- Email: [email protected]
Sequencing and expression of the aroA gene from Dichelobacter nodosus
Gene, 145 (1994) 97-101 0 1994 Elsevier Science B.V. All rights reserved.
97
0378-l 119/94/$07.00
GENE 07971
Sequencing and expression of the aroA gene from Dichelobacter nodosus (Aromatic
amino acid; protein
production;
phylogeny;
Richard
A. Alma, Brian P. Dalrympleb
phosphatase;
complementation)
and John S. Mattick”
“Centre for Molecular Biology and Biotechnology, University of Queensland, St. Lucia, Qld 4072. Australia; and bCSIRO Division of Tropical Animal Production. Longpocket Laboratories, Brisbane, Qld 4068, Australia. Tel. (61-7) 377-0711 Received by P.A. Manning:
17 November
1993; Revised/Accepted:
17 January
1994; Received at publishers:
21 March
1994
SUMMARY
The aroA locus of the Grampathogen Dichelobacter nodosus, which encodes 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, has been sequenced and expressed in Escherichia coli. The gene is located on a 1.48-kb DraI-Hind111 fragment located directly upstream and in opposite transcriptional orientation to the gene encoding the fimbrial structural subunit. The deduced open reading frame is 1329 nucleotides in length, which encodes a protein of 443 amino acids (aa) with a calculated M, of 47413, which was visualized in E. coli minicells, under the control of its native promoter. This derived aa sequence displays significant similarities with the sequences of the aroA gene products from a variety
of microorganisms.
INTRODUCTION
The fastidious is the etiological
Gram- anaerobe Dichelobacter nodosus agent of ovine footrot (Egerton, 1977).
Nine major serogroups of D. nodosus have been identified based on the K-agglutination test, and the complexity of the serological hierachy extends to a total of some 18 subsidiary serotypes (Claxton, 1989). The antigenic structures that are the basis for the serogroup specificity are the polar type-4 fimbriae. The genes encoding the structural subunit of the fimbriae (JimA) representative of all nine major serogroups, including both class-I and class-II strains, have been sequenced (Mattick et al., 1991). Analysis of the fimbrial subunit gene region identified a partial ORF situ-
Correspondence to: Dr. J.S. Mattick,
Centre for Molecular
Biotechnology. University of Queensland, St. Lucia, Australia. Tel. (61-7) 365-4446; Fax (61-7) 365-4388: e-mail: [email protected]
Biology Qld
and 4072,
Abbreviations: aa, amino acid(s); Ap, ampicillin; aroA, gene encoding EPSP synthase; bp, base pair(s); D., Dichelobacter: E., Escherichia; EPSP, 5enolpyruvylshikimate 3-phosphate; CCC, Genetics Computer Group (Madison, WI. USA); kb. kilobase or 1000 bp; nt. nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; RBS. ribosome-binding site(s); SDS, sodium dodecyl sulfate. SSDI 0378-1119(94)00182-R
ated upstream from theJimA gene in all D. nodosus strains that displayed similarity to the aroA gene from a number of species, and was able to complement an Escherichia coli aroA mutant strain AB2829 (Hobbs et al., 1991). In E. coli the aroA gene encodes the biosynthetic enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, which is essential for the synthesis of a spectrum of aromatic compounds which include p-aminobenzoic acid, 2,3_dihydroxybenzoate and the aromatic amino acids, phenylalanine, tyrosine and anthranilic acid, a precursor of tryptophan. The aroA genes belonging to a number of both Gram- and Gram+ organisms have been sequenced, and many show areas of substantial conservation (Duncan et al., 1984; Stalker et al., 1985; Maskell et al., 1988; O’Gaora et al., 1989; Maskell, 1993; O’Connell et al., 1993). Here we report the complete sequence and expression of an aroA gene from D. nodosus, and its comparison to related genes in other species.
EXPERIMENTAL
AND DISCUSSION
(a) Subcloning, nt sequence and analysis of the am4 locus The pBA122 plasmid, which contains the aroA gene, the fimbrial genesjimA and$mB, and the clpB gene from
98 D. nodosus strain VCSlOOl (class I, serotype A) (Hobbs et al.. 1991; Fig. 1A) was digested with HindIII+ DraI, and the 1.5kb fragment that represented the aroA locus was cloned into the HincII-Hind111 sites within the multiple cloning site of plasmid pOK12 (Vieira and Messing, 1991) generating plasmid pRIC201 (Fig. 1A). The nt sequence of this fragment was determined using the dideoxy chain termination method (Sanger et al., 1977), and is shown in Fig. 1B. One significant ORF was identified in this region that encoded a protein of 443 aa with a calculated M, of 47413 (Fig. 1B). This ORF showed significant similarity at the aa level to several other bacterial AroA proteins (Fig. 2A). The predicted aa sequence of AroA from D. nodosus is approx. 55% similar to that of other Gram- species, which is significantly lower than the general level of similarity among these species (Fig. 2A). Curiously however, the D. nodosus AroA appears to be most similar to that of the Gram+ pathogen Staphylococcus aweus (59% similar) and to
A
have diverged significantly from the other Gram- organisms (Fig. 2B). Two regions of high similarity had been previously identified within the aroA genes of Gram- bacteria, LGNAGTA centered around aa95, and DHRMAMCF around aa (Maskell, 1993). It was adjacent to this first region that a single aa change in the S. typhimurium AroA protein rendered the enzyme inactive (Stalker et al., 1985), suggesting a crucial domain for enzyme activity. Within the D. nodosus uroA gene, sequences containing identical or conservative aa in these regions were identified: MQNSGTS (aa 92-98) and DHRIAMSL (aa 389-396) (Fig. 1B). We have also identified two further domains, not previously recognised, one at the N-terminal and one in the centre of the molecule, that exhibit significantly higher similarity between the various species including S. aureus (Fig. 3). These domains may represent important structural or functional areas of the EPSP synthase as they appear to have maintained strong conservation.
