folA, a new member of the TyrR regulon in Escherichia coli K-12.

The folA gene was identified as a new member of the TyrR regulon by genomic SELEX. Binding of TyrR to two sites in folA activated its transcription. Mutations in the N-terminal or central domain of TyrR, the alpha subunit of RNA polymerase, or integration host factor all abolished activation of the folA promoter.

Interaction of initiator proteins with the origin of replication of an IncL/M plasmid.

The origin of replication of the IncL/M plasmid pMU604 was analyzed to identify sequences important for binding of initiator proteins and origin activity. A thrice repeated sequence motif 5'-NANCYGCAA-3' was identified as the binding site (RepA box) of the initiator protein, RepA. All three copies of the RepA box were required for in vivo activity and binding of RepA to these boxes appeared to be cooperative. A DnaA R box (box 1), located immediately upstream of the RepA boxes, was not required for recruitment of DnaA during initiation of replication by RepA of pMU604 unless a DnaA R box located at the distal end of the origin (box 3) had been inactivated. However, DnaA R box 1 was important for recruitment of DnaA to the origin of replication of pMU604 when the initiator RepA was that from a distantly related plasmid, pMU720. A mutation which scrambled DnaA R boxes 1 and 3 and one which scrambled DnaA R boxes 1, 3 and 4 had much more deleterious effects on initiation by RepA of pMU720 than on initiation by RepA of pMU604. Neither Rep protein could initiate replication from the origin of pMU604 in the absence of DnaA, suggesting that the difference between them might lie in the mechanism of recruitment of DnaA to this origin. DnaA protein enhanced the binding and origin unwinding activities of RepA of pMU604, but appeared unable to bind to a linear DNA fragment bearing the origin of replication of pMU604 in the absence of other proteins.

Role of RepA and DnaA proteins in the opening of the origin of DNA replication of an IncB plasmid.

The replication initiator protein RepA of the IncB plasmid pMU720 was shown to induce localized unwinding of its cognate origin of replication in vitro. DnaA, the initiator protein of Escherichia coli, was unable to induce localized unwinding of this origin of replication on its own but enhanced the opening generated by RepA. The opened region lies immediately downstream of the last of the three binding sites for RepA (RepA boxes) and covers one turn of DNA helix. A 6-mer sequence, 5'-TCTTAA-3', which lies within the opened region, was essential for the localized unwinding of the origin in vitro and origin activity in vivo. In addition, efficient unwinding of the origin of replication of pMU720 in vitro required the native positioning of the binding sites for the initiator proteins. Interestingly, binding of RepA to RepA box 1, which is essential for origin activity, was not required for the localized opening of the origin in vitro.

Putative interhelical interactions within the PheP protein revealed by second-site suppressor analysis.

Highly conserved glycine residues within span I and span II of the phenylalanine and tyrosine transporter PheP were shown to be important for the function of the wild-type protein. Replacement by amino acids with increasing side chain volume led to progressive loss of transport activity. Second-site suppression studies performed with a number of the primary mutants revealed a tight packing arrangement between spans I and II that is important for function and an additional interaction between spans I and III. We also postulate that a third motif, GXXIG, present in span I and highly conserved within different members of the amino acid-polyamine-organocation family, may function as a dimerization motif. Surprisingly, other highly conserved residues, such as Y60 and L41, could be replaced by various residues with no apparent loss of activity.

Out of our reach? Assessing the impact of introducing a critical care outreach service.

An outreach service was introduced in three surgical wards and the surgical high dependency unit in a large teaching hospital. A modified early warning score and callout algorithm were used to facilitate referrals to the team. Changes in unplanned admission rate to intensive care, length of stay, mortality rate and number of re-admissions following the introduction of outreach were sought. Following the introduction of the outreach service the emergency admission rate to intensive care fell from 58% to 43% (p = 0.05). These emergency patients had shorter lengths of stay (4.8 days vs. 7.4 days) and had a lower mortality (28.6% vs. 23.5%, p = 0.05). The re-admission rate also fell from 5.1% to 3.3% (p = 0.05). The outreach service had a significant impact on critical care utilisation.

Interaction of the initiator protein of an IncB plasmid with its origin of DNA replication.

The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5'AANCNGCAA3'. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to ori(B) interact with one another.

