Applied and Environmental Microbiology, September 2003, p . 5627-5635, Vol . 69, No . 9

There is no real consensus for the UAS of ⁵⁴-dependent systems except that they involve repeat sequences and are located between about bases -70 and -200 upstream of the transcriptional start (29) . Two such well-studied systems for the catabolism of aromatic compounds in Pseudomonas strains are controlled by the regulators DmpR and XylR, where the UAS in each case is an inverted repeat (IR) sequence (22, 28) .

Two overlapping IRs are present between bases -79 and -140 upstream of the areCBA transcriptional start (12) and were suggested as possible UAS (Fig . 1) .

 
The creation of site-directed mutations is very important for understanding gene function . Here we report the development of a reliable and efficient method for generating site-directed chromosomal mutations by allelic replacement (homologous recombination) based on the two-step marker exchange-eviction mutagenesis strategy (23) . This has been developed for use in the naturally transformable bacterium Acinetobacter sp . strain ADP1, which has been used as a model for understanding the organization and integration of catabolism of carbon compounds, particularly arising from plants (21) . We use a newly constructed cassette containing a gene conferring kanamycin resistance and the sacB gene from Bacillus subtilis that codes for levan sucrase and confers lethality when strains are grown in the presence of sucrose (7, 27) . The sacB gene is used as a counterselectable marker when homologous mutated DNA fragments, created by overlap extension PCR (OEP) (9), are used to transform Acinetobacter sp . strain ADP1, which leads to the eviction of the sacB gene by homologous recombination . This mutational strategy was used in this study to create a variety of specific mutations within the putative UAS upstream of the areCBA promoter region as a means of confirming its position and better understanding the role of the two overlapping IR sequences .

 
Bacterial strains and growth conditions.
Aromatic substrates were obtained from Sigma-Aldrich Co . Luria-Bertani medium (LB) (25) was used to cultivate bacteria unless noted otherwise . For growth on minimal medium, single carbon sources were added to the minimal salts medium (3) at the following concentrations: benzyl acetate at 2.5 mM and succinate at 10 mM . Where appropriate, ampicillin at 100 µg/ml and kanamycin at 50 µg/ml for Escherichia coli and at 10 µg/ml for Acinetobacter were used . Sucrose at 50 g liter^-1 was added to LB when used for sacB counterselection .

PCR amplification.
PCR amplifications were performed in 50-µl reaction mixture volumes containing 10 ng of template DNA, 100 pmol of each primer, 2.5 nmol of each deoxynucleoside triphosphate, and 1 U of Pfu polymerase (Promega) in the reaction buffer supplied by the manufacturer . Reaction components were the same for OEP, except that the template DNA consisted of the two overlapping PCR-amplified fragments purified by excision from an 0.8% agarose gel . The PCR mixtures were subjected to a 2-min hot start at 94°C and then to 35 cycles, with one cycle consisting of 30 s at 94°C, 1 min at 56°C, and 2 min per kilobase at 72°C . PCR fragments to be cloned into pGEM-TEasy cloning vector (Promega Inc.) were incubated at 72°C for 10 min in the presence of Taq DNA polymerase, polymerase buffer, and 0.5 mM dATP prior to ligation .

Construction of pRMJ1 carrying the sacB-Km cassette.
EcoRI digestion and religation of pKOK6.1 (17) created pKOKD, a 6.85-kb ampicillin- and kanamycin-resistant derivative of pKOK6.1 but with the lacZ reporter gene and its adjacent BamHI, PstI, and SalI sites deleted (Fig . 2) . The vector pKNG101 contains sacB and its promoter region (14), and primers were designed to amplify both on a single fragment . The forward primer sacB-1 (5'-AGATATCGGATCCGTCGACCTGCAGGCCCATGCAACAGAAAC-3') contained adjacent engineered BamHI, PstI, and SalI sites (underlined), and the reverse primer sacB-2 had the sequence 5'-GGTTAGGAATGATATCAGCCATTTGCCTGCTTTTA-3' . The amplification product was A-tailed and cloned into the pGEM-TEasy cloning vector (Promega Inc.), creating pSacB . The inserted sacB PCR fragment in pSacB was flanked by the vector EcoRI sites . This enabled it to be excised as an EcoRI fragment after partial digestion to take account of the additional EcoRI site within sacB . This fragment was then cloned into the single EcoRI site within pKOKD, creating pRMJ1 . Multiple restriction digests determined that sacB is in the orientation opposite that of the kanamycin resistance gene in pRMJ1 and that the two genes are transcribed divergently . Restriction digests also showed that the sacB-Km cassette thus created could be excised on a single BamHI, PstI, or SalI fragment .

