The effect of transposon inserts, on the ability o f CHIL208 to complement Out' mutants was found to be extremely complex. The apparent variable effect of the mutations, combined with the variability of complementation by CHIL208 itself (4.4.2) meant that no information on the existence and possible location of a repressor could be obtained. It is possible that limitations were imposed on the experiment by the use of X. In retrospect, a more conventional technique might have proved more successful.
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5.3 SCREENING FOR GENE FUSIONS.
The seven transposon inserts chosen for complementation analysis (3.2.4) were to be screened for the formation of gene fusions in the Ecc strains: GS2001 and GS4000 (PhoA" and LacZ' respectively, due to Tn70 insertions). The control strains GS2001(cHIL208) and GS4000(cHIL208) were required, but could not be generated directly as the recipients and cosmid had the same resistance markers: Ap and Tc. GS2001 and GS4000 were cured o f Tn70 using fusaric acid selection (2.12). Two rounds of growth on fusaric acid resulted in 100% loss of TniO. Screening for PhoA and LacZ activity (on XP and XGal plates: 2.2), showed that the activity of each enzyme had been lost. Previous work showed that approximately half of excision events involving T n /0 caused alterations in the chromosome very close to the original insertion site. Other events caused more extensive alterations, mainly inversions, with 10% of all excisions creating extensive deletions (Bochner et al 1980). The PhoA' and LacZ' colonies obtained from GS2001 and GS4000 were therefore likely to be due to a variety of inversion and deletion events.
GS2001(a/j/jo/4) and GS4000(alacZ) were transduced with cHIL208 and the 7 Tn- insertion derivatives (3.2.4), selecting for Tc resistance. Colonies were screened for PhoA and LacZ activity on the relevant chromogenic substrates (2.2), using HC131 as a positive control for both enzymes. Both GS2001(cHIL208) and GS4000(cHIL208) produced only white colonies, as expected for the negative controls.
All the chosen insert-containing derivatives of CHIL208, when transduced into GS2001 or GS4000 produced occasional blue colonies in a white background, rather than the blue only colonies expected if functional gene fusions had been formed. It was possible that gene fusions had been formed, which were extremely unstable, but it seemed more likely that secondary transposition had created rare fusions with chromosomal genes. This hypothesis was tested by the extraction, and restriction analysis of cosmid DNA from blue colonies. Since the mechanism o f Tn5 transposition is conservative rather than replicative (Berg 1977, Berg 1983), secondary transposition
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into the chromosome should be characterized by the loss of the transposon from the cosmid. Cosmids extracted from blue colonies were found to contain the original insertions and there was no evidence for a mixed population of cosmids, only some of which carried transposons. However, further evidence for secondary transposition was obtained. White colonies (taken from mixed plates), were streaked out to give single colonies: some of which were blue. It was concluded that none of the transposon inserts tested had generated useful protein fusions with enzyme activity.
TnlacZ, used to generate transcriptional fusions, must be inserted in the correct orientation, within an expressed gene, if LacZ activity is to be detected. Although sequence data (Reeves et al in press) suggested that the insertion in cHIL208/L5 was in the correct orientation relative to the direction of transcription of the out cluster, it might have been in a non-coding or poorly expressed region of DNA. Alternatively, the insert could have formed a fusion with an exported or trans-membrane protein. Such fusions tend not to be functional, as discussed previously (5.1). Work done subsequently (discussed earlier: 1.7, 1.9) suggested that the out cluster encodes predominantly periplasmic and/ or membrane-associated proteins. Considering this, functional LacZ fusions would not be expected.
To yield a functional PhoA fusion, an insertion must be in the correct reading frame of an expressed gene, since these fusions are translational. It is possible that of the three insertions (cHIL208/P5, P6 and P I2) which were apparently correctly orientated relative to the direction of transcription, none were in the correct reading frame of transcribed regions. Since PhoA is only active when transported to the periplasm or beyond (5.1), and the out cluster is thought to encode periplasmic and/ or membrane- associated proteins, functional PhoA fusions would be expected. It is possible that in frame fusions were formed within cytoplasmic or trans-membrane domains of integral membrane proteins, making them inactive for PhoA.
Further information on the locations of the Tn inserts could have been obtained by DNA sequence analysis of the junction sites. Since the inserts did not yield either functional gene fusions, or information on the putative repressor, no such analysis was
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performed, although information on the precise location o f the insertions might have been useful for the prediction o f open reading frames and the organization of protein domains across the cytoplasmic membrane.
It might have been possible to identify gene fusions by performing the same analysis on each of the other insert-containing derivatives of cHIL208 which were shown by sequence analysis to be in the correct orientation relative to the direction of transcription of the out cluster. Generating a homogeneous lysate for each insert, transductions and screening for fusions would have been very laborious: work which did not seem justified. It was clear that no useful information on the ability o f each cHIL208 derivative to complement Out' mutants would be obtained, due to variable phenotypes (S.2.S). Although information on the topology of transmembrane proteins might have been obtained, far more efficient methods are available for this (Broome- Smith and Spratt 1986), and are currently being used to study the Out proteins (Reeves pers. comm.).
5.4 GENE FUSIONS IN THE STUDY O F GENE EXPRESSION.
5.4.1 INTRODUCTION.
It was hoped that the work described above (5.2-5.3) would generate gene fusions, in which reporter enzymes (LacZ or PhoA) were produced under the control of out gene promoters. The intention was to use these fusions to investigate the factors responsible for the regulation o f particular out genes. Since the expression of a reporter gene in a multi-copy plasmid may be different from that of the same insertion in the chromosome (reporter activity being affected by the vector promoter and copy number), it was proposed to introduce the fusions into the Ecc chromosome by reverse genetics. Since this technique was causing problems at the time (Kell pers. comm.) and no gene fusions were identified (5.3), an alternative strategy was found.
Gene fusions had been generated previously by \ : :TnphoA mutagenesis o f GS2001 (Hinton and Salmond 1987). Among the strains generated were two Pel'Cel' mutants: