A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol

AIMS: The minimum inhibitory concentration (MIC) of oregano essential oil (OEO) and two of its principle components, i.e. thymol and carvacrol, against Pseudomonas aeruginosa and Staphylococcus aureus was assessed by using an innovative technique. The mechanism of action of the above substances was also investigated. METHODS AND RESULTS: The applied technique uses 100-well microtitre plate and collects turbidimetric growth data. To produce the inhibition profiles, a wide range of concentrations were tested for each of the three compounds, as well as for carvacrol-thymol mixtures. Following a specific mathematical analysis of the observed inhibition profiles from all compounds, it was suggested that mixtures of carvacrol and thymol gave an additive effect and that the overall inhibition by OEO can be attributed mainly to the additive antimicrobial action of these two compounds. Addition of low amounts of each additive: (a) increased permeability of cells to the nuclear stain EB, (b) dissipated pH gradients as indicated by the CFDA-SE fluorescent probe irrespective of glucose availability and (c) caused leakage of inorganic ions. CONCLUSION: Mixing carvacrol and thymol at proper amounts may exert the total inhibition that is evident by oregano essential oil. Such inhibition is due to damage in membrane integrity, which further affects pH homeostasis and equilibrium of inorganic ions. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of extent and mode of inhibition of specific compounds, which are present in plant extracts, may contribute to the successful application of such natural preservatives in foods, since certain combinations of carvacrol-thymol provide as high inhibition as oregano essential oil with a smaller flavour impact.     

A Novel indole compound that inhibits Pseudomonas aeruginosa growth by targeting MreB is a substrate for MexAB-OprM

Drug efflux systems contribute to the intrinsic resistance of Pseudomonas aeruginosa to many antibiotics and biocides and hamper research focused on the discovery and development of new antimicrobial agents targeted against this important opportunistic pathogen. Using a P. aeruginosa PAO1 derivative bearing deletions of opmH, encoding an outer membrane channel for efflux substrates, and four efflux pumps belonging to the resistance nodulation/cell division class including mexAB-oprM, we identified a small-molecule indole-class compound (CBR-4830) that is inhibitory to growth of this efflux-compromised strain. Genetic studies established MexAB-OprM as the principal pump for CBR-4830 and revealed MreB, a prokaryotic actin homolog, as the proximal cellular target of CBR-4830. Additional studies establish MreB as an essential protein in P. aeruginosa, and efflux-compromised strains treated with CBR-4830 transition to coccoid shape, consistent with MreB inhibition or depletion. Resistance genetics further suggest that CBR-4830 interacts with the putative ATP-binding pocket in MreB and demonstrate significant cross-resistance with A22, a structurally unrelated compound that has been shown to promote rapid dispersion of MreB filaments in vivo. Interestingly, however, ATP-dependent polymerization of purified recombinant P. aeruginosa MreB is blocked in vitro in a dose-dependent manner by CBR-4830 but not by A22. Neither compound exhibits significant inhibitory activity against mutant forms of MreB protein that bear mutations identified in CBR-4830-resistant strains. Finally, employing the strains and reagents prepared and characterized during the course of these studies, we have begun to investigate the ability of analogues of CBR-4830 to inhibit the growth of both efflux-proficient and efflux-compromised P. aeruginosa through specific inhibition of MreB function.     

Susceptibility of compound 48/80-sensitized Pseudomonas aeruginosa to the hydrophobic biocide triclosan

Pseudomonas aeruginosa is intrinsically resistant to the hydrophobic biocide triclosan, and yet it can be sensitized to low concentrations by permeabilization of the outer membrane using compound 48/80. A selective plating assay revealed that compound 48/80-permeabilized YM64, a triclosan-recognizing efflux pump-deficient variant, was unable to initiate growth on a medium containing triclosan. Macrobroth dilution assay data revealed that treatment with compound 48/80 synergistically decreased minimal inhibitory concentrations of the hydrophobic antibacterial agents rifamycin SV and chloramphenicol for all cell envelope variant strains examined. A low concentration of triclosan exerted a transient bactericidal effect on permeabilized wild-type strain PAO1, after which exponential growth resumed within 4 h. Permeabilized strain YM64 was unable to overcome the inhibition; yet, both strains remained susceptible to chloramphenicol for as long as 6 h, thereby suggesting that the outer membrane remained permeable to nonpolar compounds. These data support the notion that the transitory nature of compound 48/80 sensitization to triclosan in P. aeruginosa does not involve obviation of the hydrophobic diffusion pathway through the outer membrane. The inability of strain YM64 to overcome the synergistic effect of compound 48/80 and triclosan strongly suggests that triclosan-recognizing efflux pumps are involved in maintaining viability in wild-type cells whose outer membranes are otherwise compromised.     

Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a

The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P. fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics.     

Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes three types of xenobiotic efflux pumps, MexAB-OprM, MexCD-OprJ, and MexEF-OprN, which are regulated by the nalB, nfxB, and nfxC genes, respectively, and their high expression renders the cells resistant to multiple species of antibiotics. We evaluated the role of the outer membrane permeability barrier and the efflux pump in lowering the intracellular concentration of fluorescent probes. The wild-type, nalB, nfxB, and nfxC strains with an intact outer membrane showed equally high capability in draining out intracellular fluorescent dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium and ethidium bromide. When the outer membrane barrier was dismantled by the EDTA treatment, wild-type, nfxC, nfxB, and nalB strains showed significantly different levels of dye accumulation. The polymyxin B-treated cells showed an even more pronounced difference in dye accumulation among the nfxC, nfxB, and nalB mutants. We concluded from these results that the xenobiotic extrusion pumps interplay with the outer membrane permeability barrier in lowering the intracellular substrate concentration. Among three extrusion pumps in P. aeruginosa, MexAB-OprM was the most efficient, followed by MexCD-OprJ and MexEF-OprN pumps for the fluorescent dye extrusion.     

Genes related to chromate resistance by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1

Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon-insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted in the mutants with the PAO1 genome revealed that the genes affected in three mutant strains were oprE (ORF PA0291), rmlA (ORF PA5163), and ftsK (ORF PA2615), respectively. A relationship of these genes with chromate tolerance has not been previously reported. No other phenotypic changes were observed in the oprE mutant but its resistance to chromate was not fully restored by expressing the ChrA protein, which extrudes chromate ions from the cytoplasm to the periplasmic space. These data suggest that OprE participates in the efflux of chromate from the periplasm to the outside. Increased susceptibility of the rmlA mutant to the metals cadmium and mercury and to the anion-superoxide generator paraquat suggests a protective role of LPS against chromate toxicity. A higher susceptibility of the ftsK mutant to compounds affecting DNA structure (ciprofloxacin, tellurite, mitomycin C) suggests a role of FtsK in the recombinational repair of DNA damage caused by chromate. In conclusion, the P. aeruginosa genome contains diverse genes related to its intrinsic resistance to chromate. Systems pertaining to the outer membrane (OprE), the cell wall (LPS), and the cytoplasm (FtsK) were identified in this work as involved in chromate protection mechanisms.     

Efflux as a mechanism of resistance to antimicrobials in Pseudomonas aeruginosa and related bacteria: unanswered questions

Pseudomonas aeruginosa is an opportunistic human pathogen exhibiting innate resistance to multiple antimicrobial agents. This intrinsic multidrug resistance is caused by synergy between a low-permeability outer membrane and expression of a number of broadly-specific multidrug efflux (Mex) systems, including MexAB-OprM and MexXY-OprM. In addition to this intrinsic resistance, these and three additional systems, MexCD-OprJ, MexEF-OprN and MexJK-OprM promote acquired multidrug resistance as a consequence of hyper-expression of the efflux genes by mutational events. In addition to antibiotics, these pumps export biocides, dyes, detergents, metabolic inhibitors, organic solvents and molecules involved in bacterial cell-cell communication. Homologues of the resistance-nodulation-division systems of P. aeruginosa have been found in Burkholderia cepacia, B. pseudomallei, Stenotrophomonas maltophilia, and the nonpathogen P. putida, where they play roles in resistance to antimicrobials and/or organic solvents. Despite intensive studies of these multidrug efflux systems over the past several years, their precise molecular architectures, their modes of regulation of expression and their natural functions remain largely unknown.     

The MexA-MexB-OprM multidrug efflux system of Pseudomonas aeruginosa is growth-phase regulated

Intrinsic antibiotic resistance in Pseudomonas aeruginosa is attributed to low outer membrane permeability and drug efflux mediated by the products of mexAmexBoprM efflux operon. Using a mexA-phoA fusion, expression of the efflux genes was assessed as a function of growth in a variety of strains. The efflux operon was growth-phase regulated in both wild-type and nalB strains, being minimally expressed in lag phase and increasing in log to late log phase. MexR, the only known regulator of MexAMexBOprM and target of mutation in nalB strains, was not involved in the growth-phase regulation. The las cascade regulates genes based on increased cell-density, but a deletion in lasR had no effect on mexAmexBoprM expression. Putative recognition sequences for AlgT/U and RpoN were identified upstream of mexA, but algT/U and rpoN null mutants also had no effect on mexAmexBoprM expression.     

