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Factors influencing synonymous codon and amino acid usage biases in

Aromonas salmonicida phage 44RR

A.P.Ghosh, A. Deb, and K. Sau*

Department of Biotechnology, Haldia Institute of Technology, PO-HIT, Dist-Purba-Medinipur, Haldia, W. B.

721657.

To reveal the structure-function of the genes ofA. salmonicida phage 44RR, synonymous codon and

amino acid usage biases in this phage have been investigated at length. As expected for an AT-rich phage,

third codon position of the synonymous codons of 44RR carries mostly A and/or T base. Analyses on the

RSCU values of 44RR genes reveal that synonymous codon usage bias in 44RR is strongly dictated by the

mutational pressure. Further analysis reveals that 44RR-specifc tRNAs preferentially influence the codon

usage of putatively lowly expressed genes of 44RR, whereas, codon usage of its putatively highly expressed

genes are influenced mainly by the abundant cellular tRNAs. The data suggest that translational selection

also plays a role in shaping the codon usage variation in 44RR. Additional analysis reveals that the three

factors such as hydropathy, aromaticity and cysteine content are mostly responsible for the variation of

amino acid usage in 44RR proteins.

Key words:Relative synonymous codon usage (RSCU); correspondence analysis; amino acid usage; phage 44RR.

. Introduction

Synonymous codon and amino acid

usages in living organisms had been shown tovary inter- as well as intra-genomically.

While mutational pressure (Levine et al.,

2000; Jenkins et al., 2001; Jenkins and

Holmes, 2003), translational selection(Grantham et al., 1981; Ikemura, 1985; Sharp

and Cowe, 1991; Lesniket al., 2000; Ghoshet al ., 2000; Gupta and Ghosh, 2001),secondary structure of proteins (Oresisc and

Shalloway, 1998; Xie and Ding, 1998;

Chiusano et al., 2000; Gupta et al., 2000;DOnofrio et al ., 2002), replicational &

transcriptional selection (McInerney, 1998;

Romero et al., 2000) environmental factors

(Lynn et al., 2002; Basak et al., 2004) etcinfluence the codon usage in various

organisms, amino acid usage

*Corresponding author:

(K. Sau): keya_sau@yahoo.co.in

Tel: +91-3224-252900. Ext. 234.Fax: +91-3224-252800

was shown to be governed by hydrophobicity,aromaticity, mean molecular weight, cysteine

content, etc. (Lobry and Gautier, 1994; Garat

and Musto, 2000; Zavala et al., 2002;Banerjee et al., 2004; Naya et al., 2004).

Apart from elucidating the structure-function

and evolution of genes / genomes, codon

usage in particular has number of practicalapplications.

Synonymous codon and amino acid usage

biases had been studied in details in a fewphage genomes mainly belonging to different

coliphages, mycobacteriophages, S. aureus

phages, and P. aeruginosa phages (Sharp et al.,1984-85; Gouy, 1987; Holm, 1987; Kunisawa,

1992; Kunisawa et al., 1998, Sahu et al., 2004;

Sahu et al., 2005, Sau et al., 2005a; Sau et al.,

2005b). Synonymous codon usage of highlyand lowly expressed genes in T4 were shown

to be influenced by the abundant host tRNAs

and own tRNAs, respectively, (Kunisawa,1992). In phage T7, codon usage was also

suggested to be influenced mainly by the

abundant host tRNAs (Sharp et al., 1984-85).It was described that codon usage in both T4

and T7 are influenced mainly by mutational

pressure (Kunisawa et al., 1998).

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Table 1. Overall codon usage analysis in 44RR.

