. The open reading frame consisted of 1,434 bp, encoding a polypeptide of 477 amino acids with a molecular mass of 53,325 Da. The predicted amino acid sequence of V. furnissii PMM showed high similarity with PMMs from other enteric bacteria, such as V. cholerae, Salmonella sp. and Escherichia coli. The PMM protein was overexpressed in E. coli as a His6-tagged recombinant protein. The estimated apparent K m and k cat values of the purified recombinant protein for mannose 1-phosphate were about 60 μM and 800 min−1, respectively. To investigate the biochemical functions and the role of pmm in the virulence of V. furnissii, a pmm knock-out mutant was constructed by homologous recombination mutation. Under the various physical conditions, cell numbers of the wild-type and the mutant did not differ. Oral introduction of bacterial suspensions to a mouse model showed that the pmm-deficient mutant decreased in viability at the intestine. Microscopy of the isolated intestines from mice revealed significant damage after 3 days in intestinal mucosa infected with the wild-type as compared with the mutant. The pmm-deficient mutant caused a reduction of virulence in mice and the loss of O-antigen polysaccharide, and showed low resistance relative to the wild-type when incubated with normal human serum.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2. A
Fig. 3.
Fig. 4A–C.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8.
Fig. 9.

Similar content being viewed by others

References

  • Alberti S, Alvarez D, Merino S, Casado MT, Vivanco F, Tomas JM, Benedi VJ (1996) Analysis of complement C3 deposition and degradation on Klebsiella pneumoniae. Infect Immun 64:4726–4732

    CAS  PubMed  Google Scholar 

  • Apicella MA, Westerink MA, Morse SA, Schneider H, Rice PA, Griffiss JM (1986) Bactericidal antibody response of normal human serum to the lipopolysaccharide of Neisseria gonorrhoeae. J Infec Dis 153:520–526

    CAS  Google Scholar 

  • Banemann A, Deppisch H, Gross R (1998) The lipopolysaccharide of Bordetella bronchiseptica acts as a protective shield against antimicrobial peptides. Infect Immun 66:5607–5612

    CAS  PubMed  Google Scholar 

  • Brenner DJ, Hickman-Brenner FW, Lee JV, Steigerwalt AG, Fanning GR, Hollis DG, Farmer JJ 3rd, Weaver RE, Joseph SW, Seidler RJ (1983) Vibrio furnissii (formerly aerogenic biogroup of Vibrio fluvialis), a new species isolated from human feces and the environment. J Clin Microbiol 18:816–824

    CAS  PubMed  Google Scholar 

  • Chiang SL, Mekalanos JJ (1998) Use of signature-tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization. Mol Microbiol 27:797–805

    Article  CAS  PubMed  Google Scholar 

  • Cortés G, de Astorza B, Benedí VJ, Albertí S (2002) Role of the htrA gene in Klebsiella pneumoniae virulence. Infect Immun 70:4772–4776

    Article  PubMed  Google Scholar 

  • Godfroid F, Taminiau B, Danese I, Denoel P, Tibor A, Weynants V, Cloeckaert A, Godfroid J, Letesson JJ (1998) Identification of the perosamine synthetase gene of Brucella melitensis 16 M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages. Infect Immun 66:5485–5493

    CAS  PubMed  Google Scholar 

  • Goldberg JB, Hatano K, Pier GB (1993) Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase. J Bacteriol 175:1605–1611

    CAS  PubMed  Google Scholar 

  • Griffiss JM, O'Brien JP, Yamasaki R, Williams GD, Rice PA, Schneider H (1987) Physical heterogeneity of neisserial lipooligosaccharides reflects oligosaccharides that differ in apparent molecular weight, chemical composition, and antigenic expression. Infect Immun 55:1792–1800

    CAS  PubMed  Google Scholar 

  • Joiner KA, Grossman N, Schmetz M, Leive L (1986) C3 binds preferentially to long-chain lipopolysaccharide during alternative pathway activation by Salmonella montevideo. J Immunol 136:710–715

    CAS  PubMed  Google Scholar 

  • Kawahara K, Tsukano H, Watanabe H, Lindner B, Matsuura M (2002) Modification of the structure and activity of lipid A in Yersinia pestis lipopolysaccharide by growth temperature. Infect Immun 70:4092–4098

