High-sensitivity detection of newly induced LamB protein on the Escherichia coli cell surface.

The kinetics of the appearance at the cell surface of the outer membrane LamB protein after induction were determined by using specific antibodies and radioiodinated protein A as a probe. This was done in two different induction systems. First, LamB protein was induced in a wild-type strain by the simultaneous addition of cyclic AMP and maltose. Second, an operon fusion strain in which the lamB gene is expressed under lac promoter control was used; in this system, LamB protein can be induced by isopropyl-beta-D-thiogalactopyranoside. When uninduced cells were grown in glucose minimal medium, background expression of the lamB gene was found to be ca. 10-fold lower in lac-lamB cells than in wild-type cells. The level of LamB protein present in uninduced wild-type cells could, however, be reduced by supplementing the growth medium with Casamino Acids. After induction, the LamB protein appeared at the cell surface of both strains within a few minutes, and then the LamB level per cell increased linearly. The time lag in cell surface exposure of LamB protein differed slightly under both induction conditions: the LamB protein appeared at the surface of lac-lamB cells within 3 min of induction, whereas in wild-type cells it could not be detected earlier than after 4 to 5 min of induction.     

LamB-mediated adherence of enteropathogenic Escherichia coli to HEp-2 cells

Aims:  To establish the role of maltoporin (LamB) in adherence of enteropathogenic Escherichia coli (EPEC) to epithelial cells in vitro.
Methods and Results:  Three strains, wild type (WT) EPEC, a maltoporin (LamB) mutant ΔlamB , and DH5α were used to study adherence to cultured HEp-2 cells. Mutant ΔlamB was found to be deficient in adherence compared to WT EPEC. Adherence of ΔlamB was restored to wild type levels when complemented with the cloned lamB gene. The non–adherent strain DH5α also adhered to HEp-2 cells when it harboured the cloned lamB gene. The LamB protein was isolated from WT EPEC by electroelution and antibodies were raised in rabbits. The specificity of the antibodies was analysed by Western blotting. Anti-LamB antiserum reduced adherence of WT EPEC to HEp-2 cells. The LamB protein was coated on latex beads and the beads adhered to HEp-2 cells. Anti-LamB antiserum prevented bead adherence to HEp-2 cells. Multiple sequence alignment showed that the L9 loop of EPEC LamB had four amino acids different from the L9 loop of LamB from several other related pathogens.
Conclusions:  LamB serves as an alternative or additional adherence factor for EPEC.
Significance and Impact of the Study:  Adherence is an important component of the pathogenesis of noninvasive pathogens like EPEC. A putative adhesin such as LamB, which has already been found to be co-expressed with virulence factor EspB may be a potential vaccine candidate for control of EPEC and related pathogens.     

Benzoate-dependent induction from the OP2 operator-promoter region of the TOL plasmid pWWO in the absence of known plasmid regulatory genes.

Expression of the lower catabolic pathway of the TOL plasmid pWWO requires an aromatic acid inducer and the product of the xylS regulatory gene. Pseudomonas putida cells transformed with a plasmid containing the operator-promoter region of the lower pathway (OP2 [or Pm]), upstream from the catechol 2,3-dioxygenase structural gene, showed enzyme induction in the absence of known TOL plasmid regulatory genes. Induction was not seen in transformed Escherichia coli cells or in a P. putida mutant lacking chromosomally encoded benzoate catabolic functions.     

Enhanced metallosorption of Escherichia coli cells due to surface display of beta- and alpha-domains of mammalian metallothionein as a fusion to LamB protein

The lamB gene was inserted at with DNA fragments encoding N-terminal beta- and C-terminal alpha-domains of human metallothionein 1A (HMT1A). The hybrid LamB proteins were expressed as full-length products. Virtually whole pool of hybrid LamB proteins was found localized in the outer membrane of E. coli to and cells expressing LamB variants retained sensitivity to lambda phage, indicating their correct folding. Expression of hybrid LamB proteins increased natural ability of E. coli accumulate bivalent heavy metals ions with the highest efficiency observed for cadmium. The order of amount of cadmium accumulated is alpha-domain of HMT1A > HMT1A > beta-domain of HMT1A. This correlates with affinity for cadmium and stability of metallothionein and its individual domains. This confirms suitability of LamB vehicle for surface display of various bioactive molecules and suggests possibility of engineering of cell surface for bioremediation of heavy metals.     