Study of second-site suppression in the pheP gene for the phenylalanine transporter of Escherichia coli.

Site-directed mutagenesis was used to investigate a region of the PheP protein corresponding to the postulated consensus amphipathic region (CAR) in the GabP protein. Whereas some critical residues are conserved in both proteins, there are major differences between the two proteins which may reflect different functions for this region. Replacement of R317, Y313, or P341 by a number of other amino acids destroyed the PheP function. An R317E-E234R double mutant exhibited low levels of PheP transport activity, indicating that there is a possible interaction between these two residues in the wild-type protein. E234 is highly conserved in members of the superfamily of amino acid-polyamine-organocation transporters and also is critical for PheP function in the wild-type protein. Second-site suppressors were isolated for mutants with mutations in E234, Y313, R317, and P341. Most suppressor mutations were found to cluster towards the extracellular face of spans III, IX, and X. Some mutations, such as changes at M116, were able to suppress each of the primary changes at positions E234, Y313, R317, and P341 but were unable to restore function to a number of other primary mutants. The possible implications of these results for the tertiary structure of the protein are discussed.

Pseudoknot-dependent translational coupling in repBA genes of the IncB plasmid pMU720 involves reinitiation.

Replication of the IncB miniplasmid pMU720 requires synthesis of the replication initiator protein, RepA, whose translation is coupled to that of a leader peptide, RepB. The unusual feature of this system is that translational coupling in repBA has to be activated by the formation of a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. A small antisense RNA, RNAI, controls replication of pMU720 by interacting with repBA mRNA to inhibit expression of repA both directly, by preventing formation of the pseudoknot, and indirectly, by inhibiting translation of repB. The mechanism of translational coupling in repBA was investigated using the specialized ribosome system, which directs a subpopulation of ribosomes that carry an altered anti-Shine-Dalgarno sequence to translate mRNA molecules whose Shine-Dalgarno sequences have been altered to be complementary to the mutant anti-Shine-Dalgarno sequence. Our data indicate that translation of repA involves reinitiation by the ribosome that has terminated translation of repB. The role of the pseudoknot in this process and its effect on the control of copy number in pMU720 are discussed.

Effect of CIS on activity in trans of the replication initiator protein of an IncB plasmid.

RepA, the replication initiator protein of the IncB plasmid pMU720, acts preferentially in cis. The cis activity of RepA is thought to be mediated by CIS, a 166-bp region of DNA separating the coding region of repA from the origin of replication (ori) of pMU720. To investigate the trans activity of RepA, the repA gene, without its cognate ori, was cloned on a multicopy plasmid, pSU39. The ori on which RepA acts was cloned on pAM34, a plasmid whose replicon is inactive without induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). Thus, in the absence of IPTG, replication of the pAM34 derivatives was dependent on activation of the cloned ori by RepA produced in trans from the pSU39 derivatives. The effect of CIS, when present either on the RepA-producing or the ori plasmid or both, on the efficiency of replication of the ori plasmid in vivo, was determined. The presence of CIS, in its native position and orientation, on the RepA-producing plasmid reduced the efficiency of replication of the ori plasmid. This inhibitory activity of CIS was sequence specific and involved interaction with the C-terminal 20 to 37 amino acids of RepA. By contrast, CIS had no effect when present on the ori plasmid. Initiation of replication from the ori in trans was independent of transcription into CIS.

A study of AroP-PheP chimeric proteins and identification of a residue involved in tryptophan transport.

In vivo recombination has been used to make a series of AroP-PheP chimeric proteins. Analysis of their respective substrate profiles and activities has identified a small region within span III of AroP which can confer on a predominantly PheP protein the ability to transport tryptophan. Site-directed mutagenesis of the AroP-PheP chimera, PheP, and AroP has established that a key residue involved in tryptophan transport is tyrosine at position 103 in AroP. Phenylalanine is the residue at the corresponding position in PheP. The use of PheP-specific antisera has shown that the inability of certain chimeras to transport any of the aromatic amino acids is not a result of instability or a failure to be inserted into the membrane. Site-directed mutagenesis has identified two significant AroP-specific residues, alanine 107 and valine 114, which are the direct cause of loss of transport activity in chimeras such as A152P. These residues replace a glycine and an alanine in PheP and flank a highly conserved glutamate at position 110. Some suggestions are made as to the possible functions of these residues in the tertiary structure of the proteins.

Mechanism of repression of the aroP P2 promoter by the TyrR protein of Escherichia coli.

Previously, we have shown that expression of the Escherichia coli aroP P2 promoter is partially repressed by the TyrR protein alone and strongly repressed by the TyrR protein in the presence of the coeffector tyrosine or phenylalanine (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206-4212, 1997). Here we present in vitro results showing that the TyrR protein and RNA polymerase can bind simultaneously to the aroP P2 promoter. In the presence of tyrosine, the TyrR protein inhibits open complex formation at the P2 promoter, whereas in the absence of any coeffector or in the presence of phenylalanine, the TyrR protein inhibits a step(s) following the formation of open complexes. We also present mutational evidence which implicates the N-terminal domain of the TyrR protein in the repression of P2 expression. The TyrR binding site of aroP, which includes one weak and one strong TyrR box, is located 5 bp downstream of the transcription start site of P2. Results from a mutational analysis show that the strong box (which is located more closely to the P2 promoter), but not the weak box, plays a critical role in P2 repression.

Role of CIS in replication of an IncB plasmid.

Replication of the IncB plasmid pMU720 requires the synthesis of the cis-acting RepA protein and the presence of two DNA elements, ori and CIS. CIS is the 166-bp sequence separating the RepA coding sequence from ori. To investigate how this organization of the pMU720 replicon contributes to the mechanism of initiation of replication, mutations in the sequence and/or the length of CIS were introduced into the CIS region and their effects on the efficiency of replication of the pMU720 replicon in vivo was determined. The CIS region was found to be composed of two domains. The repA-proximal domain, which showed strong transcription termination activity, could be replaced by equivalent sequences from I-complex and IncL/M plasmids, whose replicons are organized in the same fashion as pMU720. Replacement by a trpA transcription terminator afforded only partial replication activity. The repA-distal domain was shown to be a spacer whose role was to position sequence(s) within ori on the correct face of the DNA helix vis-à-vis the repA-proximal portion of CIS. A model for the loading of RepA protein onto ori is discussed.

Specific contacts between residues in the DNA-binding domain of the TyrR protein and bases in the operator of the tyrP gene of Escherichia coli.

In the presence of tyrosine, the TyrR protein of Escherichia coli represses the expression of the tyrP gene by binding to the double TyrR boxes which overlap the promoter. Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051-1058, 1997). In this study, we have used missing contact probing to test the involvement of all of the bases within the tyrP operator in the binding of TyrR. Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the tyrP operator were measured by a gel shift assay. HA494 was shown to be completely defective in binding to the tyrP operator in vitro, while, in comparison with wild-Type TyrR, TA495 had only a small reduction in DNA binding. Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the +/-5 positions in the TyrR boxes.

Analysis of elements involved in pseudoknot-dependent expression and regulation of the repA gene of an IncL/M plasmid.

Replication of the IncL/M plasmid pMU604 is controlled by a small antisense RNA molecule (RNAI), which, by inhibiting the formation of an RNA pseudoknot, regulates translation of the replication initiator protein, RepA. Efficient translation of the repA mRNA was shown to require the translation and correct termination of the leader peptide, RepB, and the formation of the pseudoknot. Although the pseudoknot was essential for the expression of repA, its presence was shown to interfere with the translation of repB. The requirement for pseudoknot formation could in large part be obviated by improving the ribosome binding region of repA, either by replacing the GUG start codon by AUG or by increasing the spacing between the start codon and the Shine-Dalgarno sequence (SD). The spacing between the distal pseudoknot sequence and the repA SD was shown to be suboptimal for maximal expression of repA.

Superimposition of tyrR protein-mediated regulation on osmoresponsive transcription of Escherichia coli proU in vivo.

Osmotic regulation of proU expression in the enterobacteria is achieved, at least in part, by a repression mechanism involving the histone-like nucleoid protein H-NS. By the creation of binding sites for the TyrR regulator protein in the vicinity of the sigma70-controlled promoter of proU in Escherichia coli, we were able to demonstrate a superposed TyrR-mediated activation by L-phenylalanine (Phe), as well as repression by L-tyrosine, of proU expression in vivo. Based on the facts that pronounced activation in the presence of Phe was observed even at a low osmolarity and that the affinity of binding of TyrR to its cognate sites on DNA is not affected by Phe, we argue that H-NS-mediated repression of proU at a low osmolarity may not involve a classical silencing mechanism. Our data also suggest the involvement of recruited RNA polymerase in the mechanism of antirepression in E. coli.

