©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Two New Genes in the Pseudomonasaeruginosa Amidase Operon, Encoding an ATPase (AmiB) and a Putative Integral Membrane Protein (AmiS) (*)

(Received for publication, March 30, 1995)

Stuart A. Wilson (§) Rachel J. Williams (¶) Laurence H. Pearl Robert E. Drew (**)

From theDepartment of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The nucleotide sequence of the amidase operon of Pseudomonas aeruginosa has been completed and two new genes identified amiB and amiS. The complete gene order for the operon is thus amiEBCRS. The amiB gene encodes a 42-kDa protein containing an ATP binding motif that shares extensive homology with the Clp family of proteins and also to an open reading frame adjacent to the amidase gene from Rhodococcus erythropolis. Deletion of the amiB gene has no apparent effect on inducible amidase expression and it is thus unlikely to encode a regulatory protein. A maltose-binding protein-AmiB fusion has been purified and shown to have an intrinsic ATPase activity (K = 174 ± 15 mM; V(max) = 2.4 ± 0.1 mM/min/mg), which is effectively inhibited by ammonium vanadate and ADP. The amiS gene encodes an 18-kDa protein with a high content of hydrophobic residues. Hydropathy analysis suggests the presence of six transmembrane helices in this protein. The AmiS sequence is homologous to an open reading frame identified adjacent to the amidase gene from Mycobacterium smegmatis and to the ureI gene from the urease operon of Helicobacter pylori. AmiS and its homologs appear to be a novel family of integral membrane proteins. Together AmiB and AmiS resemble two components of an ABC transporter system.


INTRODUCTION

The amidase enzyme of Pseudomonas aeruginosa is produced in response to short chain aliphatic amides such as acetamide (1) . Expression of the enzyme is positively regulated by the amiR gene, which lies 2 kb (^1)downstream from the amiE gene and is transcribed in the same direction(2) . Positive control of amidase expression is mediated by a transcription antitermination mechanism, whereby AmiR is thought to allow RNA polymerase to read through a rho-independent transcription terminator between the amiE promoter and gene(3) . A second regulatory gene has been identified, amiC, which negatively regulates amidase expression(4) , and inducible amidase expression can be reconstituted in Escherichia coli with the amiE, -C, and -R genes alone. In addition, AmiC binds inducing amides, and this relieves AmiC repression of AmiR(5) . The AmiC protein has been crystallized, and the structure has been solved at 2.1-Å resolution(6, 7) . The AmiC protein is structurally homologous to the periplasmic binding proteins, in particular the leucine-isoleucine-valine binding protein (LivJ) from E. coli(8) , and consists of two beta-alpha-beta domains with a central cleft in which the amide binds.

The amidase genes were originally cloned on a 5.3-kb HindIII-SalI fragment from PAC433, a constitutive amidase mutant (9) and more recently from the wild-type strain PAC1 (4) . The nucleotide sequence of amiE, amiC, and amiR have been determined(10, 4, 11) , and transcriptional analysis indicates that all of the amidase genes are translated from a single mRNA, approximately 5.0 kb in length, following induction. (^2)We have now completed the DNA sequence between amiE and amiC and downstream of amiR, and identified two further genes in this operon, amiB and amiS, thus completing the 5.3-kb nucleotide sequence and gene organization of the amidase operon.


EXPERIMENTAL PROCEDURES

DNA Manipulations

Plasmid purifications, transformations, mobilizations, and cloning were carried out as described previously (4) . Polymerase chain reaction amplification was carried out using Vent DNA Polymerase (New England Biolabs) according to the manufacturer's instructions. Two oligonucleotides were used for the amplification of amiB with the following sequences 5`-ATCCGAATTCGCAGAAGGAGTTTCATCCAT and 5`-TGCTGGATCCTCAGCGCCGTACGAAGCG.25 cycles of amplification were performed using the following temperatures: 95 °C for 3 min; 55 °C for 1 min; 73 °C for 2 min. Plasmid pAS20 was used as a template(4) .