Multidrug resistance mechanisms: drug efflux across two membranes

A set of multidrug efflux systems enables Gram-negative bacteria to survive in a hostile environment. This review focuses on the structural features and the mechanism of major efflux pumps of Gram-negative bacteria, which expel from the cells a remarkably broad range of antimicrobial compounds and produce the characteristic intrinsic resistance of these bacteria to antibiotics, detergents, dyes and organic solvents. Each efflux pump consists of three components: the inner membrane transporter, the outer membrane channel and the periplasmic lipoprotein. Similar to the multidrug transporters from eukaryotic cells and Gram-positive bacteria, the inner membrane transporters from Gram-negative bacteria recognize and expel their substrates often from within the phospholipid bilayer. This efflux occurs without drug accumulation in the periplasm, implying that substrates are pumped out across the two membranes directly into the medium. Recent data suggest that the molecular mechanism of the drug extrusion across a two-membrane envelope of Gram-negative bacteria may involve the formation of the membrane adhesion sites between the inner and the outer membranes. The periplasmic components of these pumps are proposed to cause a close membrane apposition as the complexes are assembled for the transport.     

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

Broad-specificity efflux pumps have been implicated in multidrug-resistant strains of Pseudomonas aeruginosa and other Gram-negative bacteria. Most Gram-negative pumps of clinical relevance have three components, an inner membrane transporter, an outer membrane channel protein, and a periplasmic protein, which together coordinate efflux from the cytoplasmic membrane across the outer membrane through an unknown mechanism. The periplasmic efflux proteins (PEPs) and outer membrane efflux proteins (OEPs) are not obviously related to proteins of known structure, and understanding the structure and function of these proteins has been hindered by the difficulty of obtaining reasonable multiple alignments. We present a general strategy for the alignment and structure prediction of protein families with low mutual sequence similarity using the PEP and OEP families as detailed examples. Gibbs sampling, hidden Markov models, and other analysis techniques were used to locate motifs, generate multiple alignments, and assign PEP or OEP function to hypothetical proteins in several species. We also developed an automated procedure which combines multiple alignments with structure prediction algorithms in order to identify conserved structural features in protein families. This process was used to identify a probable alpha-helical hairpin in the PEP family and was applied to the detection of transmembrane beta-strands in OEPs. We also show that all OEPs contain a large tandem duplication, and demonstrate that the OEP family is unlikely to adopt a porin fold, in contrast to previous predictions.     

Inner membrane efflux components are responsible for beta-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa.

A major feature of the MexAB-OprM multidrug efflux pump which distinguishes it from the MexCD-OprJ and MexEF-OprN multidrug efflux systems in Pseudomonas aeruginosa is its ability to export a wide variety of beta-lactam antibiotics. Given the periplasmic location of their targets it is feasible that beta-lactams exit the cell via the outer membrane OprM without interaction with MexA and MexB, though the latter appear to be necessary for OprM function. To test this, chimeric MexAB-OprJ and MexCD-OprM efflux pumps were reconstituted in delta mexCD delta oprM and delta mexAB delta oprJ strains, respectively, and the influence of the exchange of outer membrane components on substrate (i.e., beta-lactam) specificity was assessed. Both chimeric pumps were active in antibiotic efflux, as evidenced by their contributions to resistance to a variety of antimicrobial agents, although there was no change in resistance profiles relative to the native pumps, indicating that OprM is not the determining factor for the beta-lactam specificity of MexAB-OprM. Thus, one or both of inner membrane-associated proteins MexA and MexB are responsible for drug recognition, including recognition of beta-lactams.     