Hi&III
DraI
Hi&II
jimA
aroA
clpB
j%nB
DraI lHincI1
Hi&II
pRIc201
%zi$ aroA
B
.
TTTAARAACAATGAGTT
. AAiAAA
.
.
.
.
.
.
TTATTTTTCTGGCACACGCGCTTTTTTTGCATTTTTTCTCCCATTTTTCCGGCACAATAACGTTGGTTTTAT
&TGATG~TGACGAATA~A RR.9 MetMetThrAsnIle
121 5
TGGCACACCGCGCCCGTCTCTGCGCTTTCCGGCGAAATAACGATATGCGATATGCGGCGAT~TC~TGTCGCATCGCGCCTTATTATTAGCAGCGTTAGCAGAAGGACAAACGGATCCGCGGC TrpHisThrAlaProValSerAlaLeuSer~GlnThrGluIleArgGly
241 4s
TTTTTAGCGTGCGCGGATTGTTTGGCGACGCGGC~GCATTGCGCGCATTAGGCGTTGATATTC~GAG~~TAGT~CGATTCGCGGTGTGGGATTTCTGGGTTTGCAGCCG PheLeuAlaCysAlaAspCysLeuAlaThrArgG1nAlaLeuArgAlaLeuGlyValAspIleGlnArgGluLysGluIleValThrIleArgGlyValGlyPheLauGlyLeuGlnPro
361 85
CCGAAAGCACCGTTAAATATGCAAAACAGTGGCAGTGGCACTAGCATGCGTTTATTGGCAG~TTTTGGCAGCGCAGCGCTTTGAGAGCGTGTTATGCGGCGATG~TCATTAG~CGTCCG ProLysAlaProLeuAsnMetGlnAsnSerGlyThrSerMetArgLeuLeuAlaGlyIleLeuAlaAlaGlnArgPheGluSerValLeuCysGlyAspGluSerLeuGluLysArgPro
481 125
ATGCAGCGCATTATTACGCCGCTTGTGCAAATGGGGGGC~TTGTCAGTCACAGC~TTTTACGGCGCCGTTACATATTTCAG~CGCCCGCTGACCGGCATTGATTACGCGTTACCG MetGlnArgIleIleThrProLeuValGlnMetGlyAlaLysIleValSerHisSerAsnPheThrAlaPro~uHisIleSerGlyArgProLeuThrGlyIleAspTyrAlaLeuPro
601 165
CTTCCCAGCGCGCAATTAAAAAGTTGCCTTGCCTTATTTTGGCAGGATTATT~CT~CGGTACCACGCGGCTGCATACTTGCGGCATCAGTCGCGACCACACGG~CGCATGTTGCCGCTTTTT LeuPtoSerAlaGlnLeuLysSerCysLeuIlaLeuAlaGlyLeuLeuAlaAspGlyThrThrArgLeuHisThrCysGlyIleSerArgAspHisThrGluAsgMatLeuProLeuPha
721 205
GGTGGCGCACTTGAGATCAAGAAAGAGCAAATAATCGTCACCGGTGGAC~TTGCACGGTTGCGTGCTTGATATTGTCGGC~TTTGTCGGCGGCGGCGTTTTTTATGGTTGCGGCT GlyGlyAlaLeuGluIleLysLysGluGlnIleIleValThrGlyGlyGlnLysLeuHisGlyCysValLe~
841 245
TTGATTGCGCCGCGCGCGGRTCGTTATTCGTMTGTCGGCATTMTCCGACGCGGGCGGC~TCATTACTTTGTTGC~ TGGGCGGACGGATTGAATTGCATCATCAGCGCTTT ~roArgAlaGluValValIleArgAsnValGlyIleAsnProThrArgAl~laIleIleThrLeuLeuGlnLys~tGlyGlyArgIleGluLeuH~sH~sGl~rgPhe
961 285
TGGGGCGCCGMCCGGTGGCAGATATTGTTGTTTATCATTC~TTGC~GGCATTACGGTGGCGCCGG~TGGATTGCCMCGCGATTGATGMTTGCCGATTTTTTTTATTGCGGCA TrpGlyAlaGluProValAlaAspIleValValTyrHisSerLysLeuArgGlyIleThrValAlaProGluTrpIleAlaAsnAlaIleAspGluLeuProIlePhePheIleAlaAla
1081 325
GCTTGCGCGGAAGGGACGACTTTTGTGGGCAATTTGTCAGAATTGCGTGTGAAAGAATCGGATCGTTTAGCGGCGATGGCGC~TTTAC~CTTTGGGCGTGGCGTGCGACGTTGGC AlaCysAlaGluGlyThrThrPheValGlyAsnLeuSerGluLeuArgValLysGluSerAspArgLeuAlaAlaMetAlaGlnAsnLeuGlnThrLeuGlyValAlaCysAspValGly
1201 365
GCCWLTTTTATTCATATATATGGMGMGCGATCGGC~TTTTTACCGGCGCGGGTG~CAGTTTTGGCGATCATCGGATTGCGATGAGTTTGGCGGTGGCAGGTGTGCGCGCGGCAGGT AlaAspPheIleHisIleTyrGlyArgSerAspArgGlnPheLeuProAlaArgValAsnSerPheGlyAspHisArgIleAlaMetSerLeuAlaValAlaGlyValArgAlaAlaGly
1321 405