Protein Purification

E. coli strain JM109 carrying the maltose binding protein-AmiB fusion vector pSW81 was grown to an A of 0.5 and then induced with 1 mM IPTG. Growth was continued for a further 14 h, and then the cells were harvested. Cell pellets were resuspended in buffer A (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM dithiothreitol, 15% (v/v) glycerol) and sonicated as described previously(4) . Cell-free extracts were prepared by centrifugation of the sonicated cells at 15,000 g in a Sorvall RC2B centrifuge for 30 min. The cell-free extract was loaded onto an amylose affinity column at a flow rate of 1 ml/min. The column was subsequently washed with buffer A until a steady base line was achieved. The maltose-binding protein (MBP)-AmiB fusion protein was eluted from the column using elution buffer (buffer A + 10 mM maltose). A single large peak was collected and stored at 4 °C. Gel filtration of the amylose affinity purified protein was carried out using a Superdex 200 16/10 gel filtration column (Pharmacia Biotech Inc.). 10 mg (5 ml) of protein was loaded onto this column equilibrated in buffer A minus NaCl. The flow rate was 0.5 ml/min. Soluble and insoluble crude protein extracts were prepared by sonication of cells in buffer A as described previously (4) followed by centrifugation at 15,000 g. The soluble supernatant was used directly and the insoluble material was resuspended in buffer A prior to analysis. SDS-polyacrylamide gel electrophoresis (PAGE) of crude extracts and purified protein samples was carried out as described previously(4) , and all gels were stained with Coomassie Blue.

Assay of Amidase Activity

Amidase activity in intact cells was measured by the transferase assay (12) with acetamide as substrate. Specific activities presented correspond to 1 mmol of acethydroxamate formed per min/mg of bacteria.

ATPase Assay

The ATPase activity was quantitated by a colorimetric assay(13) . Routinely, MBP-AmiB protein was diluted with double concentrated assay buffer (100 mM Tris-HCl, pH 8.0, 4 mM MgCl(2), 15% (v/v) glycerol, 10% (v/v) dimethyl sulfoxide) to a final volume of 250 ml. To initiate the reaction, an equal volume of ATP solution was added, to give a final concentration of 1 mM. After incubation at 37 °C for 5 min, the reaction was stopped by the addition of 2 ml of malachite green reagent. Liberated P(i) was quantitated as described previously(14) . Assays were carried out in triplicate, and absorbances were measured at 630 nm. The values obtained were corrected for nonenzymatic release of P(i) from ATP, GTP, CTP, and ADP and for P(i) contamination. Protein concentrations were determined using the Bradford assay(15) .

DNA Sequencing

Restriction enzyme fragments from pAS20 were isolated from agarose gels as described previously (4) and ligated into appropriately cut M13mp18 or M13mp19 vectors. Single-stranded DNA from M13 recombinants for use as sequencing templates was purified by standard methods(16) . DNA sequences were determined using the chain termination method for M13 recombinants using universal sequencing primer, and both strands of the DNA were sequenced. An 18-base pair oligonucleotide primer was synthesized to complete the sequencing of the amiS gene. Sequencing reactions utilized standard and 7-deaza-dGTP premixes (Pharmacia) and [S]dATP (DuPont NEN).

Sequence Analysis

Codon preference and third position GC bias statistics were calculated to identify open reading frames using programs of the GCG package(17) . Sequence similarity searches were performed using the FASTA and TFASTA program (18) implemented in the GCG package. Hydropathy analyses were performed according to the method of Kyte and Doolittle (19) and Stirk et al.(20) . Primary sequence threading was performed as described previously(21) , using a set of 104 unique protein folds. Trans-membrane helix identification was performed using the method of Jones et al.(22) . Protein sequences were aligned using the programs GAP and PILEUP in the GCG package.


RESULTS

Nucleotide Sequence of amiB

The organization of the amidase operon and locations of amiB and amiS are shown in Fig.1. Plasmid pAS20 carrying the wild-type amidase genes in pBR322 (4) was used as a source of DNA for sequencing. The nucleotide sequence of amiB is shown in Fig.2, together with the deduced amino acid sequence. The sequence shown extends from the 3` end of the amidase gene (amiE). There is a short intergenic region between amiE and amiB that has an inverted repeat and acts as a partial transcription terminator blocking approximately 50% of the transcripts arising from the amiE promoter.^2 The correct ORF for amiB was identified using third position GC bias and codon preference plots (17) . The amiB ORF is preceded by a ribosome binding site (underlined) and encodes a 371-amino-acid protein (42 kDa), which contains the boxes A and B of the Walker ATP binding motif (23) (Fig.2). A FASTA search of the SwissProt protein sequence data base indicates that AmiB shows extensive homology with an ORF downstream of the amidase gene from Rhodococcus erythropolis(24) (39% identity, 60% similarity) (Fig.3). The role of the R. erythropolis ORF has not been described. In addition, the FASTA search reveals AmiB is homologous to the large Clp family of proteins. AmiB shows homology with all the sub-families of the Clp proteins (A, B, and C(25) ), and representatives of each subfamily are shown in the multiple alignment with AmiB in Fig.3. The Clp proteins normally contain two ATP binding domains with a variable length spacer between them. Consequently, the Clp family proteins are approximately twice the size of AmiB and its homolog from R. erythropolis. Although AmiB shows homology with both ATP binding domains in the Clp proteins, the homology with the C-terminal ATP binding motif is highest and is shown in Fig.3.