44RR A.salmonicida tRNA

Amino Overall HEG* LEG** HEG copy

Acid Codon RSCU RSCU RSCU RSCU 44RR

Phe UUU 0.88 0.77 0.94 0.46

UUC 1.12 1.23 1.06 1.54 1

Leu UUA 0.58 0.66 0.72 0.03 1

UUG 1.39 1.1 1.43 0.31

CUU 1.32 0.62 1.39 0.17

CUC 0.92 0.72 0.85 0.65

CUA 0.39 0.31 0.49 0.00

CUG 1.4 2.59 1.12 4.84

Ile AUU 1.19 1.1 1.12 0.54

AUC 1.27 1.5 1.08 2.36 1

AUA 0.54 0.4 0.8 0.11

Met AUG 1 1 1 1 2

Val GUU 2.02 2.2 1.98 0.94

GUC 0.77 0.54 0.8 1.11

GUA 0.75 0.72 0.78 0.62

GUG 0.45 0.54 0.44 1.34

Ser UCU 1.31 1.33 1.26 1.04

UCC 0.74 1.42 0.52 2.78

UCA 1.49 1 1.7 0.11 1

UCG 0.89 0.67 1.04 0.16

AGU 0.68 0.71 0.62 0.38AGC 0.89 0.88 0.86 1.53 1

Pro CCU 1.04 1.15 1.01 0.48

CCC 0.25 0.2 0.19 0.85

CCA 1.43 0.85 1.59 0.24 1

CCG 1.29 1.8 1.21 2.42

Thr ACU 1.25 1.46 1.22 0.62

ACC 1.18 1.76 0.92 3.01

ACA 1.06 0.46 1.31 0.29 1

ACG 0.51 0.33 0.55 0.07

Ala GCU 1.5 1.98 1.5 1.11GCC 0.74 0.65 0.66 1.90

GCA 1.09 1.02 1.05 0.36

GCG 0.67 0.35 0.79 0.63

Tyr UAU 1.15 0.91 1.22 0.52

UAC 0.85 1.09 0.78 1.48 1

His CAU 1.12 0.88 1.11 0.37

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CAC 0.88 1.12 0.89 1.63 1

Gln CAA 1.29 1.45 1.18 0.51

CAG 0.71 0.55 0.82 1.49

Asn AAU 0.93 0.84 0.94 0.24

AAC 1.07 1.16 1.06 1.76 1

Lys AAA 1.38 1.37 1.34 0.78 1

AAG 0.62 0.63 0.66 1.22

Asp GAU 1.15 1.01 1.18 0.79

GAC 0.85 0.99 0.82 1.21 1

Glu GAA 1.5 1.7 1.45 1.31

GAG 0.5 0.3 0.55 0.69

Cys UGU 0.79 0.85 0.9 0.73

UGC 1.21 1.15 1.1 1.27

Trp UGG 1 1 1 1 1

Arg CGU 1.89 1.89 1.63 3.04

CGC 1.63 1.89 1.37 2.39CGA 1.16 0.94 1.31 0.07

CGG 0.64 0.67 0.68 0.29

AGA 0.49 0.33 0.78 0.07 1

AGG 0.19 0.28 0.23 0.14

Gly GGU 1.68 2.1 1.38 1.75

GGC 1.3 1.31 1.33 1.98

GGA 0.74 0.44 0.93 0.05

GGG 0.28 0.15 0.36 0.22

Note- HEG and LEG denote highly and lowly expressed genes. The * and ** indicate putatively highly and lowlyexpressed genes of phage 44RR, which have been categorized respectively on the basis of lowest 10% and highest

10% of the genes according to their Nc values.

In phages lambda, N15, P2, and P4,

synonymous codon usage patterns were

found nearly similar to that of the lowly

expressed genes of E. coli (Holm, 1987;Gouy, 1987; Kunisawa, 1992; Kunisawa et

al., 1998). Contrary to above, codon usages

of mycobacteriophages and S. aureus phageswere found almost identical to their

respective bacterial hosts (Kunisawa, 2000;Sahu et al., 2004; Sahu et al., 2005; Sau et

al., 2005a). It was shown that mutational pressure and translational selection mainly

influence the codon usages in mycobacterial

and S. aureus phages (Sahu et al., 2004;Sahu et al., 2005; Sau et al., 2005a). While

tRNAs of mycobacteriophage Bxz1 wassuggested to regulate the expression of both

its highly and lowly expressed genes (Sahuet al., 2004), tRNAs present in phages D29and L5 were suggested to affect their amino

acid usage to some extent (Kunisawa, 2000).