 
Creation of lacZ reporter gene fusions.
To generate the areA::lacZ fusion, the promoterless lacZ-Km cartridge of pKOK6.1 (17) was isolated as a PstI fragment and inserted into the compatible NsiI site in areA of plasmid pADPW36, creating pADPW38 (12) . A linear DNA fragment carrying the areA::lacZ fusion was generated by digestion of pADPW38 with restriction endonucleases KpnI and XbaI and used to transform ADP1, thereby generating ADPW61 . The same plasmid was linearized and transformed into each of the constructed ADP1 mutants, thereby creating a secondary set of mutants with mutations in the UAS and lacZ reporter genes in areA . Induction of the areCBA operons in these mutants was measured by ß-galactosidase activities of whole cells (19) after growth to late log phase on succinate in the presence of benzyl alcohol as described above .

OEP.
PCR for OEP (9) was routinely performed by using Pfu DNA polymerase (Promega) . The same two external primers, OEP-1 (5'-CACTTACTTGAATTCGAACTTTTCGGCTATG-3') and OEP-2 (5'-TGGTACCTGCAGGCAGTAATCCCTTTGG-3'), were used for all the mutants and were complementary to regions of about 800 bp on either side of the UAS region . The mutations were designed on two overlapping internal primers (Table 2) which were routinely about 30 mers . At least 20 bases at the 5' end of each were complementary to each other and constituted the region into which the designed mutations were incorporated . The 10 bases at the 3' end of each were identical to the chromosomal sequence .

 
Natural transformation of ADP1 strains.
Recipient strains of ADP1 were transformed as described previously (6) .

Cultures and preparation of cell extracts.
Overnight cultures of ADPW P_are mutants were prepared and inoculated into 1 liter of minimal medium containing 10 mM succinate and 1 mM benzyl alcohol . Following overnight growth on a shaking incubator at 30°C, additional succinate (to 10 mM) and benzyl acetate (to 1 mM) were added . The cells were grown for a further 4 h and harvested by centrifugation at 4°C, and the pellets were stored at -20°C . When required for assay, the pellets were resuspended in 50 mM phosphate buffer, pH 7.4, and the cells were disrupted ultrasonically . Disrupted cell suspension was clarified by centrifugation at 116,000 x g for 30 min . Samples of supernatants were stored at -20°C .

Enzyme assays.
Benzyl alcohol dehydrogenase (AreB) activity was measured as previously described (11) . Assays were done in triplicate at two enzyme concentrations for two independently prepared cell extracts of each mutant .

 
(ii) Marker exchange-eviction of the sacB-Km cassette from ADPW112.
The sacB-Km cassette from ADPW112 was evicted by marker exchange . The protocol was tested by transforming ADPW112 with pADPW112 (containing the novel BamHI site in the putative UAS region) . Transformation mixtures of ADPW112 and linearized pADPW112 were grown overnight at ambient temperatures and then plated onto LB agar containing sucrose and incubated at ambient temperatures . Single colonies of transformants appeared after 48 h of incubation; a single transformant was designated ADPW114 . No transformants were obtained when the same transformation was undertaken at 37 or 30°C . The transformants which were no longer sucrose sensitive were found to have lost kanamycin resistance . The adjacent DNA was amplified by PCR from genomic DNA from a representative transformant by using primers OEP-1 and OEP-2 . A 1.6-kbp product was amplified that, on digestion with BamHI restriction endonuclease, showed the presence of two bands of 0.75 and 0.85 kbp, proving the presence of the created BamHI restriction site in ADPW114 by comparison with the product of PCR amplification of the same region in wild type ADP1, which failed to yield two fragments following BamHI digestion (data not shown) .

