Liferay

4.3.6.1 A-HIS1 vs A-HIS2

When the genomes of phages A-HIS1 and A-HIS2 were compared to each other a high degree of synteny was observed and in general, the relative positions of genes are conserved. The amino acid sequences of predicted ORFs from each phage were compared against each other by standalone

Chapter 4. Phage genomic and proteomic characterisation 76

blastp to find the number of genes with significant identity between the two phages. The A-HIS1 genes were queried against the A-HIS2 genes as the database and vice versa. Shared geneswere found by considering only those gene pairs which were top hits for each other in both sets of blastp results which could mutually identify each other.

In total, phages A-HIS1 and A-HIS2 share 48 such genes (Table 4.5). Other gene pairs, termed similar genes were identified by considering the lesser hits. All assignments were based on all percent identity matrix (PIM) val- ues betweens ORFs being greater than 20 % when pairs of genes were aligned. This was observed for example for the AP2 endonucleases (Ta- ble 4.6, discussed further in § 4.3.7.2). It is worth noting that though these phages share nearly half their genes, they remain highly divergent from each other with % identity ranging from the least similar shared gene 20 % for A-HIS1 ORF 51 and A-HIS2 ORF 66 up to the most similar gene, the carboxypeptidase with 70 % identity between the two phages (A-HIS1 ORF 88 and A-HIS2 ORF 97).

Using NCBI blastp only ORF 59 from A-HIS2 had a significant hit (e- value 6e-5_{) to an Acaryochloris gene which encodes hypothetical protein}

AM1 2888. A-HIS1 ORF39 also had a hit to AM1 2888 but the e-value was 0.6. This lone result indicated there was no strong evidence for lat- eral gene transfer between phages A-HIS1 and A-HIS2 and the host strain MBIC11017.

A further three ORFs could be assigned a virion structural function by virtue of their similarity (from the analysis of shared genes, Table 4.6) to ORFs identified as VSPs by mass spectrometry. As a result virion struc- tural function was assigned to A-HIS1 ORFs 37 and 63 which are similar to A-HIS2 ORFs 57 and 73, respectively. Similarly, this applied to A-HIS2 ORF 101, which is a homologue of A-HIS1 ORF 93. In total, the combi- nation mass spectrometry, blastp analyses and ORF comparisons, allowed

Table 4.5: Shared genes. Summary of gene similarities between A-HIS1 and A-HIS2 based on the standalone blastp (default parameters) of each set of phage genes first as query against the other, then as the subject (i.e. database) for the other. Results

where e > 1e-3 _{were removed. Shared gene pairs were the top hits in both blastp}

searches which could mutually identify one another. % identity values taken from PIM calculated in ClustalX (1.83). * denotes ORFs with nucleotide sequences which are inverted with respect to each other. Abbreviations as in Table 4.2 and Table 4.3.

A-HIS1 A-HIS2 % identity Putative function

ORF Size (aa) ORF Size (aa)

1 486 1 478 63 TerL 4 86 9 86 37 - 6 236 12 137 42 - 12 112 17 97 35 - 14 993 20 959 57 DNA polγ 16 605 22 610 47 DNA pri 19 283 25 238 40 - 20 191 27 199 47 RNase T 22 57 28 63 37 - 24 209 30 210 49 - 25 59 95 58 36 - 26 108 31 141 50 End 27 269 32 251 36 DNA Mtase 28 259 33 345 42 -

30 365 35 361 52 DNA pol I-like FEN

31 265 36 235 35 - 33 263 38 243 32 - 34 73 44 73 31 - 36 169 55 168 61 VSP 37 688 57 687 66 VSP 41 55 58 50 50 - 43 83 54 83 64 - 46 149 45 133 27 - 47 61 63 76 33 - 51 267 66 276 20 - 53 77 67 95 42 - 55 539 68 278 49 min head 57 137 69 128 53 VSP 58* 176 104 180 56 End 63 460 73 443 57 VSP 64 190 74 201 36 - 65 61 76 59 30 - 66 214 77 208 43 - 70 78 79 78 31 - 71 54 81 51 31 - 73 359 82 358 60 maj head 75 173 84 174 62 - 77 156 85 146 57 VSP 79 737 87 1041 52 TMP 82 216 90 199 56 dNK 84 89 91 95 30 - 86 160 94 158 50 - 88 371 97 371 70 Carbpep 89* 260 99 239 59 VSP 91 96 42 86 53 - 93 147 101 142 28 VSP 94 376 102 378 45 - 95 955 103 1431 43 tail fibre

Chapter 4. Phage genomic and proteomic characterisation 78

Table 4.6: Similar genes. Similar gene pairs listed are those which could also mutually identify one another, but were not mutual top hits in the blastp searches. +, with the exception of A-HIS2 ORF 36. Details as in Table 4.5.

A-HIS1 A-HIS2 % identity Putative function

ORF Size (aa) ORF Size (aa)

13 215 36/104 235/180 12/52 End+ 17 182 14/48 58 176 36 235 15 41 55 4 60 29 - 43 83 42* 86 46 - 76 127 45 133 24 - 91 96 54* 83 44 -

23 ORFs of each phage to be assigned a putative function (Table 4.2 and Table 4.3), i.e. 24 % (A-HIS1) and 22 % (A-HIS2).

4.3.6.2 A-HIS1/2 vs other siphoviruses

Phage taxonomy has aroused much discussion in recent years and there has been growing support for a comparative genomics led-approach to un- derstandingCaudoviralesdiversity (Nelson, 2004). However, new method- ologies for taxonomy are being highlighted along with the dilemmas of ex- isting methods, since there does not appear to be a unifying taxonomic method (Proux et al., 2002). In fact, comparative analyses of the Acary-

ochlorisphage genomes showed they have a unique composition and organ-

isation compared to other siphoviruses. Seguritan et al. and Br ¨ussow and Desiere have shown that other siphoviruses appear to have some conserva- tion of gene order which is not the same as that found in theAcaryochloris phages (Br ¨ussow and Desiere, 2001; Seguritan et al., 2003). Another fea- ture of other siphoviruses from these genome comparisons is that each has a cluster of genes comprising most or all of the following genes: a large ter- minase, a small terminase, a portal protein, a protease and a major head protein. These genes occur very close or next to each other in more or less the same order in the examples presented.

also contain novel genes found in no other phages like an RNase T gene. In light of all these observations it would be premature to place them into an existingSiphoviridaegenus.

4.3.6.3 A-HIS1/2 vs strain MBIC11017

The genome of strain MBIC11017 was recently sequenced and there are a number of phage-related genes in the A. marina MBIC11017 genome, mainly consisting of tail-related proteins, a phage lysozyme and many in- tegrases which are commonly associated with temperate phage. However, from a blastp analysis it seems that none of these genes are related to genes in the phages A-HIS1 and A-HIS2. Moreover, unlike the situation

withSynechococcusandProchlorococcusphages, no photosynthesis-related

genes were found in phages A-HIS1 and A-HIS2. This property may ex- tend to otherAcaryochlorisphages and may be because they do not infect a host which is found in high-light conditions where genes such aspsbA are thought to provide increased fitness under high irradiance levels (Bragg and Chisholm, 2008; Hellweger, 2009).