Figure 1: Genetic organization of the amidase operon of P. aeruginosa. amiC and amiR are involved in the regulation of amidase (amiE) expression. amiB and amiS are newly identified genes described in this paper. The stem-loop structures represent transcription terminators. Restriction enzyme sites are as follows: H, HindIII; X, XhoI; S= SalI.




Figure 2: Nucleotide sequence and deduced amino acid sequence for the amiB gene. The sequence is numbered from the unique HindIII target upstream of amiE (Fig.1). The sequence displayed includes the 3` end of the amiE gene. The arrowsunderneath the sequence represent a transcription terminator that partially blocks transcription from upstream(12) . The ribosome binding site is underlined. Boxes A and B of the ATP binding motif (24) in the deduced amino acid sequence are overlined.




Figure 3: Multiple alignment of the AmiB sequence with homologs. adpr_rhoer, an ORF identified adjacent to the amidase gene from R. erythropolis(25) ; clpa_ecoli, ClpA from E. coli(34) ; clpb_ecoli, ClpB from E. coli(27) ; clp_trybb, Clp protein from Trypanosoma brucei(27) ; cd4a_lyces, CD4A protein from Lycopersicon esculentum(27) ; h104_yeast, HSP104 from Saccharomyces cerevisiae(35) . Highly conserved residues are shown in boldface. The AmiB sequence is shown in full, whereas for the Clp proteins, the C-terminal ATP binding region is shown.



Threading analysis (21) has been used to investigate the tertiary structure of the AmiB protein and to determine whether it is similar to known ATP binding proteins. The AmiB sequence was threaded onto a data base of 104 unique folds, and the results of this analysis (Fig.4) showed that adenylate kinase had a significantly higher score than any other fold. This suggests that AmiB would adopt a similar three-dimensional structure to adenylate kinase.


Figure 4: Threading analysis for the AmiB sequence. AmiB was threaded (21) onto 104 unique folds from the Brookhaven data base. Adenylate kinase (ADK) shows the lowest threading energy, indicating the highest structural homology with AmiB.



Amidase Activity with an amiB Deletion

To confirm that AmiB is not involved in amidase induction, a plasmid was constructed with a 423-base pair in-frame ApaI deletion within amiB. The broad host range parental plasmid, pSW101, which carries the whole amidase operon from PAC1, and the amiB deletion derivative (pMW22) were mobilized into P. aeruginosa strain PAC452, which has a chromosomal deletion of the amidase locus (28) and amidase activity measured. The parental plasmid pSW101 shows high inducible amidase expression (specific activity = 1.1 when noninduced, and specific activity = 52 when induced). The amiB deletion strain gave similar high inducible amidase expression (specific activity = 1 noninduced, and specific activity = 55 induced) showing that AmiB is not involved in the induction process.

Nucleotide Sequence of amiS

The nucleotide sequence of the cloned P. aeruginosa DNA downstream of amiR in plasmid pAS20 has been determined, and an ORF (amiS) has been identified using the third position GC bias and codon preferences. The nucleotide sequence for amiS and the deduced protein sequence are shown in Fig.5. The AmiS sequence encodes a 172-amino-acid protein with a predicted molecular mass of 18 kDa. The AmiS sequence was used to search the SwissProt and EMBL libraries, and two significant matches were found. The first to an ORF adjacent to the amidase gene from Mycobacterium smegmatis(29) (40% identity; 68% similarity) and the second to the ureI gene (30) (37% identity; 63% similarity) from the urease operon of Helicobacter pylori. Neither of these proteins have been ascribed a function. The striking feature of AmiS and its two homologs is the high content of hydrophobic residues, which suggests that all three may be integral membrane proteins. To test this hypothesis, hydropathy plots were produced for all three proteins (Fig.6). In each case, the plots reveal characteristic hydrophobic regions of 18-20 amino acids, indicative of transmembrane helices, with variable length hydrophillic loop regions. AmiS and the M. smegmatis ORF appear to encode proteins with six potential transmembrane helices. The ureI gene appears to encode seven transmembrane helices. Multiple sequence alignment of the three sequences (Fig.7) indicates significant homology between all three proteins. To confirm the membrane topology of these proteins, the program MEMSAT (22) was used, and the predicted transmembrane regions of the proteins are indicated in Fig.7. MEMSAT also predicts that the N terminus would be external for each protein. The multiple sequence alignment indicates that relative insertions in the ureI sequence occur in nonmembrane loop regions and that these are least conserved between the proteins. The high level of homology between these proteins suggests that they represent a novel family of integral membrane proteins. A sequence template was generated from the aligned sequences and used to search sequence libraries; however, this failed to identify any additional family members.