(iii) Creation of ADP1 mutants with deletions or substitutions in the UAS.
ADP1 mutants with designed deletions or substitutions in the UAS were created essentially by the method described above but with different complementary primer pairs spanning the UAS region and carrying the designed mutation within each primer sequence (Table 2) . The same flanking primers, OEP-1 and OEP-2, were used for all amplifications, and pADPW47 (cloned wild-type ADP1 DNA) (Table 1) was used as template DNA . The amplified OEP fragments were A-tailed and cloned into pGEMeasy, and each plasmid was linearized and used to transform ADPW112, where the eviction of the sacB marker gene was monitored by the selection on LB-sucrose plates of sucrose-resistant, kanamycin-sensitive transformants . The successful creation of the designed mutation was confirmed by sequencing a PCR fragment amplified from the genomic DNA of a transformant colony . Once the successful integration of the mutated fragment was confirmed, the mutant was transformed with a reporter lacZ gene . This was done by linearizing pADPW38 (areA::lacZ-Km), using it for transformation, and selecting for growth in the presence of kanamycin, thus creating a double mutant with a mutation in the UAS and a reporter lacZ gene in areA .

The effect of the mutation on are gene expression was tested (i) by using biochemical assays of AreB (benzyl alcohol dehydrogenase) activity of the primary mutant and (ii) by measuring ß-galactosidase activity in the corresponding areA::lacZ derivative strain .

Structure of IR sequences at the UAS.
The UAS region has two IRs which overlap (Fig . 1) . One is a near-perfect IR (16 of 17 bp) covering bases -105 to -139 from the +1 transcription start site and centered on base -122 . This IR is referred to as IR1 (comprising IR1L and IR1R) .

The other (IR2) stretches from -79 to -132 of the P_are UAS region . Each arm (IR2L and IR2R) consists of two stretches which are perfect 9- and 7-bp repeats and are separated by a 3-bp mismatch . The three mismatched bases in IR2L, ATT (bases -121 to -123), are centered between the two repeats of IR1 and would lie at the apex of any hairpin loop structure formed by IR1 (Fig . 1) .

The central bases of the inner 7-bp repeats of IR2 (bases -94 and -117) are separated by 22 bp, and the central bases of the two 9-bp arms of IR2 (bases -83 and -128) are separated by 44 bp (Fig . 1) . This is characteristic of regions shown to be involved in regulator binding in other ⁵⁴-dependent regulatory systems in which the IRs are separated by complete turns of the DNA helix, i.e., multiples of 11 bp (26) .

Deletion mutations of the P_are UAS region.
Three mutants were created with major deletions in the UAS region . The deletions in mutants ADPW151 and ADPW152 were both 26 bp, that in mutant ADPW153 was 19 bp, and in all three cases the deletions were located in IR1 (Table 1; Fig . 3) . Each mutation deleted nearly all of IR1 and the whole of IR2L (Fig . 1) . Expression of the are genes was reduced by 85 to 90% by each mutation .

A further series of five mutants with deletions in IR2R was created . The mutants (ADPW164, ADPW165, ADPW197, ADPW198, and ADPW199) contained 3-, 6-, 9-, 10-, and 11-bp deletions, respectively, that were centered on base -94 at the center of the 7-bp arm of IR2R (Fig . 4A) . Whereas the shorter 3- and 6-bp deletions caused major (90 to 98%) reductions in are gene expression, the longer the deletion the less was its effect: the 9- and 10-bp deletions caused 80% loss of expression, whereas the 11-bp deletion in ADPW199, which constituted one complete turn of the double helix, caused only a 50% loss of expression .