Mechanisms of bacterial biocide and antibiotic resistance

Resistance to antibiotics is increasingly commonplace amongst important human pathogens. Although the mechanism(s) of resistance vary from agent to agent they typically involve one or more of: alteration of the drug target in the bacterial cell, enzymatic modification or destruction of the drug itself, or limitation of drug accumulation as a result of drug exclusion or active drug efflux. While most of these are agent specific, providing resistance to a single antimicrobial or class of antimicrobial, there are currently numerous examples of efflux systems that accommodate and, thus, provide resistance to a broad range of structurally unrelated antimicrobials--so-called multidrug efflux systems. Resistance to biocides is less common and likely reflects the multiplicity of targets within the cell as well as the general lack of known detoxifying enzymes. Resistance typically results from cellular changes that impact on biocide accumulation, including cell envelope changes that limit uptake, or expression of efflux mechanisms. Still, target site mutations leading to biocide resistance, though rare, are known. Intriguingly, many multidrug efflux systems also accommodate biocides (e.g. triclosan) such that strains expressing these are both antibiotic- and biocide-resistant. Indeed, concern has been expressed regarding the potential for agents such as triclosan to select for strains resistant to multiple clinically-relevant antibiotics. Some of the better characterized examples of such multidrug efflux systems can be found in the opportunistic pathogen Pseudomonas aeruginosa where they play an important role in the noted intrinsic and acquired resistance of this organism to antibiotics and triclosan. These tripartite pumps include an integral inner membrane drug-proton antiporter, an outer membrane- and periplasm-spanning channel-forming protein and a periplasmic link protein that joins these two. Expression of efflux genes is governed minimally by the product of a linked regulatory gene that is in most cases the target for mutation in multidrug resistant strains hyperexpressing these efflux systems. Issues for consideration include the natural function of these efflux systems and the therapeutic potential of targeting these systems in combating acquired multidrug resistance.     

Cross-resistance to antibiotics of Escherichia coli adapted to benzalkonium chloride or exposed to stress-inducers

AIMS: To study the effects of adaptation and stress on the resistance to benzalkonium chloride (BC) and cross-resistance to antibiotics in Escherichia coli. METHODS AND RESULTS: Precultivation of E. coli ATCC 11775 and E. coli DSM 682 in the presence of subinhibitory concentrations of BC or stress inducers (salicylate, chenodeoxycholate and methyl viologen) resulted in higher minimum inhibitory concentration (MIC) of BC and chloramphenicol (CHL). Adaptation to growth in sixfold of the initial MIC of BC resulted in stable BC resistance and enhanced tolerance to several antibiotics and ethidium bromide (EtBr). The MIC of CHL increased more than 10-fold for both strains. Enhanced efflux of EtBr in adapted E. coli ATCC 11775 indicated that the observed resistance was due to efflux. Changes in outer membrane protein profiles were detected in the BC-adapted cells. There were no indications of lower membrane permeability to BC. CONCLUSIONS: Induction of stress response or gradual adaptation to BC or CHL results in acquired cross-tolerance between BC and antibiotics in E. coli. Enhanced efflux was one of the observed differences in adapted cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Provided not taking due precautions, extensive use of disinfectants could lead to emergence of antibiotic-resistant isolates.     

Expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco is not associated with either enhanced citrate accumulation or efflux

Aluminum (Al) toxicity and poor phosphorus (P) availability are factors that limit plant growth on many agricultural soils. Previous work reported that expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco (Nicotiana tabacum; CSb lines) resulted in improved Al tolerance (J.M. de la Fuente, V. Ramírez-Rodríguez, J.L. Cabrera-Ponce, L. Herrera-Estrella [1997] Science 276: 1566-1568) and an enhanced ability to acquire P from alkaline soils (J. López-Bucio, O. Martínez de la Vega, A. Guevara-García, L. Herrera-Estrella [2000] Nat Biotechnol 18: 450-453). These effects were attributed to the P. aeruginosa citrate synthase increasing the biosynthesis and efflux of citrate from roots. To verify these findings we: (a) characterized citrate efflux from roots of wild-type tobacco; (b) generated tobacco lines expressing the citrate synthase gene from P. aeruginosa; and (c) analyzed selected CSb lines described above. Al stimulated citrate efflux from intact roots of wild-type tobacco and root apices were found to be responsible for most of the efflux. Despite generating transgenic tobacco lines that expressed the citrate synthase protein at up to a 100-fold greater level than the previously described CSb lines, these lines did not show increased accumulation of citrate in roots or increased Al-activated efflux of citrate from roots. Selected CSb lines, similarly, failed to show differences compared with controls in either citrate accumulation or efflux. We conclude that expression of the P. aeruginosa citrate synthase gene in plants is unlikely to be a robust and easily reproducible strategy for enhancing the Al tolerance and P-nutrition of crop and pasture species.     

Reevaluation, using intact cells, of the exclusion limit and role of porin OprF in Pseudomonas aeruginosa outer membrane permeability.