A lamB expression plasmid for extending the host range of phage λ to other enterobacteria

Abstract We have constructed a multicopy plasmid vector (pAMH62) expressing lamB , the gene coding for the phage λ receptor protein in Escherichia coli . In this construction, the lamB structural gene was fused to the ompR promoter of E. coli . The ompR promoter was employed because: (i) it can function in other gram negative bacteria; (ii) it expresses lamB in a multicopy state at a level comparable to that of maltose-induced chromosomal lamB in E. coli . The vector pAMH62 was tested in E. coli and Salmonella typhimurium . In both cases the LamB protein was produced in similar amounts, was properly integrated to the outer membrane and was functional as phage λ receptor. Thus pAMH62 should provide a useful tool for extending the host range of phage λ and λ-derived vectors to other Gram-negative bacteria.     

Dual system to reinforce biological containment of recombinant bacteria designed for rhizoremediation

Active biological containment (ABC) systems have been designed to control at will the survival or death of a bacterial population. These systems are based on the use of a killing gene, e.g., a porin-inducing protein such as the one encoded by the Escherichia coli gef gene, and a regulatory circuit that controls expression of the killing gene in response to the presence or absence of environmental signals. An ABC system for recombinant microorganisms that degrade a model pollutant was designed on the basis of the Pseudomonas putida TOL plasmid meta-cleavage regulatory circuit. The system consists of a fusion of the Pm promoter to lacI, whose expression is controlled by XylS with 3-methylbenzoate, and a fusion of a synthetic P(lac) promoter to gef. In the presence of the model pollutant, bacterial cells survived and degraded the target compound, whereas in the absence of the aromatic carboxylic acid cell death was induced. The system had two main drawbacks: (i) the slow death of the bacterial cells in soil versus the fast killing rate in liquid cultures in laboratory assays, and (ii) the appearance of mutants, at a rate of about 10(-8) per cell and generation, that did not die after the pollutant had been exhausted. We reinforced the ABC system by including it in a Deltaasd P. putida background. A P. putida Deltaasd mutant is viable only in complex medium supplemented with diaminopimelic acid, methionine, lysine, and threonine. We constructed a P. putida Deltaasd strain, called MCR7, with a Pm::asd fusion in the host chromosome. This strain was viable in the presence of 3-methylbenzoate because synthesis of the essential metabolites was achieved through XylS-dependent induction. In the P. putida MCR7 strain, an ABC system (Pm::lacI, xylS, P(lac)::gef) was incorporated into the host chromosome to yield strain MCR8. The number of MCR8 mutants that escaped killing was below our detection limit (<10(-9) mutants per cell and generation). The MCR8 strain survived and colonized rhizosphere soil with 3-methylbenzoate at a level similar to that of the wild-type strain. However, it disappeared in less than 20 to 25 days in soils without the pollutant, whereas an asd(+), biologically contained counterpart such as P. putida CMC4 was still detectable in soils after 100 days.     

Binding of basement-membrane laminin by Escherichia coli

An invasive Escherichia coli (EIEC) isolate was found to bind basement-membrane laminin in a saturable and time-dependent manner. Excess of unlabelled laminin inhibited the binding of the radioactively labelled protein. Non-invasive E. coli K-12 exhibited only low-level laminin binding but introduction of the virulence-associated plasmid from the EIEC isolate led to high-level binding. Expression of a receptor for laminin on the bacteria was therefore associated with the presence of the virulence plasmid. Scatchard plot analysis indicated approximately 1000 receptors per bacterial cell, and a Kd of high-affinity binding of 0.5 pM. A laminin-binding protein which correlated with the presence of the plasmid was isolated and characterized. Its sequence of the eight amino-terminal amino acids was identical to that of the LamB protein of E. coli, although the molecular mass of the two in sodium dodecyl sulphate/polyacrylamide gel (SDS-PAGE) appeared to be slightly different. Both proteins reacted in immunoblot assays with polyclonal antisera raised against either protein, and both proteins bound laminin. Southern-blot hybridization analysis established that both the EIEC strain and the K-12 strains with or without the virulence plasmid contained one lamB gene only, and no laminin-binding protein appeared when the virulence plasmid was introduced into bacteria deleted for the lamB gene. On the basis of these results we suggest that native LamB protein of E. coli or a modified variant of it serves as a major receptor for laminin binding and is present at an increased level in invasive E. coli containing the virulence plasmid.     

