Microbial Pathogenesis 1987 ; 3: 227-230

Mini-review

;, vehicles provide new tools for genetic analysis ofGram-negative bacteria

E . Tapio Palva,' Anu Harkki, 2 Hanna Karkku,3 Hannu Lang' andMinna Pirhonen'

'Department of Molecular Genetics, Swedish University of Agricultural Sciences, Box7003, S-750 07 Uppsala, Sweden; 2Biotechnical Laboratory, The Technical ResearchCentre of Finland, SF-02150 Espoo, Finland; and 'Department of Genetics, University ofHelsinki, SF-00 100 Helsinki, Finland

The success of Escherichia coil as a model for probing fundamental biological problemsdepends to a great extent on the advanced genetic tools available for this organism .Bacteriophage iA is one of the powerful research tools available for E. coil moleculargenetics. It provides the vehicle for vectorial translocation of exogenous DNA intorecipient cells and is commonly used as such in transposon mutagenesis, cosmidcloning and in vivo generation of gene fusions . Until recently the utility of 1 vehicleshas been largely limited to E. coil. However, recent results 1-6 strongly suggest thatthese techniques could be readily applied to many different Gram-negative bacteria .

The A, receptor

Many of the applications of A vehicles require only the expression of a functionalphage receptor in the bacterial outer membrane . The receptor function is encoded bythe lamB gene',' in the maltose regulon in E. coli. 9 This gene codes for a major outermembrane protein, LamB .' LamB is classified as a porin and facilitates the permeationof maltodextrins through the outer membrane .'"'

Expression of A receptor in Gram-negative bacteria

In nature 7 receptor activity has been only detected in E. coil and in certain Mal`strains of Shige/la and Enterobacter. 12-13 However, due to the uncomplicated natureof the A receptor, a single outer membrane protein, transfer of this function to otherGram-negative bacteria becomes feasible. An indication that the E. coli A receptorcould function in other enteric bacteria was provided by the construction of E. coli-Salmonella typhi hybrids that were susceptible to A adsorption."The lamB gene of E. coli was first introduced to S . typhimurium on an F'factor and

shown to be expressed and to provide a functional phage receptor in its new host .'This early success with extending the host range of a to Salmonella has recently ledto the construction of several lamB expression plasmids that have been introduced toother Gram-negative bacteria including S.typhimurium, Klebsiella pneumoniae, Vibriocholerae and Erwinia carotovora . 3-6 In all of these cases lamB gene is expressed andthe resulting bacterial strains produce a functional A receptor. The only exception so

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228 E . T. Palva et al.

L B

Fig. 1 . Expression of /amB gene in Gram-negative bacteria . Cell envelope fractions of the bacteriaindicated were analyzed by SDS-PAGE: E. co/i (lanes 1 and 2), S . typhimurium (lanes 3 and 4), Erwiniacarotovora (lanes 5 and 6) and V. cho/erae (lanes 7 and 8) . The bacteria indicated by + harbor the lamBexpression plasmid pAMH62," except for E. coil (lanes 1 and 2) where + indicates cells grown in thepresence of Maltose.

far has been Pseudomonas aeruginosa where no 2 receptor activity was detected . 3The ompR promoter of E. co/i that we have employed to express /amB4 appears to befunctional in several Gram-negative bacteria, including S.typhimurium,° V. cholerae, 6Erwinia carotovora (Fig. 1), and Agrobacterium tumefaciens (unpublished results fromthis laboratory) . This suggests that the /amB gene can be expressed in a wide varietyof Gram-negative bacteria, and consequently the host range of 2 could be extendedto these species .

