1. health and fitness
2. disease

791
Transfer RNA genes and their significance to
codon usage in the Pseudomonas aeruginosa
lamboid bacteriophage D3
Andrew M. Kropinski and Mary Jo Sibbald
Abstract: Using tRNAscan-SE and FAStRNA we have identified four tRNA genes in the delayed early region of the
bacteriophage D3 genome (GenBank accession No. AF077308). These are specific for methionine (AUG), glycine
(GGA), asparagine (AAC), and threonine (ACA). The D3 Thr- and Gly-tRNAs recognize codons, which are rarely
used in Pseudomonas aeruginosa and presumably, influence the rate of translation of phage proteins. BLASTN
searches revealed that the D3 tRNA genes have homology to tRNA genes from Gram-positive bacteria. Analysis of
codon usage in the 91 ORFs discovered in D3 indicates patterns of codon usage reminiscent of Escherichia coli or
P. aeruginosa.
Key words: bacteriophage, Pseudomonas, D3, tRNA, codon usage.
Résumé : Les techniques tRNA scan-SE et FAStRNA nous ont permis d’identifier quatre gènes de l’ARNt dans la
région précoce retardée du génome D3 des bactériophages (No. d’accès GenBank AF077308). Ces gènes sont
spécifiques de la méthionine (AUG), de la glycine (GGA) de l’asparagine (AAC) et de la thréonine (ACA). Les Glyet Thr-ARNt D3 reconnaissent des codons qui sont rarement utilisés chez Pseudomonas aeruginosa et qui
vraisemblablement influencent le taux de traduction des protéines du phage. Des études BLASTN révèlent que les
gènes de l’ARNt D3 ont une homologie avec les gènes de l’ARNt des bactéries à Gram positif. L’analyse de
l’utilisation des codons chez les 91 ORFs découverts dans D3 indique des profils d’utilisation qui rappellent ceux
observés chez Escherichia coli ou P. aeruginosa.
Mots clés : bactériophage, Pseudomonas, D3, ARNt, utilisation de codons.
[Traduit par la Rédaction]
Notes
796
Bacteriophages, being cellular parasites, subvert the host’s
protein synthesis mechanism and its components (tRNAs,
aminoacyl-tRNA synthetases) to synthesize viral enzymatic
and structural proteins. Certain members of the Caudovirales (Ackermann 1999) encode their own tRNAs. This has
been elegantly demonstrated with members of the Myoviridae (phages with contractile tails) including the T-even
coliphages and Haemophilus influenzae phages HP1 and S2.
Phage T4 encodes tRNAs that are acylated with arginine,
glutamate, glycine, leucine, isoleucine, proline, serine, and
threonine (Desai and Weiss 1977; Kunisawa 1992; Schmidt
and Apirion 1993). It has been claimed that phage S2 has
Lys (AAA)- and Leu (TTA)-tRNA genes (Skowronek 1998),
but our studies suggest that HP1 and S2 only contain a single copy of a tRNAlys (Kropinski, unpublished observations).
Received May 7, 1999. Revision received July 8, 1999.
Accepted July 13, 1999.
A.M. Kropinski.1 Department of Microbiology and
Immunology, Queen’s University, Kingston, ON K7L 3N6,
Canada.
M.J. Sibbald. School of Health Sciences, St. Lawrence
College, Kingston, ON K7M 1V6, Canada.
1
Author to whom all correspondence should be addressed
(e-mail: kropinsk@post.queensu.ca).
Can. J. Microbiol. 45: 791–796 (1999)
Several members of the viral family Siphoviridae (phages
with long noncontractile tails) possess tRNA genes. These
are coliphages T5 and its relative BF23 (McCordquodale
and Warner 1988), Streptomyces phage φC31 (Hendrix et al.
1999), Vibrio eltor phage e4 (Chattopadhyay and Ghosh
1988), and the mycobacterial phages L5 (Hatfull and Sarkis
1993) and D29 (Ford et al. 1998). T5 has at least 24 tRNA
genes while e4 has 12 tRNAs. The remainder of the phages
possess a more limited repertoire of tRNA genes. Phage
φC31 has Thr-tRNA genes; phage L5 has Asn- and TrptRNA genes; and phage D29 has Asn-, Trp-, Tyr-, and GlutRNA genes.