Figure 5: Nucleotide and deduced amino acid sequence for AmiS. The sequence is numbered from the unique HindIII target upstream of amiE. The 3` end of the amiR gene is shown at the begining of the sequence, and the ribosome binding site for amiS is underlined. An inverted repeat has been identified in the sequence downstream of amiS, which may represent the operon transcription terminator.




Figure 6: Hydropathy analysis for AmiS and homologs. A window of 25 amino acids was used for the plots. The romannumerals indicate the potential transmembrane helix regions. The abscissus scale indicates amino acids.




Figure 7: Multiple alignment of the AmiS sequence with homologs. M.s.ORF, an open reading frame in the Mycobacteria smegmatis amidase operon (30); ureI, the ureI gene from Helicobacter pylori(31) . Conserved residues in all three sequences are indicated by an asterisk, and residues conserved in two sequences are shown by :. The transmembrane regions predicted by MEMSAT (23) are indicated by the shadedregions, and each potential transmembrane helix is numbered. The orientation (IN, internal; OUT, external) of the nonmembrane loop regions, predicted by MEMSAT is shown above the sequence.



Expression and Purification of AmiB

The amiB gene was polymerase chain reaction amplified with EcoRI and XbaI restriction sites at the 5` and 3` ends, respectively, and subcloned into the broad host range expression vector pMMB66EH(4) . The resulting plasmid pRW303 was mobilized into the P. aeruginosa strain PAC452, which carries a chromosomal deletion for the amidase locus(28) . Soluble and insoluble fractions were prepared from PAC452 pRW303 after growth under noninducing and inducing conditions (3 mM IPTG). The samples were analyzed for AmiB over-expression by SDS-PAGE, and the results are shown in Fig.8A. The soluble fractions (lanes2 and 3) show no evidence of AmiB in either noninduced or induced samples. However, clear overexpression of a band with a calculated molecular weight of 40 kDa is seen in the insoluble induced sample (lane5). This molecular weight agrees closely with the expected molecular weight for AmiB from the nucleotide sequence (42 kDa). Thus in P. aeruginosa, AmiB appears to aggregate when overexpressed. To circumvent the problems of protein refolding and purification, the amiB gene was subcloned into the protein fusion expression vector pMALCR1 to create a MBP-AmiB fusion. The fusion protein was overexpressed in E. coli JM109 following IPTG induction and purified by amylose affinity chromatography. The affinity purification of MBP-AmiB results in fusion protein substantially free of contaminants as shown in Fig.8B. The fusion protein showed less tendency to form insoluble aggregates than AmiB. However, gel filtration chromatography of the amylose-purified fusion indicates that a significant proportion of the purified protein forms soluble aggregates of high molecular weight, which elute shortly after the void volume on a Superdex 200 column (separation range, 600-10 kDa) and only a small proportion of the fusion is monomeric (Fig.8, C and D). The fusion protein contains a Factor Xa cleavage site between the MBP and AmiB. However, cleavage of the fusion with Factor Xa led to rapid aggregation and precipitation of the AmiB protein, and thus it was not possible to generate soluble stable native AmiB by this route. Consequently enzymatic analysis of the AmiB protein was carried out using the purified fusion protein.


Figure 8: SDS-PAGE analysis of protein extracts containing AmiB. A, SDS-PAGE analysis of crude extracts of P. aeruginosa containing an amiB expression vector (pRW303). Lane1, protein markers; lane2, soluble fraction of PAC452 pRW303, noninduced; lane3, soluble fraction of PAC452 pRW303, induced with 3 mM IPTG; lane4, insoluble fraction of PAC452 pRW303, noninduced; lane5, insoluble fraction of PAC452 pRW303, induced with 3 mM IPTG. AmiB is seen in lane5 and is indicated by an arrow. B, SDS-PAGE analysis of amylose affinity column purified MBP-AmiB fusion. Lane1, purified MBP-AmiB; lane2, molecular weight markers. C, Superdex 200 gel filtration analysis of the amylose affinity column-purified MBP-AmiB. Gel filtration chromatography was carried out as described under ``Experimental Procedures.'' The majority of the fusion protein eluted shortly after the void volumn of the column (38 ml) and thus has a molecular mass in excess of 600 kDa and is likely to represent protein aggregates. A small peak is seen eluting between 72 and 78 ml, which corresponds to monomeric MBP-AmiB. D, SDS-PAGE analysis of MBP-AmiB fractions eluting from the gel filtration column (Fig.8C). Lane1, molecular weight markers; lane2, pooled fractions corresponding to the peak eluting immediatley after the void volume; lane3, pooled fractions 72-78, corresponding to monomeric MBP-AmiB.