GMTTATTGATTGATGACGGCGCGGTGGCGGCGGTTTCTATGCCGC~TTTCGCGATTTTGCCGCCGC~TTGGTATG~TGTAG~ GAAAAAGATGCGAAAAATTGTCACGATTG GluLeuLeuIleAspAspGlyAlaValAlaAlaValSerMetProGlnPheArgAspPheAlaAl~laIleGly~tAsnValGlyGluLysAspAlaLysAsnCysHisAsp*~*
ATG Met
GTCCTAGCGGTGTTGGAAAAGGCACGGTGGCGCAAGCTT ValLeuAlaValLeuGluLysAlaArgTrpArgLysLeu
1440 443 1 1479 14
Fig. 1. Localizationand nt sequence of aroA. (A) Schematic representation of plasmids pBA122 and pRIC201 showing the localization of the uroA, JimA, fimZ and clpB genes. (B) The nt sequence of the pRIC201 fragment that encodes D. nodosus aroA and derived aa sequence. The putative RBS is underlined and labeled. These data have been submitted to the GenBank/EMBL DNA sequence database and have accession No. 229339.
99
A Dn Dn (443) 100 su (430) Bs (3.58) t?@ Ye
Sa
Bs
59 44 100 44 100
Bp
St
EC
Ye
Pm
Hi
As
Mt
!% 53 44 100
55 56 44 69 100
55 54 45 94 69 100
52 51 43 71 88
54 52 45 70 80 82
53 51 46 69 83
51 50 4s 73 72 64
47 43 44 47 43 45
100 81
85
69
45
71 100 87 100 70
44 45
(427)
L:
I$
2
$Z]
100 44 100
I
81
Direction of divergence
Fig. 2. Analysis of the AroA protein. (A) Comparison of AroA protein sequences using the GAP alignment program available from the GCG analysis software, using a gap weight of 3.0, and a gap length weight of 0.1. The matrix shows percent similarities which includes conservative aa changes. The length of the respective AroA proteins are shown bracketed on the left edge of the figure. Abbreviations and references for the sequences are as follows: St: Salmonella typhimurium (Stalker et al., 1985); Hi: Haemophilus injuenzae (Maskell, 1993); EC: Escherichia coti (Duncan et al., 1984); Ptn: P~reurel~a mu~~ocida(Homchampa et al., 1992); Ye: Yersinia enterocu~jfjcu (O’Gaora et al., 1989); As: Aeromonas salmonieida (Vaughan et al., 1993); Bp: Bordetella perussis (Maskell et al.. 1988); Dn: Dichelobacter nodosus; Sa: Staphylococcus aureus (O’Connell et al., 1993); Bs: Bacillus subtitis (Henner et al., 1986); Mr: Mycobacterium trtbercutosis (Garbe et al., 1991). (B) A phylogenetic tree comparing the evolutionary relationships of the translated AroA proteins. The tree was generated by a branch and bound analysis using Version 3.1.1 of the PAUP software (Swofford, 1993) after the proteins were aligned with the ChrstalV multiple alignment program. The numbers at each node represent the percentage the divergence occurred during 100 bootstrap replicas. Vertical separation is included for the purpose of clarity only; the internal branch points emerge at nodes and are able to be rotated, and the direction of divergence is indicated by an arrow.