Very recently, it was shown that in AT-rich P. aeruginosa phage PhiKZ, codon usage

bias is dictated mainly by mutational biasand, to an extent, by translational selection.

Analysis also revealed that amino acid usagein PhiKZ proteins are mainly dictated by

mean molecular weight, aromaticity and

cysteine content (Sau et al., 2005b).

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Complete genome sequences of threeAeromonas phages, namely, 44RR (synonym44RR2.8t), phage 31, and Aeh1, are available

in public databases at present. While former

two phages grow in A. salmonicida, Aeh1grows in A. hydrophila (Ttart et al., 2001).

The %GC content is nearly identical in all

three phages. Thus far, codon and amino acidusage biases have not been studied in

Aeromonas phages at length though such

Fig. 1. Nc plot of phage 44RR genes. See text for

details.

study may reveal the structure-function ofprotein-coding genes, evolution etc. In this

communication, We have studied bothsynonymous codon and amino acid usage

biases in A. salmonicida phage 44RR (Ttartet al ., 2001), determined the factorsresponsible for codon and amino acid usage

biases in 44RR, and discussed the data

elaborately. My data show that synonymouscodon usage of 44RR genes are influenced by

both mutational bias and translational

selection, whereas, amino usage of the 44RRproteins are mainly dictated by hydropathy,

aromaticity and cysteine content.

Materials and Methods

The genome sequence of bacteriophage

44RR (synonym 44RR2.8t) was down loaded

from GenBank (USA) and its two hundred

forty nine protein coding sequences (carrying50 or more codons) have been extracted from

the genome by coderet

(http:bioweb.Pasteur.fr/seqanal/interfaces/codret.html) program. The relative synonymous

codon usage (RSCU) in all protein coding

sequences was determined to study the overallcodon usage variation among the genes.

RSCU is defined as the ratio of the observed

frequency of codons to the expected

frequency if all the synonymous codons forthose amino acids are used equally (Sharp and

Li, 1987). RSCU values greater than 1.0

indicate that the corresponding codon is more

frequently used than expected, whereas thereverse is true for RSCU values less than 1.0.

RSCU values in seven putatively highlyexpressed genes (e. g. genes encoding

chaperonins, detoxification, and outer

membrane proteins etc.) of A. salmonicida

were also determined in this paper forcomparison.

GC3S is the frequency of (G+C) and A3S,

T3S, G3S, and C3S are the frequencies of A, T,G and C at the synonymous third positions of

codons. Nc, the effective number of codons

used by a gene, is generally used to measurethe bias of synonymous codons and

independent of amino acid compositions and

codon number (Wright, 1990). The values ofNc range from 20 (when one codon is used per

amino acid) to 61 (when all the codons are

used with equal probability). Nc values were

calculated according to the method ofBanerjee et al. (2005). The putatively highly

and lowly expressed genes have been

categorized respectively on the basis oflowest 10% and highest 10% of the genes

according to their Nc values. The program

CodonW 1.3 (available atwww.molbio.ox.ac.uk/cu) was used for

calculating most of the parameters including

correspondence analysis (CA) on the relative

synonymous codon and amino acid usages.

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In correspondence analysis, the data are

plotted in a multidimensional space of 59 axes

(excluding Met, Trp and stop codons) andthen it determines the most prominent axes

contributing the codon usage variation among

the genes. In the present study RSCU valueshave been used for CA in order to minimize

the amino acid composition.

Results and Discussion

Overall codon usage analysis in A.

salmonicida phage 44RR.

The relative synonymous codon usage

(RSCU) values determined in all the 249

protein coding genes of 44RR shows that A

and / or T ending codons are predominant inthis phage (Table 1). This is expected as %GC

content in 44RR is 43.66. As analysis ofoverall RSCU alone is not sufficient to reveal

the heterogeneity of codon usage in 44RR

genes, WE also determined the effective

numbers of codons used by gene (Nc) and(G+C) percentage at the synonymous third

positions of codons (GC3s). It was observed

that in 44RR, Nc values range from 26.486 to55.241 with a mean of 40.025 and standard

deviation (s.d.) 6.248, whereas, GC3s ranges

from 0.234 to 0.577 with a mean of 0.414 ands.d. 0.056. Taking together the results suggest

that apart from the mutational bias, other

factors might have some influences in thecodon usage variation among 44RR genes.