ATPase Activity of the MBP-AmiB Fusion

The ATP binding motif (23) within AmiB and the proposed structural homology with adenylate kinase suggested that AmiB may be capable of ATP hydrolysis. To test this hypothesis, ATPase assays were carried out using the purified MBP-AmiB fusion, and the results of such an assay are shown in Fig.9together with an assay for GTPase activity. K and V(max) determinations were carried out using hyperbolic regression analysis, and the values for ATP were: K = 174 ± 15 mM; V(max) = 2.4 ± 0.1 mM/min/mg. Similar values were obtained for the monomeric and aggregated forms of MBP-AmiB. MBP-AmiB was also capable of GTP hydrolysis, although the rate was much lower than for ATP (Fig.9), and MBP-AmiB was unable to hydrolyze CTP. To confirm that the observed ATPase activity was not due to the maltose binding protein nor any E. coli contaminant, ATPase assays were also carried with an affinity-purified MBP-KorB fusion, KorB is a DNA binding protein (31) that has no ATPase activity. This fusion displayed no activity in the assay, confirming that the observed ATPase activity was due to AmiB and not MBP nor any of the minor contaminants observable by SDS-PAGE.


Figure 9: MBP-AmiB catalyzed hydrolysis of ATP and GTP. The enzyme assay reactions were initiated by the addition of ATP (bullet) or GTP () to the final concentration indicated. The data represent the mean values of three independent experiments with standard deviations shown.



Magnesium-ATP Is the Preferred Substrate

The ability of various metal ions to replace magnesium in the ATPase assays was investigated (Fig.10). In the absence of any added metal in the assay buffer, AmiB showed little residual ATPase activity. Addition of magnesium strongly stimulated the ATPase activity; however, the stimulation of activity decreased at higher magnesium concentrations. Manganese was able to substitute for magnesium, but it only gave 60% of the magnesium-ATP hydrolysis rate. Zinc and calcium-ATP could not replace magnesium as substrates for MBP-AmiB. These findings are in contrast to results obtained with other ATPases for example most F-type ATPases, which hydrolyze ATP in the presence of any one of the above divalent cations(32) .


Figure 10: Dependence of MBP-AmiB ATPase activity on Mg. MBP-AmiB fusion protein was diluted with assay buffer lacking Mg ions, and ATPase activity was monitored in the presence of 1 mM ATP and increasing concentrations of MgCl(2) (bullet), MnCl(2) (), ZnCl(2) (), and CaCl(2) (black square).



ATPase Activity of MBP-AmiB Is Inhibited by ADP and Vanadate

The ATPase activity of MBP-AmiB was measured in the presence of increasing concentrations of ADP, and the results are shown in Fig.11a. ADP strongly inhibits the ATPase activity with up to 50% inhibition at concentrations as low as 0.2 mM with an ATP concentration of 1 mM in the assay. It seems most likely that this inhibition occurs by competition for the substrate binding site(s). Similarly, nanomolar concentrations of ammonium vanadate, which forms a pentacovalent bypyramidal structure like that of a phosphate ester undergoing hydrolysis, inhibit the ATPase activity of MBP-AmiB.


Figure 11: Inhibition of MBP-AmiB ATPase activity by ADP and ammonium vanadate. MBP-AmiB was diluted in assay buffer and ddH(2)O and incubated for 5 min at room temperature in the indicated final concentrations of inhibitor. Subsequently, ATP hydrolysis was monitored as described under ``Experimental Procedures.'' a, inhibition by ADP; b, inhibition by ammonium vanadate.