The D. nodosus aroA gene sequence contains three consecutive potential start codons which are preceded by an ideal RBS (Fig. 1B). It is likely that the second of these is the start point due to the optimal spacing between the RBS and this AUG codon. The (G + C)% of 49.1%, and the codon usage and third position bias in the aroA gene was comparable to that in other LXnodosus genes present in the databases which include proteases, fimbrial genes and c&B (data not shown). There is another putative ORF downstream from aroA whose start codon overlaps
the TGA stop codon of the aroA gene, and which extends beyond the Hind111 site. This phenomenon would suggest that aroA and the downstream ORF are translationally coupled (Normark et al., 1983). However, we have not yet been able to clone these downstream sequences in E. coli, suggesting that this ORF may have deleterious effects on this host. (b) Expression of the amA gene in E. c&i In order to examine whether the aroA gene was being expressed from its native promoter, the aroA gene was cloned from pRIC201 into both pBluescriptKS+ and SK’ using unique restriction sites in the polylinker either side of the aroA gene. These recombinant plasmids, designated pRIC230 (KS+) and pRIC231 (SK’) were introduced into the E. coli strain AB2829 which carries an aroA mutation (Pittard and Wallace, 1966). Both pRIC230 and pRIC231 were able to complement the aroA mutation and permitted growth on defined minimal media, indicating that expression of the aroA gene was occurring from its native promoter rather than being dependent on the external lac promoter of the vector. In order to visualize the product of the aroA gene expressed from its native promoter, plasmid pRIC230 was transformed into the E. co/i minicell producing strain DS410. A protein product corresponding to the expected molecular weight of AroA was produced (Fig. 4). (c) Conclusions (I) The aroA genes from a number of other pathogens have been sequenced, and show a high degree of overall similarity with the D. nodosus sequence, especially in two newly identified conserved domains. The aroA gene from several other Gram- species forms an operon with serC, whereas in ~aenzop~ilus in~~enz~e the aroA gene is translationally coupled to a purl\r-like gene involved in purine biosynthesis (Maskell, 1993). This is not the case in D. nodosus, where no ORFs lie upstream from aroA in the same transcriptional orientation. (2) The I). no~osus AroA protein was shown to have diverged significantly from the AroA proteins of other Gram- organisms, and indeed the closest relatives found using the PAUP phylogeny program were the Gram+ organisms S. aureus and B. subtilis. This close evolutionary relationship was not seen for B. su~~i~iswhen looking purely at overall aa similarity, as the level of similarity was lower than for the other Gram- bacteria (Fig. 2A). This program may indicate closer evolutionary relationships than just simple aa conservation as it infers the phylogenies based on the maximum parsimony principle. These data confirm the evolutionary groupings of AroA enzymes put forward by Griffin and Griffin (1991) where
100
Dn Sa BP St EC Ye Pm Hi As
Dn Sa BP St EC Ye Pm Hi AS
237 237 248 241 239
LVEGDASSASYFLAAXAIK LVEGDASSASYFLAAAAIK LVEGDASSASYFLAAAAIK
255 255 266 259 257
8
LVEGDASSASYFLAAGAIK LVEGDASSASYFLAAGAIK
Fig. 3. Two highly conserved domains of AroA proteins from both Gramand Gram+ species. The circled aa residues represent a conservative substitution, whereas the boxed aa residues are neither identical or conserved. The following definition of conserved aa groups have been used:
M,L,V.I; T,S,G.A,P; R,K,H; D,E; Q.N; F.W,Y. The bold numbers
represent
AroA protein
in Fig. 1B. The sequences
programs
sequences,
(Needleman
and the domains
and Wunsch.
are shown
1970; Higgins
1
underlined
and Sharp,
AroA +
T
+
45.0
t
31.0
t
21.5
Fig. 4. Minicell analysis of E. co/i strain DS410 carrying plasmids pRIC230 (lane 1) or pBluescriptKS+ (lane 2). The production of a 47-kDa AroA protein by plasmid pRIC230 is indicated, and molecular mass standards
are shown.
Methods:
Minicells
were purified
that precede
were analysed
these domains
using the ClustalV
in the respective
and GAP
aligment
paralogues or isozymes have been found in other genes (uroK and aroL) that encode enzymes involved in the biosynthesis of aromatic aa from E. coli (Lobner-Olesen and Marinus, 1992). (3) Not widely recognised as classic virulence determi-
kDa
66.2
of aa residues
1989)
2
t
the number
and la-
belled essentially as described by Achtman et al. (1979). The minicells were purified through two successivesucrose gradients followed by preincubation in minimal medium to degrade long-lived mRNAs that correspond to chromosomally encoded genes. Plasmid-encoded proteins were then pulse labelled with [?3]methionine in the presence of methionine assay medium (Difco). Samples were analyzed by separation on a 0.1% SDS-12% PAGE gel. and visualized by autoradiography.
Gram+ and Gram- bacteria, fungi and plants form four distinct evolutionary classes. Such a genetic divergence suggests the possibility that the aroA gene of D. nodosus has been acquired laterally and/or may represent a duplicate or paralogue of another aro.4 gene present elsewhere in the D. nodosus genome. Examples of such duplicated
nants, inactivation of the native aroA gene has been shown to cause loss of virulence in animal infection models for a variety of organisms including Yersinia enterocolitica, Salmonella typhimurium, Shigella jexneri, Neisseria gonorrhoeae, Bordetella pertussis, Aeromonas salmonicida and Pasteurella multocida (Bowe et al., 1989; Hoiseth and Stocker, 1981; Roberts et al., 1990; Verma and Lindberg, 1991; Homchampa et al., 1992; Vaughan et al., 1993; Chamberlain et al., 1993). In this context, it is interesting that in D. nodosus the aroA gene is not simply located adjacent to the fimbrial subunit gene, but may possibly share a complex promoter sequence with it (Hobbs et al., 1991). Furthermore, aro genes have been detected adjacent to genes involved in type-4 fimbrial biosysnthesis in other species (Martin et al., 1993). Thus, the linkage between fimbrial expression and the catabolism of aromatic compounds may be more than coincidental (Hobbs and Mattick, 1993).
