Genetic mechanisms of Escherichia coli resistance to target inactivation * Genes governing purine metabolism in enterobacteria : an unexpected sequence found via complementation selection

Using enterobacterial strains having block in 3 different genes required for GMP synthesis, 3 groups of inserts with different restriction patterns were expected. But the fragments cloned represented 5 such groups. One consisted of Salmonella typhimurium DNA fragments with 2 SacI sites available. Sequencing revealed 100 % homology of the cloned insert to the hi-term of Y protein of the hemC-hemD linkage group of E. coli chromosome (85 min locus). It is suggested that Y may represent the gpp gene, coding for guanosinepentaphosphatase. It was also shown that a Salmonella DNA fragment resulting from PGR amplification with 20~mer primers complementary to the Nand С-terms of the hpt gene of E. coli did not encode an hypoxanthine phosphoribosyltransferase (HPRTase)and some other gene complemented GMP synthesis block in de novo and salvage pathways in E. coli cells.

Introduction.If a chemical compound selectively inac tivates only a certain type of molecules, a target, and if the function of this target is pivotal for cell survival, then an effective control of cell viability could be possible.To discover such compounds as drugs or pesticides an innovative approach has recently been developed known as structure-based drug design (for rev.see [1,2]).This approach utilizes three-dimen sional structural data to design a selective inhibitor to the active site of an enzyme and aliosteric sites as well.Thus, if mutations render a target insensitive to an inhibitor to the active site, inhibitors to the ailosteric sites could be developed to overcome the problem of drug resistance.
But alongside with this target approach others are needed as well because non-target specific cell resis tance may also occur.Such resistance mechanisms include target site overproduction due to gene ampli fication or promoter changes, reduced inhibitor upta ke, induction of efflux processes and inhibited deto- xification or sequestration.Moreover, cells can also exploit some other means to survive with a pivotal metabolic target blocked.For example, Escherichia coli thermosensitive phenylalanine-tR N A synthetase could be protected at non-permissive temperatures when the level of its cognate tRNA gene expression was elevated (for rev.see [3 ]).But the enzyme could also be protected, when cells were transformed with multicopy colEl plasmids which use RNA1 resem bling the structure of tRNA Fhe for controlling plasmid DNA replication [4 J.
May protection of DNA replication targets inactivation with some unknown genes also occur?To answer this question the study of purine metabolism is of special interest.In enterobacteria, there are de novo and salvage pathways for GMP synthesis.Both have been very well studied [5,6].Different bacterial strains are available which allow the cloning via complementation selection of genes belonging to both pathways.This makes it possible to determine if genes of only these pathways are capable of com plementing the block of GMP synthesis, or if some other genes also exist which help a cell to overcome this block.
Materials and Methods Materials.Guanine was obtained from «Sigma (USA).Bacto-tryptone and vitamin-free Casamino acids were purchased from «Difco» (USA).All other reagents used in this work were from «Sigma» and of the highest purity avai lable.
Bacterial strains and growth media.For comple mentation experiments E. coli S06O9 strain {F-Dprogpt-lac, hpt, purH, J, thi, pup, ara, str A) [6 J unable to grow on selective medium containing guanine was used as a recipient in transformation experiments with the gene library.
Salmonella typhimurium GP36 (DproAB-gpt, purE66, sug) [7 ] with a changed substrate specificity of the hpt gene coding for hypoxanthine phosphoribosyltransferase and allowing cells to grow not only on hypoxanthine but on guanine as well.This strain was used for the gene library construction.
Generation of a genomic library of S. typhimurium DNA and complementation.
High-molecular weight DNA obtained from strain GP36 was partially diges ted with restriction endonuclease Sau3A and frag ments obtained were resolved in an agarose gel.Fragments of about 3 kb identified by reference to 1 Hindlll size markers were recovered from the gel using the glassmilk procedure (Bio 101 Inc).The fragments thus obtained were ligated in 1:1 ratio with BamHI digested, dephosphorylated Bluescript SK" vector (Statagene) at 4 °С overnight [10].With this ligation products, maximal efficiency competent DH5a cells (from Bethesda Res.Lab) were transformed giving rise to 80 % of white colonies on indicator LB medium containing X-gal and IPTG [10] a 10000member gene library was thus constructed with every gene present.