Vibrio cholerae phage φ149 (Ghosh and Guhathakurta
1983), a member of the Podoviridae (phages with short noncontractile tails), encodes 5 tRNAs. Unclassified coliphage
933W has an Ile-tRNA and two Arg-tRNA genes (Plunkett
et al. 1999). No tRNA genes have been found in the complete nucleotide sequence of coliphage lambda (GenBank
accession No. J02459), P2 (GenBank accession No. AF063097),
Methanobacterium thermoautotrophicum phage ΨM2 (GenBank accession No. AF065411), Pseudomonas aeruginosa
phage φCTX (GenBank accession No. AB008550), or Staphylococcus aureus phage φPVL (GenBank accession No.
AB009866; Kropinski, unpublished results).
Two general points can be made about the presence of
tRNA genes in phage genomes. They are almost always
clustered in the viral genomes, and they may function to fa© 1999 NRC Canada
792
Can. J. Microbiol. Vol. 45, 1999
Fig. 1. Physical and genetic map of the right end of the phage D3 genome (13 kb) showing the location of genes having homology
with other characterized phage genes and the major promoters (PR, and PLate). An enhanced map (1 kb) of the region containing the
four tRNA genes is shown immediately below. A line joining the two parts of the diagram shows the relative position of the tRNAcontaining fragment.
cilitate a more rapid overall translation rate, particularly the
translation of rare codons. The best examples of the latter
point are coliphages T4 and T5 in which the mole percent of
AT in the viral DNAs are significantly higher than that of
the host (65 and 60% vs. 50%). In the case of T4, the presence of isoaccepting tRNA species, which recognize rare
codons, has been shown to enhance the translation of certain
viral proteins (Kunisawa 1992). A similar situation has been
proposed for phage 933W (Plunkett et al. 1999).
Lastly, certain temperate phages use host tRNA genes as
integration (att) sites for the prophage genome (Bruttin et al.
1997; Dupont et al. 1995). The propensity for insertion
within tRNA genes may be a result of the potential for these
regions of DNA to form structures that facilitate integrasemediated site-specific recombination. (Gabriel et al. 1995;
Hauser and Scocca 1990; Hayashi et al. 1993; Inouye et al.
1991; Lindsey et al. 1989; McShan and Ferretti 1997;
McShan et al. 1997; Papp et al. 1993; Pierson and Kahn
1987; Ratti et al. 1997; Reiter et al. 1989).
We are working on the temperate serotype-converting Pseudomonas aeruginosa phage D3 (Gertman et al. 1987; Kuzio
and Kropinski 1983). Preliminary sequence and functional
analysis of this phage has shown it to be phylogenetically
related to coliphage lambda (Farinha et al. 1994; Farinha
and Kropinski 1997) (Fig. 1). This phage possesses two unusual properties. The DNA base composition (42 mol%AT)
differs markedly from that of its host bacterium (33 mol%AT),
and it possesses 3′-extended termini rather than blunt or
5′-extended termini (Sharp et al. 1996). The latter point distinguishes it from other phages that infect Gram-negative
bacteria. The complete D3 genome has been sequenced and
analyzed for putative tRNA species using tRNAscan-SE
(Lowe and Eddy 1997; Eddy and Durbin 1994) at its
website (http://www.genetics.wustl.edu/eddy/tRNAscan-SE/)
and FAStRNA (El-Mabrouk and Lisacek 1996) at its website
(http://bioweb.pasteur.fr/seqanal/interfaces/fastrna.html). We
have identified four tRNA genes in D3 (Fig. 1; GenBank accession No. AF077308). The tRNAs for which they code
range in size from 75 to 76 bp and are isoacceptors for
methionine (AUG), glycine (GGA), threonine (ACA), and
asparagine (AAC). The proposed structures of these tRNAs,
in cloverleaf form, are illustrated in Fig. 2.
In certain cases, tRNAs identified as Met-tRNAs by
tRNAScan-SE are in reality isoleucyl-tRNAs. In these
cases position C34 of the CAT anticodon is posttranscriptionally modified to a lysidine (4-amino-2-(N6-lysino)-1-β-Dribofuranosyl pyrimidine) residue. This has been shown to
occur in a number of bacterial and bacteriophage species
(Matsugi et al. 1996; Muramatsu et al. 1988; Plunkett et al.
1999). Extensive studies on the molecular recognition of
tRNAIle by the cognate isoleucyl-tRNA synthetase has shown
conserved base pairs in the D-arm (U12.A23), the anticodon
arm (C29.G41), and the acceptor arm (C4.G69) (Nureki et
al. 1994). These base pairs do not exist in the gene we have
defined as D3 tRNAMet. There is nothing obvious about the
nucleotide sequence of the tRNA genes that would lead us to
speculate on the role of tRNA-processing nucleases in the
maturation of the precursor tRNAs. The completion of the
Pseudomonas aeruginosa genome sequencing project (http://
www.pseudomonas. com) may well lead to the identification
of RNase P, PH, and T homologs (Li and Deutscher 1996;
Deutscher 1995), enzymes which play major roles in tRNA
processing in Escherichia coli.