ACKNOWLEDGEMENT
The authors would like to thank help with analysis of the phylogenic
Dr. Effie Ablett relationships.
for
101 Martin,
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97
0378-l 119/94/$07.00
GENE 07971
Sequencing and expression of the aroA gene from Dichelobacter nodosus (Aromatic
amino acid; protein
production;
phylogeny;
Richard
A. Alma, Brian P. Dalrympleb
phosphatase;
complementation)
and John S. Mattick”
“Centre for Molecular Biology and Biotechnology, University of Queensland, St. Lucia, Qld 4072. Australia; and bCSIRO Division of Tropical Animal Production. Longpocket Laboratories, Brisbane, Qld 4068, Australia. Tel. (61-7) 377-0711 Received by P.A. Manning:
17 November
1993; Revised/Accepted:
17 January
1994; Received at publishers:
21 March
1994
SUMMARY
The aroA locus of the Grampathogen Dichelobacter nodosus, which encodes 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, has been sequenced and expressed in Escherichia coli. The gene is located on a 1.48-kb DraI-Hind111 fragment located directly upstream and in opposite transcriptional orientation to the gene encoding the fimbrial structural subunit. The deduced open reading frame is 1329 nucleotides in length, which encodes a protein of 443 amino acids (aa) with a calculated M, of 47413, which was visualized in E. coli minicells, under the control of its native promoter. This derived aa sequence displays significant similarities with the sequences of the aroA gene products from a variety
of microorganisms.
INTRODUCTION
The fastidious is the etiological
Gram- anaerobe Dichelobacter nodosus agent of ovine footrot (Egerton, 1977).
Nine major serogroups of D. nodosus have been identified based on the K-agglutination test, and the complexity of the serological hierachy extends to a total of some 18 subsidiary serotypes (Claxton, 1989). The antigenic structures that are the basis for the serogroup specificity are the polar type-4 fimbriae. The genes encoding the structural subunit of the fimbriae (JimA) representative of all nine major serogroups, including both class-I and class-II strains, have been sequenced (Mattick et al., 1991). Analysis of the fimbrial subunit gene region identified a partial ORF situ-
Correspondence to: Dr. J.S. Mattick,
Centre for Molecular
Biotechnology. University of Queensland, St. Lucia, Australia. Tel. (61-7) 365-4446; Fax (61-7) 365-4388: e-mail: [email protected]
Biology Qld
and 4072,
Abbreviations: aa, amino acid(s); Ap, ampicillin; aroA, gene encoding EPSP synthase; bp, base pair(s); D., Dichelobacter: E., Escherichia; EPSP, 5enolpyruvylshikimate 3-phosphate; CCC, Genetics Computer Group (Madison, WI. USA); kb. kilobase or 1000 bp; nt. nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; RBS. ribosome-binding site(s); SDS, sodium dodecyl sulfate. SSDI 0378-1119(94)00182-R
ated upstream from theJimA gene in all D. nodosus strains that displayed similarity to the aroA gene from a number of species, and was able to complement an Escherichia coli aroA mutant strain AB2829 (Hobbs et al., 1991). In E. coli the aroA gene encodes the biosynthetic enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, which is essential for the synthesis of a spectrum of aromatic compounds which include p-aminobenzoic acid, 2,3_dihydroxybenzoate and the aromatic amino acids, phenylalanine, tyrosine and anthranilic acid, a precursor of tryptophan. The aroA genes belonging to a number of both Gram- and Gram+ organisms have been sequenced, and many show areas of substantial conservation (Duncan et al., 1984; Stalker et al., 1985; Maskell et al., 1988; O’Gaora et al., 1989; Maskell, 1993; O’Connell et al., 1993). Here we report the complete sequence and expression of an aroA gene from D. nodosus, and its comparison to related genes in other species.
EXPERIMENTAL
AND DISCUSSION
(a) Subcloning, nt sequence and analysis of the am4 locus The pBA122 plasmid, which contains the aroA gene, the fimbrial genesjimA and$mB, and the clpB gene from
98 D. nodosus strain VCSlOOl (class I, serotype A) (Hobbs et al.. 1991; Fig. 1A) was digested with HindIII+ DraI, and the 1.5kb fragment that represented the aroA locus was cloned into the HincII-Hind111 sites within the multiple cloning site of plasmid pOK12 (Vieira and Messing, 1991) generating plasmid pRIC201 (Fig. 1A). The nt sequence of this fragment was determined using the dideoxy chain termination method (Sanger et al., 1977), and is shown in Fig. 1B. One significant ORF was identified in this region that encoded a protein of 443 aa with a calculated M, of 47413 (Fig. 1B). This ORF showed significant similarity at the aa level to several other bacterial AroA proteins (Fig. 2A). The predicted aa sequence of AroA from D. nodosus is approx. 55% similar to that of other Gram- species, which is significantly lower than the general level of similarity among these species (Fig. 2A). Curiously however, the D. nodosus AroA appears to be most similar to that of the Gram+ pathogen Staphylococcus aweus (59% similar) and to
A
have diverged significantly from the other Gram- organisms (Fig. 2B). Two regions of high similarity had been previously identified within the aroA genes of Gram- bacteria, LGNAGTA centered around aa95, and DHRMAMCF around aa (Maskell, 1993). It was adjacent to this first region that a single aa change in the S. typhimurium AroA protein rendered the enzyme inactive (Stalker et al., 1985), suggesting a crucial domain for enzyme activity. Within the D. nodosus uroA gene, sequences containing identical or conservative aa in these regions were identified: MQNSGTS (aa 92-98) and DHRIAMSL (aa 389-396) (Fig. 1B). We have also identified two further domains, not previously recognised, one at the N-terminal and one in the centre of the molecule, that exhibit significantly higher similarity between the various species including S. aureus (Fig. 3). These domains may represent important structural or functional areas of the EPSP synthase as they appear to have maintained strong conservation.