Effect of mutational pressure in codon usage

variation in 44RR. It was suggested that a plot of Nc vs GC3s

could effectively be used to explore the codon

usage variation among the genes (Wright,1990). According to Wright (1990), the

comparison of actual distribution of genes,

with the expected distribution under noselection, could be indicative if codon usage

bias of genes has some other influences other

than mutational bias. If the codon usage bias

is completely dictated by GC3s, the values of

Nc should fall on the expected curve between

GC3s and Nc. In other words it can be said that

if codon usage bias is completely dictated by

Fig. 2. Positions of the 44RR genes along the two

major axes of variation in the correspondence analysison RSCU values. The genes presented by the open

circles.

GC3s composition the difference between

observed and expected Nc values should bevery small in majority of genes. To explore

the possible influence of natural selection

and mutational bias on synonymous codon

usage on 44RR genome we calculated(NcExpected- NcObserved)/NcExpected. The frequency

distributions of (NcExpected- NcObserved)/NcExpectedshown in Fig. 1 demonstrate that majority ofgenes have large deviation of NcObserved from

NcExpected. This suggests that the majority of

genes in 44RR have additional codon usage bias, which is independent of mutational

bias.

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Table 2. Relative synonymous codon usage (RSCU) values for each codon for the two groups of genes in phage44RR. The asterisk denotes the codons whose occurrences are significantly (p < 0.01) higher in the extreme left side

of axis 1 than the genes present on the extreme right of the first major axis. Superscript "a" denotes for genes of

extreme left of axis 1 and "b" for extreme right genes. Each group contains 10% of sequences at either extreme of

the major axis generated by correspondence analysis. N is the number of codons, AA represents amino acid.

AA Codon RSCUa Na RSCUb Nb AA Codon RSCUa Na RSCUb Nb

Phe UUU 0.50 ( 46) 1.10 ( 67) Ser UCU* 1.57 ( 73) 0.80 ( 22)

UUC* 1.50 (137) 0.90 ( 55) UCC* 1.91 ( 89) 0.33 ( 9)

Leu UUA 0.02 ( 1) 1.20 ( 36) UCA 0.43 ( 20) 1.87 ( 51)

UUG 0.66 ( 36) 1.47 ( 44) UCG 0.37 ( 17) 1.39 ( 38)

CUU 0.66 ( 36) 1.17 ( 35) Pro CCU 1.36 ( 44) 1.00 ( 25)

CUC 0.77 ( 42) 0.67 ( 20) CCC 0.28 ( 9) 0.36 ( 9)

CUA 0.07 ( 4) 0.80 ( 24) CCA 0.71 ( 23) 1.44 ( 36)CUG* 3.82 (208) 0.70 ( 21) CCG 1.64 ( 53) 1.20 ( 30)

Ile AUU 0.94 ( 92) 1.07 ( 69) Thr ACU* 1.46 ( 83) 0.88 ( 37)

AUC* 2.02 (199) 0.82 ( 53) ACC* 2.13 (121) 1.00 ( 42)

AUA 0.04 ( 4) 1.10 ( 71) ACA 0.26 ( 15) 1.67 ( 70)

Met AUG 1.00 (145) 1.00 ( 83) ACG 0.14 ( 8) 0.45 ( 19)

Val GUU 2.07 (171) 1.87 ( 77) Ala GCU* 1.92 (194) 1.30 ( 47)

GUC 0.78 ( 64) 0.70 ( 29) GCC 0.85 ( 86) 0.72 ( 26)

GUA 0.92 ( 76) 0.68 ( 28) GCA 0.87 ( 88) 1.32 ( 48)

GUG 0.23 ( 19) 0.75 ( 31) GCG 0.36 ( 36) 0.66 ( 24)

Tyr UAU 0.73 ( 47) 1.20 ( 71) Cys UGU 0.40 ( 7) 0.69 ( 10)

UAC* 1.27 ( 82) 0.80 ( 47) UGC 1.60 ( 28) 1.31 ( 19)