Between the Met-tRNA gene and the glycine, asparagine,
and threonine tRNA cluster are two small putative genes
(ORF3O and ORF104) encoding hypothetical polypeptides
of 30 and 104 amino acids, respectively. These have no obvious homologues in protein databases.
Nucleotide comparison searches of trimmed D3 Met-,
Asn-, and Thr-tRNA genes using ungapped BLASTN
(Altschul et al. 1990) showed homology to similarly functioning tRNA genes derived from Gram-positive bacteria
(Firmicutes) and their viruses (Table 1). The glycyl tRNA
gene shows homology to tDNAgly from Aquifex, a deeprooted member of the Kingdom Bacteria. It has been
proposed that tailed phages have evolved through recombinational events between different viral genomes (Schmieger
1999; Hendrix et al. 1999). The presence of tRNA genes
© 1999 NRC Canada
Notes
793
Fig. 2. The sequence of the four tRNAs in cloverleaf structures.
with homology to genes from Gram-positive bacteria suggests that this recombinantional evolution may extend outside the γ-subdivision of the Proteobacteria.
The D3 nucleotide sequence was analyzed for potential
genes using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/
gorf.html) and WebGeneMark.HMM (http://genemark.biology.gatech.edu/GeneMark/whmm.cgi) revealing 91 ORFs
(Kropinski, unpublished results). Codon usage was assessed
using DNAMAN (Lynnon BioSoft, Vaudreuil, Que.) and the
Codon Usage Database (http://www.dna.affrc.go.jp/~nakamura/
countcodon.html), while http://www.dna.affrc.gojp/nakamura/
CUTG.html was used as the source of codon usage data for
E. coli and Pseudomonas aeruginosa. Three clear examples
of differential codon preference can be seen in the data presented in Table 2. In certain cases there is a quasilinear relationship between codon preference and the overall genomic
mole percent of GC content. This is seen most clearly in the
case of all codons for Asn, Asp, and Gln. In addition, it can
be seen for specific codons for certain other amino acids.
Good examples include AGC and UCC (Ser), AUU and
AUC (Ile), and CCC (Pro). In other cases there is a clear
bias in favour of E. coli or Pseudomonas aeruginosa codon
usage. The former case includes two of the Ala codons
(GCU, GCA), Arg (GCG), Gly (GGC), Pro (CCU, CCA),
Ser (UCU), Val (GUU) and all the Thr codons. In the latter,
the stop codons, Cys, Glu, His, Lys, and Phe, are all Pseudomonas-like. In addition, subsets of codons for Arg (CGU,
CGG), Gly (GGU), Ser (UCG), and Val (GUC) also resemble Pseudomonas codon preference. Lastly, in certain cases
there is no apparent correlation between D3 codon usage and
either bacterium. This is particularly evident in the case of
Ala (GCG), Arg (CGA, AGG), Gly (GGA, GGG), Leu
(CUU, CUA), Val (GUG), and Pro (CCG).
Rare codons, which we define as codons that contribute
>10% of the total coding capacity and are present in D3 at a
level ≥ twofold higher than that of host, Pseudomonas
aeruginosa, include Ala (GCU, GCA), Arg (AGG), Gly
(GGA), Leu (CUU), Pro (CCU, CCA), Ser (UCU), Thr
(ACA), and Val (GUU). Transfer RNA genes exist for 2 out
of the 10 operationally defined rare codons in D3. We con© 1999 NRC Canada
794
Can. J. Microbiol. Vol. 45, 1999
Table 1. Homology between phage D3 tRNA genes and sequences in GenBank.
D3 tRNA gene
BLASTN hit
% identity
Met-tRNA
Mycoplasma capricolum fMet-tRNA (X16759)
Mycoplasma mycoides fMet-tRNA (K00312)
Mycobacteriophage D29 Asn-tRNA (AF022214)
Mycobacteriophage L5 Asn-tRNA (Z18946.1)
Aquifex aeolicus Gly-tRNA (AE000763)
Acholeplasma laidlawii Thr-tRNA (X61068.1)
Thermotoga maritima Thr-tRNA (Z11839.1)
80
78
77
74
71
77
75
Asn-tRNA
Gly-tRNA
Thr-tRNA
Note: The GenBank accession numbers are in parentheses.
Table 2. Codon usage in E. coli, Pseudomonas aeruginosa, and
bacteriophage D3.
Table 2 (concluded).