Hi&III
DraI
Hi&II
jimA
aroA
clpB
j%nB
DraI lHincI1
Hi&II
pRIc201
%zi$ aroA
B
.
TTTAARAACAATGAGTT
. AAiAAA
.
.
.
.
.
.
TTATTTTTCTGGCACACGCGCTTTTTTTGCATTTTTTCTCCCATTTTTCCGGCACAATAACGTTGGTTTTAT
&TGATG~TGACGAATA~A RR.9 MetMetThrAsnIle
121 5
TGGCACACCGCGCCCGTCTCTGCGCTTTCCGGCGAAATAACGATATGCGATATGCGGCGAT~TC~TGTCGCATCGCGCCTTATTATTAGCAGCGTTAGCAGAAGGACAAACGGATCCGCGGC TrpHisThrAlaProValSerAlaLeuSer~GlnThrGluIleArgGly
241 4s
TTTTTAGCGTGCGCGGATTGTTTGGCGACGCGGC~GCATTGCGCGCATTAGGCGTTGATATTC~GAG~~TAGT~CGATTCGCGGTGTGGGATTTCTGGGTTTGCAGCCG PheLeuAlaCysAlaAspCysLeuAlaThrArgG1nAlaLeuArgAlaLeuGlyValAspIleGlnArgGluLysGluIleValThrIleArgGlyValGlyPheLauGlyLeuGlnPro
361 85
CCGAAAGCACCGTTAAATATGCAAAACAGTGGCAGTGGCACTAGCATGCGTTTATTGGCAG~TTTTGGCAGCGCAGCGCTTTGAGAGCGTGTTATGCGGCGATG~TCATTAG~CGTCCG ProLysAlaProLeuAsnMetGlnAsnSerGlyThrSerMetArgLeuLeuAlaGlyIleLeuAlaAlaGlnArgPheGluSerValLeuCysGlyAspGluSerLeuGluLysArgPro
481 125
ATGCAGCGCATTATTACGCCGCTTGTGCAAATGGGGGGC~TTGTCAGTCACAGC~TTTTACGGCGCCGTTACATATTTCAG~CGCCCGCTGACCGGCATTGATTACGCGTTACCG MetGlnArgIleIleThrProLeuValGlnMetGlyAlaLysIleValSerHisSerAsnPheThrAlaPro~uHisIleSerGlyArgProLeuThrGlyIleAspTyrAlaLeuPro
601 165
CTTCCCAGCGCGCAATTAAAAAGTTGCCTTGCCTTATTTTGGCAGGATTATT~CT~CGGTACCACGCGGCTGCATACTTGCGGCATCAGTCGCGACCACACGG~CGCATGTTGCCGCTTTTT LeuPtoSerAlaGlnLeuLysSerCysLeuIlaLeuAlaGlyLeuLeuAlaAspGlyThrThrArgLeuHisThrCysGlyIleSerArgAspHisThrGluAsgMatLeuProLeuPha
721 205
GGTGGCGCACTTGAGATCAAGAAAGAGCAAATAATCGTCACCGGTGGAC~TTGCACGGTTGCGTGCTTGATATTGTCGGC~TTTGTCGGCGGCGGCGTTTTTTATGGTTGCGGCT GlyGlyAlaLeuGluIleLysLysGluGlnIleIleValThrGlyGlyGlnLysLeuHisGlyCysValLe~
841 245
TTGATTGCGCCGCGCGCGGRTCGTTATTCGTMTGTCGGCATTMTCCGACGCGGGCGGC~TCATTACTTTGTTGC~ TGGGCGGACGGATTGAATTGCATCATCAGCGCTTT ~roArgAlaGluValValIleArgAsnValGlyIleAsnProThrArgAl~laIleIleThrLeuLeuGlnLys~tGlyGlyArgIleGluLeuH~sH~sGl~rgPhe
961 285
TGGGGCGCCGMCCGGTGGCAGATATTGTTGTTTATCATTC~TTGC~GGCATTACGGTGGCGCCGG~TGGATTGCCMCGCGATTGATGMTTGCCGATTTTTTTTATTGCGGCA TrpGlyAlaGluProValAlaAspIleValValTyrHisSerLysLeuArgGlyIleThrValAlaProGluTrpIleAlaAsnAlaIleAspGluLeuProIlePhePheIleAlaAla
1081 325
GCTTGCGCGGAAGGGACGACTTTTGTGGGCAATTTGTCAGAATTGCGTGTGAAAGAATCGGATCGTTTAGCGGCGATGGCGC~TTTAC~CTTTGGGCGTGGCGTGCGACGTTGGC AlaCysAlaGluGlyThrThrPheValGlyAsnLeuSerGluLeuArgValLysGluSerAspArgLeuAlaAlaMetAlaGlnAsnLeuGlnThrLeuGlyValAlaCysAspValGly
1201 365
GCCWLTTTTATTCATATATATGGMGMGCGATCGGC~TTTTTACCGGCGCGGGTG~CAGTTTTGGCGATCATCGGATTGCGATGAGTTTGGCGGTGGCAGGTGTGCGCGCGGCAGGT AlaAspPheIleHisIleTyrGlyArgSerAspArgGlnPheLeuProAlaArgValAsnSerPheGlyAspHisArgIleAlaMetSerLeuAlaValAlaGlyValArgAlaAlaGly
1321 405