Earlier studies that used model membrane reconstitution methods have come to different conclusions regarding the exclusion limit of the outer membrane of Pseudomonas aeruginosa and whether OprF is the major channel-forming protein in the outer membrane. In this study, a 6.2-kbp SalI fragment, encoding only two cytoplasmic enzymes, alpha-galactosidase and sucrose hydrolase, and the inner membrane raffinose permease, was cloned behind the m-toluate-inducible tol promoter of vector pNM185 to create plasmid pFB71. P. aeruginosa strains harboring pFB71, when grown with inducer, produced both enzymes encoded by the insert and had acquired the ability to grow on the disaccharide melibiose and the trisaccharide raffinose. The rate of growth was dependent on the concentration and size of the saccharide and was decreased three- to fivefold by the absence of OprF, as examined by measuring the growth on melibiose and raffinose of an isogenic OprF-deficient omega insertion derivative, H636(pFB71). At high concentrations, di-, tri-, and tetrasaccharides could pass across the outer membrane to plasmolyze P. aeruginosa, as measured by light scattering and confirmed by electron microscopy. The initial rate kinetics of light-scattering changes were dependent on the size of the saccharide being used. Furthermore, the rates of change in light scattering due to raffinose and stachyose uptake across the outer membrane for strain H636 were fivefold or more lower than for its OprF-sufficient parent H103. These data are consistent with model membrane studies showing that OprF is the most predominant porin for compounds larger than disaccharides in P. aeruginosa and suggest that the exclusion limit for this porin and the outer membrane is greater than the size of a tetrasaccharide. In addition, these data confirmed the existence of other porins with a predominant function in monosaccharide uptake and a more minor function in the uptake of larger saccharides.     

Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes.

Pseudomonas aeruginosa is usually resistant to a wide variety of antibacterial agents, and it has been inferred, on the basis of indirect evidence, that this was due to the low permeability of its outer membrane. We determined the permeability of P. aeruginosa outer membrane directly, by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm. The permeability to these compounds was about 100-fold lower than in the outer membrane of Escherichia coli K-12. Also, we found that the apparent Km values for active transport of various carbon and energy source compounds were typically higher than 20 microM in P. aeruginosa, in contrast to E. coli in which the values are usually lower than 5 microM. These results also are consistent with the notion that the P. aeruginosa outer membrane indeed has a low permeability to most hydrophilic compounds and that this membrane acts as a rate limiting step in active transport processes with high Vmax values.     

Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms

Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobial agents. A major contribution to this intrinsic multidrug resistance is provided by a number of broadly-specific multidrug efflux systems, including MexAB-OprM and MexXY-OprM. In addition, these and two additional tripartite efflux systems, MexCD-OprJ and MexEF-OprN, promote acquired multidrug resistance as a result of mutational hyperexpression of the efflux genes. In addition to antibiotics, these pumps promote export of numerous dyes, detergents, inhibitors, disinfectants, organic solvents and homoserine lactones involved in quorum sensing. The efflux pump proteins are highly homologous and consist of a cytoplasmic membrane-associated drug-proton antiporter of the Resistance-Nodulation-Division (RND) family, an outer membrane channel-forming protein [sometimes called outer membrane factor (OMF)] and a periplasmic membrane fusion protein (MFP). Homologues of these systems have been described in Stenotrophomonas maltophilia, Burkholderia cepacia, Burkholderia pseudomallei and the non-pathogen Pseudomonas putida, where they play a role in export of and resistance to multiple antimicrobial agents and/or organic solvents. Although the natural function of these multidrug efflux systems is largely unknown, their contribution to antibiotic resistance and their conservation in a number of important human pathogens makes them logical targets for therapeutic intervention.     

Amino acids at positions 3 and 4 determine the membrane specificity of Pseudomonas aeruginosa lipoproteins

Escherichia coli lipoproteins with Asp at position 2 remain in the inner membrane, whereas those having other amino acids are targeted to the outer membrane by the Lol system. However, inner membrane lipoproteins without Asp at position 2 are found in other Gram-negative bacteria. MexA of Pseudomonas aeruginosa, an inner membrane-specific lipoprotein involved in multidrug efflux, has Gly at position 2. To identify the residue or region of MexA that functions as an inner membrane retention signal, we constructed chimeric lipoproteins comprising various regions of MexA and an outer membrane lipoprotein, OprM, and analyzed their membrane localization. Lys and Ser at positions 3 and 4, respectively, were found to be critical for the inner membrane localization of MexA in P. aeruginosa. Substitution of these residues with Leu and Ile, which are present in OprM, was sufficient to target the chimeric lipoprotein to the outer membrane and to abolish the ability of MexA to confer drug resistance. The membrane specificity of a model lipoprotein, lipoMalE, a lipidated variant of the periplasmic maltose-binding protein of E. coli, was also determined by the residues at positions 3 and 4 in P. aeruginosa. In contrast to the widely accepted "+2 rule" for E. coli lipoproteins, these results suggest a new "+3, +4 rule" for lipoprotein sorting in P. aeruginosa, namely, the final destination of lipoproteins is determined by the residues at positions 3 and 4.