(Received for publication, March 30, 1995)
From the
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
= 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.
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 ()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
-
-
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. ()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.
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.
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.
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.
Figure 9:
MBP-AmiB catalyzed hydrolysis of ATP and
GTP. The enzyme assay reactions were initiated by the addition of ATP
() or GTP (
) to the final concentration indicated. The data
represent the mean values of three independent experiments with
standard deviations shown.
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
(
), MnCl
(
),
ZnCl
(
), and CaCl
(
).
Figure 11:
Inhibition of MBP-AmiB ATPase activity by
ADP and ammonium vanadate. MBP-AmiB was diluted in assay buffer and
ddHO 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.
From a transcriptional analysis of the amidase system 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.
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
= 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. ()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.
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].