Identification of oprG, a gene encoding a major outer membrane protein of Pseudomonas aeruginosa

J Antimicrob Chemother 1999; 43: 607–608

Karl Gensberga, Anthony W. Smithb, Fiona S. L. Brinkmanc and Robert E. W. Hancockc,*

a Department of Surgery, Queen Elizabeth Hospital, Birmingham, UK b Department of Pharmacy and Pharmacology, University of Bath, Bath, UK c Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Sir,

Pseudomonas aeruginosa expresses between five and nine major outer membrane proteins (OMPs), depending on theconditions of growth; 1,2 the genes encoding all but one of these, OprG, have been identified. OprG has an apparentmolecular weight of 25,000 when outer membranes are heated to 88°C for 10 min insolubilization buffer before being subjected to SDS–PAGE and 19,000 when they are notpre-heated. 3 Its appearance in the outer membrane is highly dependent on growth conditions. In particular,Yates et al. 4 observed a direct relationship between the iron concentration in the medium and expression of OprG and suggested that this OMP is involved in low-affinity iron uptake. Other conditions, including growth into the stationary phase, higher growth temperatures, Mg 2+ deficiency, certain lipopolysaccharide alterations and the presence of certain carbon sources, alsoresult in the expression of varying concentrations of OprG. 1 Such apparently broad regulation of this OMP has frustrated efforts to assign a function to it.However, in addition to the potential role of OprG in iron uptake, two groups of investigatorshave suggested that it is involved in quinolone uptake/ susceptibility. 4,5 In view of this uncertainty, we undertook to identify the oprG gene.

Outer membranes were prepared according to the method described by Hancock & Carey, 3 from a 500 mL overnight culture of P. aeruginosa IA1 in L-broth (1% Bacto-Tryptone, 0.5% yeast extract; Difco Chemical Co., St Louis, MO,USA). The OMPs were electroblotted on to an Immobilon P polyvinylidinedifluoridemembrane (Millipore, Bedford, MA, USA) which was then dried and stained. The OprGband was excised from the blot and subjected to N-terminal amino acid sequencing by Dr P.Williams (Department of Pharmaceutical Sciences, University of Nottingham, Nottingham, UK). The procedure was repeated, yielding identical N-terminalsequences of DIQGHKAGD, and the sequence was used to search a release of P. aeruginosa genomic sequences (http://www. pseudomonas.com). This identified a single homologous open reading frame encoding a pre-protein with a predicted 22 amino acid signal sequence, followed by a peptide of 210 amino acids that included the above N-terminal sequence as the first nine amino acids. This sequence (Figure) was found in contig 216 (from bases 51,972 to 51,277 on the complementary strand; 15 September 1998 release of genomic sequences), located at about 1000 kbp on the physical genomic map on macrorestriction fragment DpnI-L, SpeI-A (http://www.bit.uq.edu.au/pseudomonas/ map.html).



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Figure. Sequence alignment by Clustal Wb of the deduced amino acid sequence of P. aeruginosa OprG and its closest homologue, V. cholerae OmpW. The experimentally determined N-terminal amino acid sequence is shown in bold letters, preceded by the deduced signal sequence. The symbols under the sequences represent identity (*) or high (:) or moderate (.) similarity at a given sequence position.

 
The predicted 210 amino acid protein is 43% identical and 58% similar to a Vibrio cholerae OMP, OmpW 6 (Figure), which is itself a member of a family of minor OMPs that are predicted to formeight-stranded ß-barrels. 7 Two of the closer homologues, DoxH 8 and AlkL, 9 are encoded in operons involved in naphthalene and alkane catabolic pathways respectively, buttheir precise functions are not known.

In conclusion, we have identified and sequenced the last of the P. aeruginos major OMPs. This will permit further studies to determine its cellular function and potential rolein quinolone uptake.

Acknowledgments

We would like to acknowledge the Pseudomonas Genome Project for the timely release of their sequence data. We acknowledge funding from the Canadian Cystic Fibrosis Foundation and through the Medical Research Council of Canada Distinguished Scientist Award to R.E.W.H. Funding for F.S.L.B. was from a special award from the Canadian Cystic Fibrosis Foundation for genomic studies.

Notes

* Corresponding author. Tel: +1-604-822-2682; Fax: +1-604-822-6041; E-mail:bob{at}cmdr.ubc.ca Back

References

1 . Hancock, R. E. W., Siehnel, R. & Martin, N. (1990). Outer membrane proteins of Pseudomonas. Molecular Microbiology 4, 1069–75.[ISI][Medline]

2 . Hancock, R. E. W. & Worobec, E. A. (1998). Outer membrane proteins. In Biotechnology Handbook, Vol. 10 (Montie, T., Ed.), p. 139–167. Plenum Press, London.

3 . Hancock, R. E. W. & Carey, A. M. (1979). Outer membrane of Pseudomonas aeruginosa. Heat- and 2-mercaptoethanol- modifiable proteins. Journal of Bacteriology 175 , 6890–1.[Abstract]

4 . Yates, J. M., Morris, G. & Brown, M. R. (1989). Effect of iron concentration and growth rate on the expression of protein G in Pseudomonas aeruginosa. FEMS Letters 49, 259–62.

5 . Chamberland, S., Bayer, A. S., Schollaardt, T., Wong, S. A. & Bryan, L. E. (1989).Characterization of mechanisms of quinolone resistance in Pseudomonas aeruginosa strains isolated in vitro and in vivo during experimental endocarditis. Antimicrobial Agents and Chemotherapy 33, 624–34.[ISI][Medline]

6 . Jalajakumari, M. B. & Manning, P. A. (1990). Nucleotide sequence of the gene ompW, encoding a 22 kDa immunogenic outer membrane protein of Vibrio cholerae. Nucleic Acids Research 18, 180.

7 . Baldermann, C., Lupas, A., Lubieniecki, J. & Engelhardt, H. (1998). The regulated outermembrane protein omp21 from Comamonas acidovorans is identified as a member of a new family of eight-stranded beta-sheet proteins by its sequence and properties. Journal of Bacteriology 180, 3741–9.[Abstract/Free Full Text]

8 . Denome, S. A., Stanley, D. C., Olson, E. S. & Young, K. D. (1993). Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway. Journal of Bacteriology 175, 6890–1.[Abstract]

9 . Van Beilen, J. B., Eggink, G., Enequist, H., Bos, R. & Witholt, B. (1992). DNA sequencedetermination and functional characterization of the OCT-plasmid-encoded alkJKL genes of Pseudomonas oleovorans. Molecular Microbiology 6,3121 –36.[ISI][Medline]





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