Functional consequences of changing proline residues in the phenylalanine-specific permease of Escherichia coli.

The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on genetic analysis involving the construction of protein fusions with alkaline phosphatase has previously been proposed in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm (J. Pi and A. J. Pittard, J. Bacteriol. 178:2650-2655, 1996). Site-directed mutagenesis has been used to investigate the functional importance of each of the 16 proline residues of the PheP protein. Replacement of alanine at only three positions, P54, P341, and P442, resulted in the loss of 50% or more activity. Substitutions at P341 had the most dramatic effects. None of these changes in transport activity were, however, associated with any defect of the mutant protein in inserting into the membrane, as indicated by [35S]methionine labelling and immunoprecipitation using anti-PheP serum. A possible role for each of these three prolines is discussed. Inserting a single alanine residue at different sites within span IX and the loop immediately preceding it also had major effects on transport activity, suggesting an important role for a highly organized structure in this region of the protein.

Demonstration that the TyrR protein and RNA polymerase complex formed at the divergent P3 promoter inhibits binding of RNA polymerase to the major promoter, P1, of the aroP gene of Escherichia coli.

In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the Escherichia coli aroP gene (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206-4212, 1997). Both P1 and P2 can direct mRNA synthesis for aroP expression, whereas P3 is a divergent promoter which overlaps with P1. The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P. Wang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213-4218, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the aroP regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.

Cloning and analysis of the shiA gene, which encodes the shikimate transport system of escherichia coli K-12.

In Escherichia coli K-12, the shiA gene is involved in the uptake of shikimate. This gene has been cloned and its nucleotide sequence determined. The gene is predicted to encode a protein of 438 amino acids and lies adjacent to the amn gene. The hydropathy profile and the amino acid sequence indicate that the ShiA protein is a polytopic membrane protein that shows a homology with members of the major facilitator superfamily of transport proteins. Recombining an inactive form of the cloned gene into the chromosome creates mutants unable to transport shikimate. Introducing a wild-type gene on a multicopy plasmid into a shiA mutant restores the ability to transport shikimate. When this multicopy shiA plasmid is introduced into an aroE strain, this strain is now able to grow with shikimate as the aromatic supplement, consistent with the notion that dehydroshikimate (DHS) accumulated in an aroE strain prevents uptake of shikimate by competition. Expression of the shiA gene does not appear to be regulated by the TyrR protein, a repressor/activator that controls the expression of other genes involved with the biosynthesis or transport of the aromatic amino acids.

Amino acid residues in the alpha-subunit C-terminal domain of Escherichia coli RNA polymerase involved in activation of transcription from the mtr promoter.

To examine the role of the amino acid residues (between positions 258 and 275 and positions 297 and 298) of the alpha-subunit of RNA polymerase in TyrR-mediated activation of the mtr promoter, we have carried out in vitro transcription experiments using a set of mutant RNA polymerases with a supercoiled mtr template. Decreases in factor-independent transcription in vitro by mutant RNA polymerases L262A, R265A, and K297A suggested the presence of a possible UP element associated with the mtr promoter. Mutational studies have revealed that an AT-rich sequence centered at -41 of the mtr promoter (SeqA) functions like an UP element. In vivo and in vitro analyses using a mutant mtr promoter carrying a disrupted putative UP element showed that this AT-rich sequence is responsible for interactions with the alpha-subunit which influence transcription in the absence of TyrR protein. However, the putative UP element is not needed for activator-dependent activation of the mtr promoter by TyrR and phenylalanine. The results from in vitro studies indicated that the alpha-subunit residues leucine-262, arginine-265, and lysine-297 are critical for interaction with the putative UP element of the mtr promoter and play major roles in TyrR-dependent transcription activation. The residues at positions 258, 260, 261, 268, and 270 also play important roles in TyrR-dependent activation. Other residues, at positions 259, 263, 264, 266, 269, 271, 273, 275, and 298, appear to play less significant roles or no role in activation of mtr transcription.