%
%
Amino acid
Ala
Ala
Ala
Ala
Arg
Arg
Arg
Arg
Arg
Arg
Asn
Asn
Asp
Asp
Cys
Cys
Gln
Gln
Glu
Glu
STOP
Gly
Gly
Gly
Gly
His
His
Ile
Ile
Ile
Leu
Leu
Leu
Codon
GCU
GCC
GCA
GCG
CGU
CGC
CGA
CGG
AGA
AGG
AAU
AAC
GAU
GAC
UGU
UGC
CAA
CAG
GAA
GAG
UAA
GGU
GGC
GGA
GGG
CAU
CAC
AUU
AUC
AUA
CUU
CUC
CUA
EC
17
27
22
34
38
38
6
10
5
3
45
55
61
39
44
56
33
67
68
32
61
34
40
11
15
55
45
50
42
8
11
10
4
λ
18
27
30
24
26
26
11
14
15
7
48
52
57
43
29
71
25
75
58
42
38
28
31
20
21
58
42
45
38
17
19
12
5
D3
20
35
19
26
12
40
11
18
7
12
30
70
41
59
21
79
27
73
40
60
14
17
45
19
20
34
66
27
65
9
17
18
7
PA
8
53
7
32
13
59
5
19
1
4
18
82
25
75
15
85
17
83
40
60
17
14
68
6
12
32
68
13
84
4
5
20
2
sider that the presence of these isoaccepting tRNA species in
virus-infected cells should facilitate the expression of D3
genes. The presence of Met- and Asn-tRNA isoacccepting
species in D3 cannot be accounted for by codon bias, but
their presence may contribute to the overall rate of protein
synthesis. The lack of tRNAs for the other underrepresented
codons poses an interesting problem that cannot be fully un-
Amino acid
Leu
Leu
Leu
Lys
Lys
Met
Phe
Phe
STOP
Pro
Pro
Pro
Pro
Ser
Ser
Ser
Ser
Ser
Ser
Thr
Thr
Thr
Thr
Trp
Tyr
Tyr
Val
Val
Val
Val
STOP
Codon
CUG
UUA
UUG
AAA
AAG
AUG
UUU
UUC
UAG
CCU
CCC
CCA
CCG
UCU
UCC
UCA
UCG
AGU
AGC
ACU
ACC
ACA
ACG
UGG
UAU
UAC
GUU
GUC
GUA
GUG
UGA
EC
50
13
12
74
26
100
55
45
9
16
12
19
53
16
15
13
14
15
26
18
43
14
25
100
56
44
27
21
16
36
30
λ
45
11
8
65
35
100
57
43
11
21
13
24
42
11
16
20
13
16
25
16
32
23
29
100
58
42
31
17
16
36
51
D3
45
2
11
24
76
100
22
78
11
20
19
18
44
13
19
9
21
8
31
18
45
13
25
100
33
67
26
32
13
29
75
PA
62
1
10
19
81
100
11
89
11
8
24
7
62
3
22
3
24
7
41
8
73
5
15
100
25
75
8
39
7
45
73
Notes: EC, Escherichia coli (51 mol%GC); λ, = phage (50 mol%GC);
D3, = phage (58 mol%GC); PA, = Pseudomonas aeruginosa
(67 mol%GC).
derstood until we know more about the genes of this phage.
In the case of coliphage lambda, which lacks tRNA genes,
one sees a closer correlation between its codon usage and
that of its host than one sees with D3 and P. aeruginosa (Table 2). Two codons, AGA (Arg) and AUA (Ile) are rarely
used in E. coli and yet are employed more frequently in
coliphage λ. It has been noted that the λ integrase has a
© 1999 NRC Canada
Notes
higher proportion of the rare arginine codons, AGA and
AGG, and that this influences expression of this gene (Zahn
and Landy 1996).
In the case of the two completely sequenced genomes of
the Mycobacterium phages, the mole percent of AT is very
close to that of the host bacterium (27 vs. 24), and indeed
there is no specific codon bias in the phage genes relative to
those of the host. Deletion of these tRNA genes had no affect on phage replication (Ford et al. 1998). Since these apparently nonessential genes have been retained, it has been
speculated that their function is to enhance protein synthesis
in mycobacteriophage-infected cells.
Acknowledgements
This research was funded by a grant from the Natural Sciences and Engineering Research Council of Canada. Our
thanks is extended to Brad Cooney at the Guelph Molecular
Supercentre (Guelph, Ont.) for the DNA sequencing and to
the staff at DNAStar Inc. (Madison, Wisc.) for technical
help with SeqMan II.
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