Transposon mutagenesis

The most immediate advantage of using lamB+ derivatives of Gram-negative bacteriahas been in the generation of transposon mutants .' 3,5 This has been succesfullyaccomplished in all bacteria expressing lamB.'- 3,5 The frequencies of transposition havebeen considerably lower than comparable frequencies in E. co/i, but still fully sufficientto generate transposon pools in these bacteria . The lower frequencies are probably dueto restriction of the incoming DNA . This can be partly compensated for by increasingthe multiplicity of infection .' As 2 cannot grow in most Gram-negative bacteria thekilling effect on the host is reduced .' Therefore A can be used as a true suicide vector .Another way to compensate for the lower transposition frequency is the use of thehigh hopper derivatives of 2 . 15 Using these tricks comparable or nearly comparablefrequencies to those in E. coli have been obtained .`

Other methods for delivery of transposons to Gram-negative bacteria have beendeveloped and succesfully employed, e .g . in Erwinia, Rhizobium and Agrobacterium,mostly by employing suicide plasmids . 16-19 However, the use of A vehicles fortransposon introduction offers several advantages: (1) a wide variety of transposonswith different selectable markers carried on ., vehicles are available ; 15 (2) themutagenesis is extremely easy to perform as this only involves 2 phage infection andselection for the transposon mutants; (3) the mutagenesis is clean as no unwantedinsertions of additional elements, i .e. Mu in PJB4JI mediated mutagenesis, takes place

Genetic analysis of Gram-negative bacteria

229

that would hamper the genetic analysis of the insertion mutants ; (4) it allows the useof other applications of the transposon technology such as gene fusions (see below)and generation of deletions ; (5) it should be generally applicable to all Gram-negativebacteria that can express lamB .

Cosmid cloning

Another important feature of the widened host range of 2 is that I vehicles can beemployed for cosmid cloning and transduction in these bacteria .""' Although thisapplication has not yet been fully realized it should offer important advantages inconstruction of gene libraries and cloning genes by complementation of mutants aftercosmid transduction . All of the LamB producing derivatives of Gram-negative bacteriaconstructed so far have been shown to function as recipients for cosmidtransduction ." 3.6 Consequently direct cosmid cloning into these bacteria becomesfeasible and the use of E. coil as a primary host is no more required . We are employingthis application of 2 technology to clone chromosomal genes of Erwinia and Vibrio bycomplementation of mutants in these bacteria .

Gene fusions

Gene fusions generated in vivo have proven to be of importance in analysis of geneexpression and protein localization . 20 Again, some of the more recent fusion vectorsavailable are based on 1 vehicles or utilize A vehicles to provide for transpositionfunctions."

Consequently lamB+ strains of Gram-negative bacteria could be used as recipientsfor fusion mutagenesis . This application has not yet been utilized to any great extentexcept for S.typhimurium (Harkki, Karkku and Palva, in preparation) . The reason beingpartly that several fusion vectors have been developed that employ other bacteriophagesor plasmid vehicles, and the host range of these techniques has been expanded toinclude many Gram-negative bacteria . 22 .23 In addition, the A fusion vectors result inthe integration of a transducing phage next to the gene fusion thus requiring hoststhat .? can lysogenize .

Growth and lysogeny of A is dependent on certain host functions of E. coli, the bestcharacterized being the function provided by the nusA gene prod uct . 2a-26 The NusAprotein appears to be involved in the antitermination of A transcription .21-2' Therefore,introduction of lamB alone is not sufficient but e .g . in S.typhimurium also the E. co/inusA gene is required . 2 Whether these two genes would be sufficient in other Gram-negative bacteria remains to be seen . The advantage, again, of the A vehicle approachis the simplicity by which the fusions could be generated and isolated . An inherentproperty of the A fusion vectors is that the gene/promotor of interest can be directlycloned by isolation of a 2 transducing phage .

Conclusions

In conclusion, the available data demonstrates that extension of the host range ofbacteriophage 2 to many Gram-negative bacteria is feasible . This should open up newavenues for the molecular genetic analysis of these bacteria as the powerful researchtools provided by the use of 2 vehicles become available .

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E. T. Palva et al.

We thank Rose-Marie Andersson for typing this manuscript. This research has been supportedby the Academy of Finland and the Swedish Medical Research Council (project 07487) .

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