GMTTATTGATTGATGACGGCGCGGTGGCGGCGGTTTCTATGCCGC~TTTCGCGATTTTGCCGCCGC~TTGGTATG~TGTAG~ GAAAAAGATGCGAAAAATTGTCACGATTG GluLeuLeuIleAspAspGlyAlaValAlaAlaValSerMetProGlnPheArgAspPheAlaAl~laIleGly~tAsnValGlyGluLysAspAlaLysAsnCysHisAsp*~*
ATG Met
GTCCTAGCGGTGTTGGAAAAGGCACGGTGGCGCAAGCTT ValLeuAlaValLeuGluLysAlaArgTrpArgLysLeu
1440 443 1 1479 14
Fig. 1. Localizationand nt sequence of aroA. (A) Schematic representation of plasmids pBA122 and pRIC201 showing the localization of the uroA, JimA, fimZ and clpB genes. (B) The nt sequence of the pRIC201 fragment that encodes D. nodosus aroA and derived aa sequence. The putative RBS is underlined and labeled. These data have been submitted to the GenBank/EMBL DNA sequence database and have accession No. 229339.
99
A Dn Dn (443) 100 su (430) Bs (3.58) t?@ Ye
Sa
Bs
59 44 100 44 100
Bp
St
EC
Ye
Pm
Hi
As
Mt
!% 53 44 100
55 56 44 69 100
55 54 45 94 69 100
52 51 43 71 88
54 52 45 70 80 82
53 51 46 69 83
51 50 4s 73 72 64
47 43 44 47 43 45
100 81
85
69
45
71 100 87 100 70
44 45
(427)
L:
I$
2
$Z]
100 44 100
I
81
Direction of divergence
Fig. 2. Analysis of the AroA protein. (A) Comparison of AroA protein sequences using the GAP alignment program available from the GCG analysis software, using a gap weight of 3.0, and a gap length weight of 0.1. The matrix shows percent similarities which includes conservative aa changes. The length of the respective AroA proteins are shown bracketed on the left edge of the figure. Abbreviations and references for the sequences are as follows: St: Salmonella typhimurium (Stalker et al., 1985); Hi: Haemophilus injuenzae (Maskell, 1993); EC: Escherichia coti (Duncan et al., 1984); Ptn: P~reurel~a mu~~ocida(Homchampa et al., 1992); Ye: Yersinia enterocu~jfjcu (O’Gaora et al., 1989); As: Aeromonas salmonieida (Vaughan et al., 1993); Bp: Bordetella perussis (Maskell et al.. 1988); Dn: Dichelobacter nodosus; Sa: Staphylococcus aureus (O’Connell et al., 1993); Bs: Bacillus subtitis (Henner et al., 1986); Mr: Mycobacterium trtbercutosis (Garbe et al., 1991). (B) A phylogenetic tree comparing the evolutionary relationships of the translated AroA proteins. The tree was generated by a branch and bound analysis using Version 3.1.1 of the PAUP software (Swofford, 1993) after the proteins were aligned with the ChrstalV multiple alignment program. The numbers at each node represent the percentage the divergence occurred during 100 bootstrap replicas. Vertical separation is included for the purpose of clarity only; the internal branch points emerge at nodes and are able to be rotated, and the direction of divergence is indicated by an arrow.