 
Control mutants.
Four control mutants were constructed (Fig . 4B): ADPW167, with a 4-bp substitution to the right of the entire IR region; ADPW200 (3 bp) and ADPW201 (6 bp), with substitutions to the left of IR1L; and ADPW166, with substitutions between the two repeats of IR2L . In none of these mutants was expression significantly lowered from that of the wild type .

Substitution mutations in IR1L.
Ten substitution mutants, designated ADPW171 to ADPW179, were created with substitutions in IR1L . The bases were systematically replaced in 4-, 8-, 12-, and 16-bp segments (Fig . 4C) . In all cases, the substitutions were such that CG was replaced by AT and vice versa, thus destroying the degree of complementarity of the IRs . All of the mutations also affected the 9-bp arm of IR2L, except that in ADPW171 and ADPW174 only the left base of this arm was altered .

Replacing the inner bases of IR1, which includes the 9-bp arm of IR2L, reduced expression by up to 90% . However, replacing the outer bases of the left arm of this IR (in ADPW171 and ADPW174) had no such drastic effect on induction, reducing activity by only around 50% .

Substitution mutations in IR1R.
A similar set of mutants (ADPW180 to ADPW189) (Fig . 4D) was created with substitutions in IR1R . As with IR1L substitution mutants, replacing the inner eight bases of the right arm of IR1, which includes the 7-bp arm of IR2L, reduced expression severely by up to 95% (in ADPW180, ADPW181, ADPW184, ADPW185, ADPW186, ADPW187, and ADPW188) . By contrast, replacing the outer bases of IR1R, which does not change the 7-bp arm of IR2L, had a much less drastic effect on induction, reducing activity by only around 10 to 30% (in ADPW182, ADPW183, and ADPW189) .

Substitution mutations in the 7-bp arm of IR2R.
The final series of mutants (ADPW190 to ADPW195) was created with substitutions in the right arm of IR2 (Fig . 4E) . The various mutations in this sequence of bases reduced gene expression by 50 to 80% . The greatest effect was from the replacement of 6 or 7 bp, which caused an 80% loss in enzyme induction, whereas replacing only 2 or 4 bp resulted in 50 to 70% loss of enzyme activity . In ADPW166 (Fig . 4B), the region between the two arms of IR2R was changed from CCCC to TTTT and this change had only a marginal effect upon expression (20%) .

We have used this methodology to investigate putative regulatory DNA upstream of the catabolic are operon by a process of systematic mutagenesis . Whereas it could easily be used for mutations within structural genes, a particular advantage of this procedure for studying regulation is that the mutations are introduced into the single chromosomal copy of the gene and thereby overcome any problems due to copy number associated with using plasmids as a vector .

Transcription of the areCBA operon from the -24, -12 ⁵⁴-dependent promoter is strongly regulated, with areCBA expression measurable only in the presence of pathway substrates (11, 12) . The sequence upstream of the -24, -12 promoter contains a 43-bp AT-rich region that is 98% AT (Fig . 1), abnormal even for an AT-rich bacterium such as Acinetobacter . This region separates the -24, -12 promoter from an upstream complex of IR sequences . This follows other ⁵⁴-dependent promoter systems (26) which have a UAS located 100 to 200 bp upstream of the -24, -12 region and separated from it by DNA which can form a loop to bring the UAS into physical proximity with the polymerase-⁵⁴ complex . This DNA has been shown to contain IHF binding sites (28) which bring about the loop formation . The AT-rich region upstream of the are promoter consists of 42 AT pairs containing a single GC pair at position -42 from the start of transcription (Fig . 1) . The sequence of bases containing the lone C (5'-AAAAACAATAATTT at -35 to -46) is consistent with the consensus sequence for IHF binding sites (http://www.bmb.psu.edu/seqscan)and is virtually identical to the identified binding region for IHF (IHF2; 5'-AAAAACAATACCTT) upstream of the ⁵⁴-dependent promoter system of the phenol-catabolic dmp operon (28) .