DISCUSSION

From a transcriptional analysis of the amidase system^2 it is evident that amiB and amiS are specifically transcribed in response to inducing amides such as acetamide in the growth medium. In addition, the amidase operon does not encode any other proteins since the full-length operon transcript is only 5.0 kb, sufficient to encode amiEBCRS.^2 Earlier studies have shown that a normal amide-inducible phenotype can be reconstructed in E. coli with only the amiE, amiC, and amiR genes(5) . In addition, in this study we have shown that a 423-base pair deletion in amiB has no effect on amide-inducible amidase expression in P. aeruginosa. Thus amiB and amiS are unlikely to have a regulatory role in amidase expression.

AmiB shows significant homology to the Clp family of proteins, although it is only half the size of the rest of this family. The ClpA protein with which AmiB shows high homology is the ATP binding subunit of a two-component protease complex known as Ti or the Clp protease(33) . Since there is no corresponding proteolytic subunit within the amidase operon, necessary for a Clp type function, it seems unlikely that AmiB is the ATP binding subunit of a Clp type protease. HSP104, a Clp family member, has been shown to have a chaperone type function, being able to refold heat-denatured proteins(34) . We cannot exclude such a function for AmiB. However, the much reduced size of AmiB compared with HSP104 and the unlikely requirement for an acetamide induced chaperone suggest that AmiB performs a different function within P. aeruginosa.

AmiS clearly shows the characteristics of an integral membrane protein, with hydrophobic segments of membrane spanning length, interspersed by hydrophillic loop regions. An obvious role for AmiS would be as a transport protein within the plasma membrane. The homology between AmiS, the M. smegmatis ORF, and the ureI gene from H. pylori is interesting since all three operons are involved in amide utilization, suggesting they may be amide transporters. A chromosomal knockout of the ureI gene in H. pylori renders the system urease negative(35) . This mutation had no effect with the cloned genes in E. coli, therefore UreI is not thought to be involved in enzyme regulation/synthesis. Instead, the urease negative phenotype observed in H. pylori could be due to the inability of urea to enter the cell and induce urease expression. The urease positive phenotype observed with the ureI mutant in E. coli may reflect membrane permeability differences between E. coli and H. pylori.

Taken together, the AmiS and AmiB proteins resemble two components of an ABC type transporter system. Such transport systems are found in many organisms and have a basic anatomy in common (for review, see (36) ). Exporters/importers comprise either a single polypeptide chain with two domains, an integral membrane domain with six to eight transmembrane helices, and a cytoplasmic domain with an ATP binding motif and in some cases ATPase activity. Alternatively the domains can function as two distinct polypeptide chains. Often there are two homologous copies of the integral membrane protein, for example MalF and MalG in the maltose regulon of E. coli, and typically they have six to eight membrane-spanning regions.(37) . Together these proteins form a tetrameric complex with two copies of the cytoplasmic ATP binding protein. In addition, bacterial importers often have a third protein that is located in the periplasm and binds and delivers the ligand to the integral membrane protein complex.

In the amidase operon, AmiS would represent the integral membrane component of an ABC type transporter and AmiB would represent the cytoplasmic ATP binding component. The threading analysis indicated that AmiB was structurally homologous to adenylate kinase, and it has been suggested previously that the ATP binding components of ABC transporters share structural homology with adenylate kinase(38) . AmiB has been shown to have an intrinsic ATPase and GTPase activity, which is sensitive to ADP and vanadate inhibition. MalK, the E. coli maltose operon ABC transporter counterpart, shows an intrinsic ATPase activity similar to that of AmiB (K = 70 mM; V(max) = 1.3 mmol/min/mg). In addition, MalK can also hydrolyze GTP but CTP acts as a very poor substrate. The MalK ATPase is similarly inhibited by ADP, but it is resistant to vanadate inhibition(39) . However, the mammalian ABC transporter P-glycoprotein shows sensitivity to vanadate(40) . When MalK is purified as part of a complex with the integral membrane proteins, ATPase activity is only seen in the presence of ligand-loaded maltose binding protein(41) . By comparison with the maltose uptake system, it may be that AmiS modulates the activity of the AmiB ATPase in response to amides. With regard to the strong inhibition of AmiB ATPase activity by ADP and the relationship between AmiB and adenylate kinase, the possibility of ATP/ADP exchange cannot be excluded at this stage, although it is most likely that ADP is acting as a competitive inhibitor.

If AmiB and AmiS form an ABC type importer complex for aliphatic amides, then they would be expected to be associated with a periplasmic binding protein. We have not identified a protein that could fulfill such a role in the amidase operon. However, the recently solved crystal structure of the AmiC protein (7) shows that although cytoplasmic, it is structurally highly homologous to LivJ of E. coli despite only 17% sequence identity. This raises the possibility that AmiC has switched roles during evolution from being an amide receptor in a transport system, to a cytoplasmic regulator of gene expression, retaining an amide receptor function.