The D. nodosus aroA gene sequence contains three consecutive potential start codons which are preceded by an ideal RBS (Fig. 1B). It is likely that the second of these is the start point due to the optimal spacing between the RBS and this AUG codon. The (G + C)% of 49.1%, and the codon usage and third position bias in the aroA gene was comparable to that in other LXnodosus genes present in the databases which include proteases, fimbrial genes and c&B (data not shown). There is another putative ORF downstream from aroA whose start codon overlaps
the TGA stop codon of the aroA gene, and which extends beyond the Hind111 site. This phenomenon would suggest that aroA and the downstream ORF are translationally coupled (Normark et al., 1983). However, we have not yet been able to clone these downstream sequences in E. coli, suggesting that this ORF may have deleterious effects on this host. (b) Expression of the amA gene in E. c&i In order to examine whether the aroA gene was being expressed from its native promoter, the aroA gene was cloned from pRIC201 into both pBluescriptKS+ and SK’ using unique restriction sites in the polylinker either side of the aroA gene. These recombinant plasmids, designated pRIC230 (KS+) and pRIC231 (SK’) were introduced into the E. coli strain AB2829 which carries an aroA mutation (Pittard and Wallace, 1966). Both pRIC230 and pRIC231 were able to complement the aroA mutation and permitted growth on defined minimal media, indicating that expression of the aroA gene was occurring from its native promoter rather than being dependent on the external lac promoter of the vector. In order to visualize the product of the aroA gene expressed from its native promoter, plasmid pRIC230 was transformed into the E. co/i minicell producing strain DS410. A protein product corresponding to the expected molecular weight of AroA was produced (Fig. 4). (c) Conclusions (I) The aroA genes from a number of other pathogens have been sequenced, and show a high degree of overall similarity with the D. nodosus sequence, especially in two newly identified conserved domains. The aroA gene from several other Gram- species forms an operon with serC, whereas in ~aenzop~ilus in~~enz~e the aroA gene is translationally coupled to a purl\r-like gene involved in purine biosynthesis (Maskell, 1993). This is not the case in D. nodosus, where no ORFs lie upstream from aroA in the same transcriptional orientation. (2) The I). no~osus AroA protein was shown to have diverged significantly from the AroA proteins of other Gram- organisms, and indeed the closest relatives found using the PAUP phylogeny program were the Gram+ organisms S. aureus and B. subtilis. This close evolutionary relationship was not seen for B. su~~i~iswhen looking purely at overall aa similarity, as the level of similarity was lower than for the other Gram- bacteria (Fig. 2A). This program may indicate closer evolutionary relationships than just simple aa conservation as it infers the phylogenies based on the maximum parsimony principle. These data confirm the evolutionary groupings of AroA enzymes put forward by Griffin and Griffin (1991) where
100
Dn Sa BP St EC Ye Pm Hi As
Dn Sa BP St EC Ye Pm Hi AS
237 237 248 241 239
LVEGDASSASYFLAAXAIK LVEGDASSASYFLAAAAIK LVEGDASSASYFLAAAAIK
255 255 266 259 257
8
LVEGDASSASYFLAAGAIK LVEGDASSASYFLAAGAIK
Fig. 3. Two highly conserved domains of AroA proteins from both Gramand Gram+ species. The circled aa residues represent a conservative substitution, whereas the boxed aa residues are neither identical or conserved. The following definition of conserved aa groups have been used:
M,L,V.I; T,S,G.A,P; R,K,H; D,E; Q.N; F.W,Y. The bold numbers
represent
AroA protein
in Fig. 1B. The sequences
programs
sequences,
(Needleman
and the domains
and Wunsch.
are shown
1970; Higgins
1
underlined
and Sharp,
AroA +
T
+
45.0
t
31.0
t
21.5
Fig. 4. Minicell analysis of E. co/i strain DS410 carrying plasmids pRIC230 (lane 1) or pBluescriptKS+ (lane 2). The production of a 47-kDa AroA protein by plasmid pRIC230 is indicated, and molecular mass standards
are shown.
Methods:
Minicells
were purified
that precede
were analysed
these domains
using the ClustalV
in the respective
and GAP
aligment
paralogues or isozymes have been found in other genes (uroK and aroL) that encode enzymes involved in the biosynthesis of aromatic aa from E. coli (Lobner-Olesen and Marinus, 1992). (3) Not widely recognised as classic virulence determi-
kDa
66.2
of aa residues
1989)
2
t
the number
and la-
belled essentially as described by Achtman et al. (1979). The minicells were purified through two successivesucrose gradients followed by preincubation in minimal medium to degrade long-lived mRNAs that correspond to chromosomally encoded genes. Plasmid-encoded proteins were then pulse labelled with [?3]methionine in the presence of methionine assay medium (Difco). Samples were analyzed by separation on a 0.1% SDS-12% PAGE gel. and visualized by autoradiography.
Gram+ and Gram- bacteria, fungi and plants form four distinct evolutionary classes. Such a genetic divergence suggests the possibility that the aroA gene of D. nodosus has been acquired laterally and/or may represent a duplicate or paralogue of another aro.4 gene present elsewhere in the D. nodosus genome. Examples of such duplicated
nants, inactivation of the native aroA gene has been shown to cause loss of virulence in animal infection models for a variety of organisms including Yersinia enterocolitica, Salmonella typhimurium, Shigella jexneri, Neisseria gonorrhoeae, Bordetella pertussis, Aeromonas salmonicida and Pasteurella multocida (Bowe et al., 1989; Hoiseth and Stocker, 1981; Roberts et al., 1990; Verma and Lindberg, 1991; Homchampa et al., 1992; Vaughan et al., 1993; Chamberlain et al., 1993). In this context, it is interesting that in D. nodosus the aroA gene is not simply located adjacent to the fimbrial subunit gene, but may possibly share a complex promoter sequence with it (Hobbs et al., 1991). Furthermore, aro genes have been detected adjacent to genes involved in type-4 fimbrial biosysnthesis in other species (Martin et al., 1993). Thus, the linkage between fimbrial expression and the catabolism of aromatic compounds may be more than coincidental (Hobbs and Mattick, 1993).
ACKNOWLEDGEMENT
The authors would like to thank help with analysis of the phylogenic
Dr. Effie Ablett relationships.
for
101 Martin,
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