Microbiology (1999), 145, 673-679 Printed in Great Britain Area de Microbiologia, Departamento de Biologia, Universidad de las lslas Baleares, Palma de Mallorca, and Unidad de Recursos Natura les, lnstituto Mediterrdneo de Estudios Avanzados, Carretera de Valldemosa Km 7.5, E-07071 Palma de Mallorca, Spain Departamento de Microbiologia, Universidad de Sevilla, Avenida Sdnchez Pizjuan dn, E-41080 Sevilla, Spain Servicio de Microbiologia, Hospital Son Dureta, Andrea Doria 55, E-07014 Palma de Mallorca, Spain Departamento de Microbiologia, Universidad de Barcelona, Avenida Diagonal 645, E-08071 Barcelona, Spain I M EDEA (CSIC-UIB), Porin expression in clinical isolates of Klebsiella pneurnoniae Santiago Hernandez-Alles,' Sebastian Albert(,' Dolores Alvarez,' Antonio Domenech-Sanchez,' Luis Martinez-Martinez,' Jose Gil,'t3 Juan M. and Vicente J. Benedil Author for correspondence: Vicente J. Benedi. Tel: +34 971 173335. Fax: +34 971 173184. e-mail: dbsjbbO@ps.uib.es Two porins, OmpK36 and OmpK35, have been described previously in Klebsiellapneumoniae, and they are homologous to the Escherichia coli porins OmpC and OmpF, respectively, at both the DNA and amino acid levels. Optimal resolution of the two K. pneumoniae porins by electrophoresis on polyacrylamide gels is not achieved using gel systems already described for E. coli and requires modifications of the bisacrylamide content of the resolving gels. Once resolved, identification of porins OmpK36 and OmpK35 cannot be based solely on their apparent molecular masses since in some strains the OmpK36 porin migrates faster than the OmpK35 porin, whilst in other strains OmpK35 is the faster-migrating porin. Expression of OmpK35 porin is increased in low-osmolarity medium and, combined with Western blot analysis, this allows for the identification of both porins. Application of this identification system showed that most isolates lacking expression of extended-spectrum /?-lactamasesexpress the two porins, whereas most isolates producing these /?-lactamasesexpress only porin OmpK36, and the OmpK35 porin is either very low or not expressed. Keywords : outer-membrane proteins, porins, Klebsiella pneumoniae, osmoregulation, antibiotic resistance INTRODUCTION The outer membrane of enterobacterial species has significant medical importance because its constituents play major roles in the permeability of antimicrobial agents and substrates, and in interactions with the host defence mechanisms (Benz, 1994; Roth, 1988). In Klebsiella pneumoniae, most studies have been devoted to the roles of LPS and capsule in pathogenicity and other biological phenomena, whereas the outer-mem- brane proteins (OMPs) have attracted less attention (Williams & Tomas, 1990). The OMPs of K. pneumoniae have been implicated in protection against lethal challenge with homologous strains (Serushago et al., 1989), in activation of the complement system (Alberti et al., 1993b), in iron Abbreviations : 2D, two-dimensional; ESBL, extended-spectrum p-lactam- ase; OMP, outer-membrane protein; TEMED, N,N,N',N'-tetramethyl- ethylenediamine. acquisition (Williams et al., 1987), and in permeability to antimicrobial agents (Martinez-Martinez et al., 1996). Compared with close relatives such as Escherichia coli, less is known regarding the OMPs of Klebsiella. However, the existence of K. pneumoniae homologues to the phosphate-starvation-inducible PhoE porin (van der Ley et al., 1987), the sucrose porin ScrY (Schmid et al., 1991),and the LamB maltoporin (Werts et al., 1992) have been shown. Concerning the non-specific pore proteins (porins), the molecular masses, pore sizes, and antimicrobial permeation of two porins, named 37kD and 39kD, have been characterized in Enterobacter cloacae strains later reidentified as K. pneumoniae (Kaneko et al., 1984; Sawai et al., 1982, 1987). These two porins are probably the equivalents of the two porins described by us in other K. pneumoniae strains : porin OmpK36, whose amino acid sequence is very homologous to that of E. coli OmpC porin (Alberti et al., 1995), and porin OmpK35 (Hernandez-Alles et al., 199.9, whose sequence (accession number AJOl1501) is closer to that of E. coli OmpF porin. 0002-3001 0 1999 SGM 673