Cloning and expression of the ponB gene, encoding penicillin-binding protein 1B of Escherichia coli, in heterologous systems.

A fragment from the ponB region of the Escherichia coli chromosome comprising the promoterless sequence encoding penicillin-binding protein 1B (PBP 1B) has been cloned in a broad-host-range expression vector under the control of the kanamycin resistance gene promoter present in the vector. The hybrid plasmid (pJP3) was used to transform appropriate strains of Salmonella typhimurium, Pseudomonas putida, and Pseudomonas aeruginosa. In all instances, the coding sequence was expressed in the heterologous hosts, yielding a product with electrophoretic mobility, protease accessibility, membrane location, and beta-lactam-binding properties identical to those of native PBP 1B in E. coli. These results indicated that PBP 1B of E. coli is compatible with the cytoplasmic membrane environment of unrelated bacterial species and support the idea that interspecific transfer of mutated alleles of genes coding for PBPs could potentially be an efficient spreading mechanism for intrinsic resistance to beta-lactams.     

Expression of the human interferon alpha F gene in the obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida

The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter. A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E. coli trp-operon intercystronic region. Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l. This plasmid was cloned in broad-host-range vector plasmid pAYC31. The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6. Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E. coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host.     

Expression of the phospholipase C gene of Pseudomonas aeruginosa in Escherichia coli and Pseudomonas

The plc gene for phospholipase of Pseudomonas aeruginosa, able to be transcribed only from its own promoter, has been introduced into Escherichia coli, Pseudomonas aeruginosa and Pseudomonas putida cells in the recombinant plasmid pPMS21 of a wide host range. The expression of plc gene in all recipient cells has been shown to be phosphate regulated. The fact emphasizes the identity of pho-regulation systems in Escherichia coli and Pseudomonas cells. The level of phospholipase activity is similar in Pseudomonas putida and Pseudomonas aeruginosa under the conditions of the gene derepression, while in Escherichia coli cells the level does not exceed 10% of activity registered in Pseudomonas cells.     

Degradation of chlorotoluenes by in vivo constructed hybrid strains: problems of enzyme specificity, induction and prevention of meta-pathway

The activities of the TOL plasmid-coded xylene oxygenase, benzylalcohol dehydrogenase, benzaldehyde dehydrogenase of Pseudomonas putida strain PaW1 were tested with substituted toluenes, benzylalcohols and benzaldehydes, respectively, as substrates. Several chlorinated toluenes were shown to induce enzymes of the xylene degradation sequence. Conjugative transfer of the TOL plasmid from Pseudomonas putida strain PaW1 to Pseudomonas sp. strain B13 and Pseudomonas cepacia strain JH230 allowed the isolation of hybrid strains capable of growing in the presence of 3-chloro-, 4-chloro- and 3,5-dichlorotoluene. Hybrid strains revealed new ways to prevent the dead-end meta-pathway for cholorocatechols.     

Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13.

The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate. The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27. A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27. In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence. The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp. strain B13 plasmid pWR1. A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.     

Linker mutagenesis in the gene of an outer membrane protein of Escherichia coli, lamB

In order to identify sequences involved in the localization of LamB, an outer membrane protein from E coli K12, mutagenesis by linker insertion has been performed on a lamB gene copy carried on a plasmid devised for this purpose. An analysis of the first set of 16 clones constructed by this technique shows that, in these clones, the lamB protein is altered either by frameshift mutations leading to abnormal COOH terminal (usually premature termination) or by in-phase deletions or small insertions. Except for two in-phase linker insertions, which only slightly changed the behavior of the protein, the modified proteins are either toxic to cell growth or unstable. In all cases examined so far, the modified proteins were in the outer membrane. We suggest that toxicity is due to incorrect folding, which leads to disruption of the outer membrane. The nature of the genetic alterations leads to the hypothesis that the first 183 amino acids of the LamB mature protein contain, together with the signal sequence, all the instructions needed for proper localization.