    Article  CAS  PubMed  Google Scholar 

  • Köplin R, Arnold W, Hotte B, Simon R, Wang G, Puhler A (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

    PubMed  Google Scholar 

  • Krasikova IN, Kapustina NV, Svetashev VI, Gorshkova RP, Tomshich SV, Nazarenko EL, Komandrova NA, Ivanova EP, Gorshkova NM, Romanenko LA, Mikhailov VV, Solov'eva TF (2001) Chemical characterization of lipid A from some marine proteobacteria. Biochemistry (Moscow) 66:1047–1054

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685

    Google Scholar 

  • Lee JV, Shread P, Furniss AL, Bryant TN (1981) Taxonomy and description of Vibrio fluvialis sp. nov. (synonym group F vibrios, group EF6). J Appl Bacteriol 50:73–94

    CAS  PubMed  Google Scholar 

  • Lu M, Kleckner N (1994) Molecular cloning and characterization of the pgm gene encoding phosphoglucomutase of Escherichia coli. J Bacteriol 176:5847–5851

    CAS  PubMed  Google Scholar 

  • Miller VL, Mekalanos JJ (1988) A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170:2575–2583

    CAS  PubMed  Google Scholar 

  • Milton DL, Norqvist A, Wolf-Watz H (1992) Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum. J Bacteriol 174:7235–7244

    CAS  PubMed  Google Scholar 

  • Naught LE, Tipton PA (2001) Kinetic mechanism and pH dependance of the kinetic parameters of Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase. Arch Biochem Biophys 396:111–118

    Article  CAS  PubMed  Google Scholar 

  • Nesper J, Lauriano CM, Klose KE, Kapfhammer D, Kraiss A, Reidl J (2001) Characterization of Vibrio cholerae O1 El tor galU and galE mutants: influence on lipopolysaccharide structure, colonization, and biofilm formation. Infect Immun 69:435–445

    Article  CAS  PubMed  Google Scholar 

  • Parkhill J, Wren BW, Thomson NR, Titball RW, Holden MT, Prentice MB, Sebaihia M, James KD, Churcher C, Mungall KL, Baker S, Basham D, Bentley SD, Brooks K, Cerdeno-Tarraga AM, Chillingworth T, Cronin A, Davies RM, Davis P, Dougan G, Feltwell T, Hamlin N, Holroyd S, Jagels K, Karlyshev AV, Leather S, Moule S, Oyston PC, Quail M, Rutherford K, Simmonds M, Skelton J, Stevens K, Whitehead S, Barrell BG (2002) Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413:523–527

    Article  Google Scholar 

  • Pepe JC, Wachtel MR, Wagar E, Miller VL (1995) Pathogenesis of defined invasion mutants of Yersinia enterocolitica in a BALB/c mouse model of infection. Infect Immun 63:4837–4848

    CAS  PubMed  Google Scholar 

  • Preston A, Mandrell RE, Gibson BW, Apicella MA (1996) The lipopolysaccharides of pathogenic gram-negative bacteria. Crit Rev Microbiol 22:139–180

    CAS  PubMed  Google Scholar 

  • Rahman MM, Qadri F, Albert MJ, Hossain A, Mosihuzzaman M (1992) Lipopolysaccharide composition and virulence properties of clinical and environmental strains of Vibrio fluvialis and Vibrio mimicus. Microbiol Immunol 36:327–338

    CAS  PubMed  Google Scholar 

  • Regni C, Tipton PA, Beamer LJ (2002) Crystal structure of PMM/PGM: an enzyme in the biosynthetic pathway of P. aeruginosa virulence factors. Structure 10:269–279

    Article  CAS  PubMed  Google Scholar 

  • Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, Loppnow H, Ulmer AJ, Zahringer U, Seydel U, Di Padova F, Brade H (1994) Bacterial endotoxin-molecular relationships of structure to activity and function. FASEB J 8:217–225

    CAS  PubMed  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

  • Shimada T, Arakawa E, Okitsu T, Yamai S, Matsushita S, Kudoh Y, Okamura N (1999) Additional O antigens of Vibrio fluvialis and Vibrio furnissii. Jpn J Infect Dis 52:124–126