The UAS of ⁵⁴-dependent promoters are normally arranged as IR sequences . In the putative are UAS there are two overlapping IR sequences, IR1 and IR2 . Unlike most NtrC-like regulators, the areR gene is upstream of and transcribed in the same direction as its regulated genes (11), whereas the norm is divergent transcription . The experimental problem in attempting to assign separate roles for the two IR regions is that the left arm of IR2 (IR2L) sits centrally over both arms of IR1 and most mutations will affect both IRs simultaneously .

The results taken as a whole show that the 60-bp region is clearly involved in the regulation of areCBA expression . Both deletions and substitutions reduced are gene expression by up to 90%, whereas control substitutions just outside of the region had no effect upon expression . Deletions in IR1 that also disrupted IR2L sequences drastically reduced activity (Fig . 4A) but could not distinguish the relative importances of the two IRs . Deletions in IR2R (Fig . 4A) also caused loss in areCBA expression of up to 90% . Again, this does not assign the function of UAS to that particular region since it is known that deletions other than complete turns of the helix (multiples of 11 bp) between the UAS and the ⁵⁴-dependent promoter have a deleterious effect on gene expression (20) . A 3- or 6-bp deletion effected the greatest reduction of expression, whereas deletions closer to one complete turn of the helix had less of an effect . The mutant ADPW199 with an 11-bp deletion retained 50% activity compared to the wild type, indicating that transcription is still activated despite the loss of one complete arm of IR2 .

Substitutions in the region where IR1L and the 9-bp arm of IR2L overlap showed that only the bases which were also part of IR2L had a substantial effect on gene expression (Fig . 4C) . Substitutions at the left end of IR1L, with little overlap with IR2L, although equally affecting the mismatch between IR1L and IR1R, decreased expression much less than similar substitutions that overlapped significantly with the 9-bp arm of IR2L .

Similarly, substitutions in IR1R (Fig . 4D) generally reduced expression more when they overlapped the 7-bp arm of IR2L than when they were at the outside end of IR1R . These results indicate that the central region of IR1, which also corresponds to IR2L, is more critical in determining expression, either when deleted or when replaced to reduce the complementarity of the two repeats, than are the two outer ends of IR1 . By contrast, all the substitutions in the 7-bp arm of IR2R (Fig . 4E) have a relatively minor effect and it is apparent that transcriptional expression is much more tolerant of substitutions in this region .

The overall emphasis of the results indicate that the DNA of IR1 and IR2L carries the major role in determining the regulation of the are operon . It also implies that IR2 is probably a much less critical factor than IR1 in determining expression: all mutations in IR2L clearly have a severe effect, but the absence of reciprocal effects from similar mutations in IR2R would indicate that the mutations in IR2L are effective because they are also in IR1 . If this implies that the repeat sequence of IR1 is the site where AreR binds, then it also follows from the data that there must be a graded reduction of critical contacts from the center to the periphery of the repeat .

It is unwise to conclude from the data presented that either of the IR elements has a direct role in terminating areR transcription . The results do not eliminate this as a possibility, but it would seem that a more convincing model would be that AreR, binding in the region of IR1, would act as a regulator of its own transcription .

Deletions within IR2R (Fig . 4A) do have a major effect on expression, but the correlation between the effect and the length of the deletion relative to the turn of the helix suggests that the explanation is due to a topological effect on the interaction of AreR bound to the UAS with the downstream -24, -12 promoter following loop formation rather than to the removal of an arm of the repeat .

A mutational study such as this cannot answer all the questions about the importance of specific residues in determining regulation . Analysis of DNA footprinting and gel shift assay studies of AreR on the DNA should lead to physical information on the protein-DNA contacts . This would then enable the design of mutants on a slightly more focused basis than we were able to use here, which should enable a tight correlation to be made between the physical binding data and the effect upon gene expression .

We thank Victor de Lorenzo for advanced viewing of his review article .

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