Trp UGG 1.00 ( 48) 1.00 ( 54)

His CAU 0.88 ( 26) 1.18 ( 36) Arg CGU* 2.71 ( 90) 0.73 ( 16)

CAC 1.12 ( 33) 0.82 ( 25) CGC* 2.38 ( 79) 0.87 ( 19)

Gln CAA 1.41 (138) 1.38 ( 64) CGA 0.54 ( 18) 1.37 ( 30)

CAG 0.59 ( 58) 0.62 ( 29) CGG 0.27 ( 9) 0.64 ( 14)

Asn AAU 0.59 ( 63) 1.06 ( 74) Ser AGU 0.60 ( 28) 0.84 ( 23)

AAC* 1.41 (151) 0.94 ( 66) AGC 1.12 ( 52) 0.77 ( 21)

Lys AAA 1.24 (210) 1.33 (110) Arg AGA 0.06 ( 2) 1.69 ( 37)

AAG 0.76 (129) 0.67 ( 55) AGG 0.03 ( 1) 0.69 ( 15)

Asp GAU 1.22 (171) 1.06 ( 75) Gly GGU* 2.21 (162) 1.43 ( 57)

GAC 0.78 (110) 0.94 ( 66) GGC 1.54 (113) 1.38 ( 55)

Glu GAA 1.58 (289) 1.40 (125) GGA 0.19 ( 14) 0.98 ( 39)

GAG 0.42 ( 76) 0.60 ( 54) GGG 0.05 ( 4) 0.20 ( 8)

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Next, we carried out correspondence analysis

(CA) on the RSCU values of the 249protein-

coding genes of 44RR phage in order todetermine the factors influencing the codon

usage bias in 44RR. Figure 2 shows the

distributions of 44RR genes on the first twomajor axes of the correspondence analysis. It

is found that the first major axis is accounted

for 10.13% of the total variation and thesecond major axis accounted for 5.20% of the

total variation. The position of the genes

along the first major axis is negatively

correlated with the A3s (r = -0.575, p

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Fig. 3. Correlation between GRAVY scores of each

amino acid residues versus the axis 1 values of

correspondence analysis. Single letter codes are used to

show the positions of 20 amino acid residues. Amino

acid residues are presented closed circles.

The cellular tRNA abundance is positively

correlated with over-represented codons ofthe highly expressed genes in several

organisms (Grantham et al., 1981; Sharp et

al., 1984-85; Gouy, 1987; Ikemura. 1992;Zhou et al., 1999; Kanaya et al., 1999;

Kanaya et al., 2001). In coliphage T4,

synonymous codon usage of the highly

expressed genes was also shown to be

positively correlated with the abundantcellular tRNAs (Kunisawa, 1992). This may

also be true for the highly expressed genes of44RR. As the status of tRNA copy number is

not known in A. salmonicida at present, we

determined the RSCU values of the putativelyhighly expressed genes of A. salmonicida and

compared the resulting data with that of

highly expressed genes of 44RR (Table 1). Itwas found that among the 19 over-represented

codons present in each of the highly

expressed genes of 44RR and A. salmonicida,

13 are common in both species. This indicatesthat nearly 68% over-represented codons of

highly expressed genes of 44RR are

recognized by abundant host tRNAs.Amino Acid Usage in 44RR.

To identify the factors influencing the

amino acid composition in 44RR, we also

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carried out CA on the relative amino acid

usage of its 249 proteins. Analysis showed

that the first and second major axes of CA areaccounted for 18.66% and 10.91% of the total

variation of the amino acid composition of

44RR proteins, respectively. Further analysisrevealed that first major axis is positively

correlated with GRAVY (r = 0.410, p < 0.01)

of each 44RR protein. A plot of GRAVYscore of each amino acid residue versus first

major axis in fact shows that charged residues

like lys, arg, his, asp, glu are located on the

negative side of first axis (Fig. 3).Further analysis has shown that the

first major axis is also significantly correlated

(r = -0.404, p

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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&term=%22Sau+S%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Search&term=%22Sau+S%22%5BAuthor%5D