    CAS  PubMed  Google Scholar 

  • Sisti F, Fernandez J, Rodriguez ME, Lagares A, Guiso N, Hozbor DF (2002) In vitro and in vivo characterization of a Bordetella bronchiseptica mutant strain with a deep rough lipopolysaccharide structure. Infect Immun 70:1791–1798

    Article  CAS  PubMed  Google Scholar 

  • Spinola SM, Kwaik YA, Lesse AJ, Campagnari AA, Apicella MA (1990) Cloning and expression in Escherichia coli of a Haemophilus influenzae type b lipooligosaccharide synthesis gene(s) that encodes a 2-keto-3-deoxyoctulosonic acid epitope. Infect Immun 58:1558–1564

    CAS  PubMed  Google Scholar 

  • Tsai CM, Frasch CE (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119:115–119

    CAS  PubMed  Google Scholar 

  • Ugalde JE, Czibener C, Feldman MF, Ugalde RA (2000) Identification and characterization of the Brucella abortus phosphoglucomutase gene: role of lipopolysaccharide in virulence and intracellular multiplication. Infect Immun 68:5716–5723

    Article  CAS  PubMed  Google Scholar 

  • Videira PA, Cortes LL, Fialho AM, Sa-Correia I (2000) Identification of the pgmG gene, encoding a bifunctional protein with phosphoglucomutase and phophomannomutase activities, in the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461. Appl Environ Microbiol 66:2252–2258

    Article  CAS  PubMed  Google Scholar 

  • Waldor MK, Colwell R, Mekalanos JJ (1994) The Vibrio cholerae O139 serogroup antigen includes an O-antigen capsule and lipopolysaccharide virulence derterminants. Proc Natl Acad Sci USA 91:11388–11392

    CAS  PubMed  Google Scholar 

  • West NP, Jungnitz H, Fitter JT, McArthur JD, Guzmán CA, Walker MJ (2000) Role of phosphoglucomutase of Bordetella bronchiseptica in lipopolysaccharide biosynthesis and virulence. Infect Immun 68:4673–4680

    Article  CAS  PubMed  Google Scholar 

  • Westphal O, Jann K (1965) Bacterial lipopolysaccharides. Methods Carbohydr Chem 5:83–91

    CAS  Google Scholar 

  • Ye RW, Zielinski NA, Chakrabarty AM (1994) Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide. J Bacteriol 176:4851–4857

    CAS  PubMed  Google Scholar 

  • Zhou D, Stephens DS, Gibson BW, Engstrom JJ, McAllister CF, Lee FK, Apicella MA (1994) Lipopolysaccharide biosynthesis in pathogenic Neisseria : cloning, identification, and characterization of the phosphoglucomutase gene. J Biol Chem 269:11162–11169

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Prof. Young-Jin Chang (Department of Aquaculture, Pukyong National University) for providing essential reagents and technical assistance with histological examinations. This work was partially supported by the Agricultural Research and Development Promotion Center (ARPC) (grant no. 2020 1031SB010). S.H. Ahn acknowledges the graduate fellowship of the BB21 Project. E.M. Lee is a recipient of a graduate student fellowships of the Brain Korea 21 Project.

Author information

Authors and Affiliations

  1. Department of Biotechnology and Bioengineering, Pukyong National University, 608–737, Busan, Republic of Korea

    Sun-Hoi Kim, Sun-Hee Ahn, Jong-Hee Lee, Eun-Mi Lee, Nam-Hyun Kim & In-Soo Kong

  2. Korea Food Research Institute, 463-420, Songnam, Republic of Korea

    Kee-Jai Park

Authors
  1. Sun-Hoi Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sun-Hee Ahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jong-Hee Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eun-Mi Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nam-Hyun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kee-Jai Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. In-Soo Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to In-Soo Kong.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, SH., Ahn, SH., Lee, JH. et al. Genetic analysis of phosphomannomutase/phosphoglucomutase from Vibrio furnissii and characterization of its role in virulence. Arch Microbiol 180, 240–250 (2003). https://doi.org/10.1007/s00203-003-0582-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-003-0582-z

Keywords

Search

Navigation