Alternative methods to clone genes capable of complementing a block in GMP synthesis.Numerous clones obtained via complementation selection of E. coli S06O9 recipient cells with the GP36 S. typhi murium DNA gene library were divided into groups on the basis of different availability of restriction sites in the insertions cloned.The clones from different groups were studied routinely as in [11 J in the PGR reaction with 20-mer primers to the N-and C-terms of E. coli hpt gene using the sequence of the gene published in [12 ].The sequence of the PCR amplified DNAs of the clones studied were confirmed with 35 S-iabelied dATP by the dideoxy chain termination method as described previously [13].
Results and Discussion.Using E. coli S06O9 competent cells and a GP36 S. typhimurium gene library (in both these strains the gpt gene is deleted) 3 groups of insertions could be cloned via com plementation selection.These groups should represent the hpt sug allele of GP36 conferring on the salvage pathway the ability to the recipient to grow on guanine, the purH, J gene of Salmonella which belongs to the de novo pathway and Salmonella pup gene making conversion of adenine, adenosine and deoxyadenosine to guanine nucleotides possible [6 ].Plating of transformed and washed S06O9 cells onto selective MOPS medium supplemented with 20 /*g/ml of guanine and 50 /-tg/ml of ampicillin produced more than 50 colonies after 2 days of incubation, while the negative control (Bluescript vector alone) produced none.The clones obtained were picked, plasmid DNA minipreps were isolated as in 110J and studied in transformation experiments and with restriction endonucleases.High efficiency of retransformation was shown for every insert cloned.
An example of the restriction patterns of some inserts complementing GMP synthesis block in E, coli  demonstrates that all these inserts could be divided into 5 such groups whereas only 3 groups were anticipated with cloning system used.
Because it was known that pur Я, J of E, coli contained an EcoRl site [141 and the hpt gene of E. coli contained both EcoRV and Sail sites [12], and even though the genes studied belong to different enterobacteria, we paid attention to the fragment containing 2 Sad sites (Fig. 2).Double-stranded DNA of this fragment was sequenced using the Sequenase 2.0 kit (U. S. Biochemical Corp. Cleveland, OH) and universal and reverse primers.A small part of this sequence obtained with the forward primer is shown in Fig. 3.
Analysis of the sequence shown in Fig. 3 by Simlran and dfastp programs did not reveal any similarity to genes involved in purine metabolism, but showed strong homology to the hypothetical protein У of E. coli [15].The sequnce coding for this protein was found in 4260 bp E. coli DNA fragment mapped at 85 min of the E. coli chromosome map and includes three genes (hemC, hemD, X) which were suggested to be a part of Uro operon.The function of У remains unknown.Because the «2 Sacl» containing fragment of Salmonella DNA is strongly homologous to У and conferred the ability to S06O9 to grow on exogenous guanine, we suggested on the basis of map position that У could be identified as the gpp gene coding for guanosinepentaphosphatase.This enzyme is respon sible for a conversion of pppGpp into ppGpp which is an important regulator molecule in bacterial cell (for rev.see [16]).In the conditions of overexpression could this enzyme be capable to the resynthesis with exogenous guanine and phosphates providing for DNA synthesis precursor?Because due to this en zyme activity also ppGp compound is formed in cells [16|, degradation of this molecule also could supply cells with a precursor needed for DNA replication.This suggestion remains to be verified and the gene identified.
Besides «2 Sacl» insert we also studied if a fragment containing an EcoRI site and fragments containing EcoRV and Sail sites could be amplified in the PCR reaction with 20-mer primers complementary to the N-and C-terms of the hpt gene of E. coli.Fig. 4 shows results of this study.The positive PCR amplification with the primers used is observed with E. coli DNA containing the wild type hpt allele, with S. typhimurium GP660 DNA containing also the wild type allele of the gene and with Salmonella GP36 DNA containing the sug mutation in the hpt gene (ability to grow on hypoxanthine and guanine).As seen from Fig. 4, lane 8, the fragment containing an EcoRI could not be amplfied by PCR with primers used.
This shows that there is no cell DNA conta mination in the plasmid DNA preparations used.The inserts of S. typhimurium.

DNA containing
EcoRV, Sail, Smal and HindiII sites were all positive in the PCR reaction with generic 20-mer primers comple mentary to the N-and C-terms of E. coli hpt gene.At present these fragments are under study, but sequencing of one of the positive fragments (data not shown) demonstrated that it did not encode an HPRTase.If this insert does not include the pup gene, then some new gene capable to complement a block in GMP synthesis might be identified.

12 3 4 Fig. 2 .Fig. 3 .Fig. 4 .
Fig. 2. Study of the Salmonella typhymurium DNA insert comp lementing GMP synthesis block on de novo and salvage pathway in E. coli cells with Sad restriction endonuclease (lanes I and 4: size markers X HindiII and a ladder appropriately; lanes 2 and 3: concentration of DNA loaded differs by the factor of 2)