We have used a strain of P. aeruginosa carrying an amiB deletion to investigate the role of amiB on acetamide-dependent growth in minimal medium, but we have failed to observe any differences in growth rate between the mutant and wild-type. A possible explanation for this may be that at the acetamide concentrations (millimolar) used to support growth in batch culture, in a reasonable period of time, the diffusion of acetamide into the cell was efficient enough to support growth. Alternatively, the intact AmiS protein may have AmiB-independent amide transport capabilities. Therefore, the effects of deletions in AmiB or AmiS may only be detectable in continuous culture where very low concentrations of amides can be used and very slow growth rates monitored.

Given the hypothesis that AmiB and AmiS work as an ABC type complex in amide transport, then the identification of an AmiB homolog in the R. erythropolis operon would suggest that this amidase operon should also possess an amiS gene. Such an amiS homolog has recently been identified in this operon upstream of the amidase structural gene. (^3)Similarly, we would expect to see amiB homologs in the M. smegmatis amidase operon once the sequence of this operon is completed. Within the H. pylori urease operon, we have not identified an AmiB homolog, although the ureG gene does possess an ATP binding motif (30) and may represent an evolutionarily divergent form of AmiB. It is interesting to note that no candidate periplasmic binding protein gene has been identified in any of these operons. It thus remains a possibility that AmiB/AmiS type systems, identified here in both Gram-positive and Gram-negative organisms, may not require a periplasmic binding protein and as such would represent a distinct and novel subfamily of ABC transporters.

Current experiments aimed at expression and purification of AmiS and the generation of amiS chromosomal mutations are expected to shed more light on these unusual proteins.


FOOTNOTES

*
This work was supported in part by a grant from the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) X77160 [GenBank]and X77161[GenBank].

§
Recipient of a Science and Engineering Research Council studentship. Present address: Retrovirus Molecular Biology Group, Dept. of Biochemistry, University of Oxford, South Parks Rd., Oxford, OX1 3QU, UK.

Recipient of a Medical Research Council studentship.

**
To whom correspondence should be addressed. Tel.: 71-387-7050 (ext. 2244); Fax: 71-380-7193.

^1
The abbreviations used are: kb, kilobase pair(s); ORF, open reading frame; MBP, maltose binding protein; ABC, ATP binding cassette; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; LivJ, leucine-isoleucine-valine binding protein from E. coli; PAGE, polyacrylamide gel electrophoresis.

^2
Wilson, S. A., and Drew, R. E. (1995) J. Bacteriol.177, 3052-3057

^3
H. Choubrier, personal communication.


ACKNOWLEDGEMENTS

We thank Tahir Malik and Simon Wachira for assistance in the early stages of the project.


