Molecular characterization of an Enterobacter cloacae outer membrane protein (OmpX)

Abstract

A chromosomal gene of Enterobacter cloacae encoding an outer membrane protein (OmpX) has been cloned. Overproduction of the OmpX protein decreased the quantity of porins in the outer membrane of the parental strain and of Escherichia coli HB101. The The outer membranes of gram-negative bacteria obtain their nonspecific permeability for hydrophilic solutes from the presence of porins, a class of proteins that form waterfilled diffusion channels (22). Lack of these proteins leads to resistance to most beta-lactam antibiotics and quinolones (1, 21). In a previous report (28), we described the cloning of a restriction fragment of Enterobacter cloacae chromosomal DNA that was associated with resistance to beta-lactam antibiotics in transformants both of Escherichia coli and of the parental E. cloacae strain. In these transformants, the amount of the porin proteins OmpF and OmpC present decreased, and an outer membrane protein of approximately 18 kDa was overproduced. In vitro transcription and translation of the recombinant plasmid showed that this protein was encoded by a gene on the cloned fragment. The function of the protein is still unknown, but its overproduction clearly interferes with the presence of OmpF and OmpC in the outer membrane. We propose the name OmpX for this protein, as long as its proper function is obscure. In the accompanying paper (29), the biological characterization of OmpX is presented. In this report, we present the nucleotide sequence of the ompX gene and the deduced amino acid sequence. From these data, some physical properties of OmpX are derived. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains, plasmids, and bacteriophages used are listed in Table 1.Growth conditions. Bacteria were grown at 37°C in brain heart infusion broth (Oxoid Ltd.) supplemented when required with chloramphenicol (34 jig/ml Insertions of the transposable element -yb (11) in plasmid pJS04 were obtained as follows. DNA of plasmid pJS04 was introduced into the F' lac+-containing E. coli CE1304 by transformation. Transformants were selected on Iso-Sensitest agar (Oxoid CM471) containing chloramphenicol (34 ,ug/ml). Several transformants were subsequently mated with E. coli W3110 by filter mating on agar plates for 18 h at 37°C. Cells were collected from the filter, and the mixture was plated on Iso-Sensitest agar plates containing chloramphenicol and nalidixic acid (80 ,ug/ml). Transconjugants were screened subsequently for growth on Iso-Sensitest agar plates containing cephalothin (10 ,g/ml). The positions of the -yb sequence insertions were localized by restriction enzyme digestions.Analysis of plasmid-encoded proteins. Plasmid-encoded proteins were synthesized in vitro in a procaryotic DNAdirected transcription-translation system (Amersham, U.K.

Biological characterization of an Enterobacter cloacae outer membrane protein (OmpX)

“…In such a separation (which is based on the different solubilities of these proteins in 2% Triton X-100), part of the total OmpX present was found in the outer membrane fraction. In combination with the physical properties of OmpX, derived from the nucleotide sequence of the ompX gene presented in the accompanying paper (28) Outer membrane vesicles were separated from those derived from the inner membrane in a sucrose density gradient. The A280 profile (Fig.…”

“…However, the resemblance of the phenomena observed in E. coli and in E. cloacae made it plausible that our data also hold true for E. cloacae. Previous work has shown that the presence of multiple copies of the ompX gene has a negative effect on the quantity of porin proteins present in the cell (27,28 Regulation of expression of the porin proteins has been extensively studied. The total cellular levels of OmpF and OmpC proteins are rather consistent, but the ratio varies according to environmental conditions (21,30).…”

“…When cloned in a multicopy plasmid, this gene caused a decrease in the level of OmpF and OmpC proteins in Escherichia coli and of a comparable porinlike outer membrane protein in E. cloacae. In the accompanying paper (28), the DNA sequence of the ompX gene is presented. ompX encodes a precursor of the OmpX protein consisting of 172 amino acid residues and containing an N-terminal signal sequence of 23 amino acid residues.…”

Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps

“…Further, when a simple threshold was applied to the combination function, such that continuous regions of more than six residues with values >0.65 are predicted to be TM b-strand, this resulted in correct assignment of 77 % of the residues from maltoporin (see Figure 10(a)), 73 % for OmpF, and 78 % for R. blastica porin. The average prediction accuracy of 76 % for these three protein families is higher than that of other described methods (Paul & Rosenbusch, 1985;Stoorvogel et al, 1991;Vogel & Ja È hnig, 1986;Gromiha et al 1997;Gromiha & Ponnuswamy, 1993). The only computational methods reporting equal or higher accuracy (of which we are aware) either include the test protein in the training procedure (Ponnuswamy & Gromiha, 1993) or use only a single test protein which has signi®cant sequence similarity to one of the training proteins (E-value 9 e À10 by BLAST search of PDB; Gromiha et al, 1997).…”

Phenotypic characterization of OmpX, an Ail homologue of Yersinia pestis KIM

“…One Omp, OmpX, was first described for Enterobacter cloacae (Stoorvogel et al, 1991), but homologues, including PagC, Lom, Rck and Ail (the attachment-invasion locus protein of Yersinia spp. ), were identified in other Gram-negative bacteria (Dupont et al, 2004;Heffernan et al, 1992a;Mecsas et al, 1995).…”