REFERENCES

  1. Kelly, M., and Clarke, P. H. (1962) J. Gen. Microbiol. 27,305-316
  2. Cousens, D. J., Clarke, P. H., and Drew, R. E. (1987) J. Gen. Microbiol. 133,2041-2052 [Medline] [Order article via Infotrieve]
  3. Drew, R. E., and Lowe, N. (1989) J. Gen. Microbiol. 135,817-823 [Medline] [Order article via Infotrieve]
  4. Wilson, S. A., and Drew, R. E. (1991) J. Bacteriol. 173,4914-4921 [Medline] [Order article via Infotrieve]
  5. Wilson, S. A., Wachira, S. J., Drew, R. E., Jones, D., and Pearl, L. H. (1993) EMBO J. 12,3637-3642 [Abstract]
  6. Wilson, S. A., Chayen, N. E., Hemmings, A. M., Drew, R. E., and Pearl, L. H. (1991) J. Mol. Biol. 222,869-871 [Medline] [Order article via Infotrieve]
  7. Pearl, L. H., O'Hara, B. P., Drew, R. E., and Wilson, S. A. (1994) EMBO J. 13,5810-5817 [Abstract]
  8. Quiocho, F. A. (1991) Curr. Opin. Struct. Biol. 1,922-933 [CrossRef]
  9. Drew, R. E., Clarke, P. H., and Brammar, W. J. (1980) Mol. & Gen. Genet. 177,311-320
  10. Brammar, W. J., Charles, I. G., Matfield, M., Cheng-Pin, L., Drew, R. E., and Clarke, P. H. (1987) FEBS Lett. 215,291-294 [CrossRef][Medline] [Order article via Infotrieve]
  11. Lowe, N., Rice, P. M., and Drew, R. E. (1989) FEBS Lett. 246,39-43 [CrossRef][Medline] [Order article via Infotrieve]
  12. Brammar, W. J., and Clarke, P. H. (1964) J. Gen. Microbiol. 37,307-319
  13. Chan, U. M., Delfert, D., and Junger, U. D. (1986) Anal. Biochem. 157,375-380 [Medline] [Order article via Infotrieve]
  14. Henkel, R. D., Vandeberg, J. L., and Walsh, R. A. (1988) Anal. Biochem. 169,312-318 [Medline] [Order article via Infotrieve]
  15. Bradford, M. (1976) Anal. Biochem. 72,248-254 [CrossRef][Medline] [Order article via Infotrieve]
  16. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  17. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12,387-395 [Abstract]
  18. Pearson, W. R., and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,2444-2448 [Abstract]
  19. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157,105-132 [Medline] [Order article via Infotrieve]
  20. Stirk, H. J., Thornton, J. M., and Howard, C. R. (1992) Intervirology 33,148-158 [Medline] [Order article via Infotrieve]
  21. Jones, D. T., Taylor, W. R., Thornton, J. M. (1992) Nature 358,86-89 [CrossRef][Medline] [Order article via Infotrieve]
  22. Jones, D. T., Taylor, W. R., Thornton, J. M. (1994) Biochemistry 33,3038-3049 [Medline] [Order article via Infotrieve]
  23. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1,945-991 [Medline] [Order article via Infotrieve]
  24. Soubrier, F., Lévy-Schil, S., Mayaux, J.-F., Pétré, Arnaud, A., and Crouzet, J. (1992) Gene (Amst.) 116,99-104 [Medline] [Order article via Infotrieve]
  25. Squires, C., and Squires, C. L. (1992) J. Bacteriol. 174,1081-1085 [Medline] [Order article via Infotrieve]
  26. Gottesman, S., Squires, C., Pichersky, E., Carrington, M., Hobbs, M., Mattick, J. S., Dalrymple, B., Kuramitsu, H., Shiroza, T., Foster, T., Clark, W. P., Ross, B., Squires, C. L., and Maurizi, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,3513-3517 [Abstract]
  27. Parsell, D. A., Sanchez, Y., Stitzel, J. D., and Lindquist, S. (1991) Nature 353,270-273 [CrossRef][Medline] [Order article via Infotrieve]
  28. Day, M. (1975) Genetic Studies with Pseudomonas aeruginosa Strains, Ph.D. thesis, University of London
  29. Mahenthiralingam, E., Draper, P., Davis, E. O., and Colston, M. J. (1993) J. Gen. Microbiol. 139,575-583 [Medline] [Order article via Infotrieve]
  30. Cussac, V., Ferrero, R., and Labigne, A. F. (1992) J. Bacteriology 174,2466-2473 [Abstract]
  31. Tai, J. T., and Cohen, S. N. (1994) Mol. Microbiol. 12,31-39 [Medline] [Order article via Infotrieve]
  32. Kagawa, Y., Sone, N., Hirata, H., and Yoshida, M. (1979) J. Bioenerg. Biomembr. 11,39-78 [Medline] [Order article via Infotrieve]
  33. Gottesman, S., Clark, W. P., and Maurizi, M. (1990) J. Biol. Chem. 265,7886-7893 [Abstract/Free Full Text]
  34. Parsell, D. A., Kowel, A. S., Singer, M. A., and Lindquist, S. (1994) Nature 372,475-478 [CrossRef][Medline] [Order article via Infotrieve]
  35. Ferrero, R. L., Cussac, V., Courcoux, P., and Labigne, A. (1991) Ital. J. Gastroenterol. 23,(Suppl. 2), 3 [Medline] [Order article via Infotrieve]
  36. Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8,67-113 [CrossRef]
  37. Dassa, E., and Hofnung, M. (1985) EMBO J. 4,2287-2293 [Abstract]
  38. Mimura, C. S., Holbrook, S. R., and Ames, G. F. (1991) Proc. Natl. Acad. Sci U. S. A. 88,84-88 [Abstract]
  39. Morbach, S., Tebbe, S., and Schneider, E. (1993) J. Biol. Chem. 268,18617-18621 [Abstract/Free Full Text]
  40. Al-Shawi, M. K., and Senior, A. E. (1993) J. Biol. Chem. 268,4197-4206 [Abstract/Free Full Text]
  41. Davidson, A. L., and Nikaido, H. (1991) J. Biol. Chem. 266,8946-8951 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.