DNA pol γ : CAMERA assemblies

Of the 12 assemblies which contained an A. marina phage mtDNA poly- merase γ-like sequence, four contained other predicted genes with signifi- cant BLAST hits besides the mtDNA polymerase, which may be of bacterial or phage origin: A01ORF4, A04ORF3, A05ORF9 and A06ORF10 (Table 4.9 and Table 4.10). It was noted in assembly A05 that ORF A05ORF8 had a blastp hit to S-PM2p158 which encodes a rare lipoprotein A, which has not been well-characterised in the literature. S-PM2 is the only phage which encodes this gene, which is predominantly found in bacteria and eukary- otes, which suggests that A05ORF8 may not be of phage origin. In addition, the top BLAST hits for A05ORF8 were bacterial, which suggests this gene and thus the assembly it belongs to, may be of bacterial origin. Assemblies A01 and A04 may be of phage origin based on the ORFs identified. For now, the four assemblies A01, A04, A05 and A06 are assigned putative bacterial or phage origin until further evidence might suggest otherwise. Unlike

assemblies A05 and A06, A01 and A04 contained mtDNA polymerase γ

sequences which did not contain the DNA pol A domain and so were not in- cluded in the phylogenetic tree (Fig. 4.8). An analysis of the accompanying assembly metadata indicated the size fraction that contained the CAMERA mtDNA polymerase sequences was the 0.1–0.8µm filter fraction which was reported to be the sample that concentrated mostly bacteria and archaea (Rusch et al., 2007; Yooseph et al., 2007). However, it cannot be ruled out that this fraction may have contained viral sequences.

This analysis suggests that these environmental fragments of DNA may have originated from either phages or extant marine bacteria which are descendants of protomitochondrion-like bacteria. Indeed, if these environ- mental sequences came from an extant lineage of bacteria, the phylogenetic

Chapter 4. Phage genomic and proteomic characterisation 94

Table 4.9: ORFs from CAMERA assemblies. Blastp analysis of ORFs from CAMERA en- vironmental assemblies. DNA polymerase gamma ORF numbers highlighted in bold. AxORFy denotes Assembly x ORF y (the assembly numbering was arbitrary).

Assembly Assembly ID Location (region, country, habitat)

ORF Comment Putative origin

A01 1096627020476 Lake Gatun

(Panama Canal, Panama, fresh- water)

A01ORF1 similar to hypo-

thetical protein S- PM2p148/gp191 phage syn9 Bacterial or phage A01ORF2 -

A01ORF3 hypothetical protein; various hits including bacterial (specifically proteobacterial), ar- chaeal, phage, eukary- ota and phycodnaviridae

A01ORF4 DNA polymerase

gamma (no DNA pol A domain)

A02 1096627024361 A02ORF1 DNA polymerase

gamma (complete DNA pol A domain, possibly incomplete gene)

Unknown

A03 1101667175471 A03ORF1 DNA polymerase

gamma (partial DNA pol A domain)

Unknown

A04 1096627138623 Punta Cor-

morant, Hyper- saline Lagoon, Floreana Island (Galapagos Is- lands, Ecuador, hypersaline)

A04ORF1 putative high light inducible protein, cyanobacterial hits

Bacterial or phage

A04ORF2 similar to hypo-

thetical protein S- PM2p148/gp191 phage syn9

A04ORF3 DNA polymerase

gamma (no DNA pol A domain)

A05 1096627359208 A05ORF1 - Bacterial or

phage A05ORF2 - A05ORF3 - A05ORF4 - A05ORF5 - A05ORF6 -

A05ORF7 DPS (DNA Protecting protein under Starved conditions) domain, superfamily of ferritin- like diiron-carboxylate proteins

A05ORF8 rare lipoprotein A (DPBB 1 superfamily); cyanobacterial and other bacterial hits, also hit cyanophage S-PM2p158

A05ORF9 DNA polymerase

gamma (complete DNA pol A domain, possibly incomplete gene)

Assembly Assembly ID Location (region, country, habitat)

ORF Comment Putative origin

A06 1096627384653 Punta Cor-

morant, Hyper- saline Lagoon, Floreana Island (Galapagos Is- lands, Ecuador, hypersaline)

A06ORF1 top hit serine/threonine protein phosphatase (Thermotoga neopoli- tana) Bacterial or phage A06ORF2 -

A06ORF3 Partial P-loop NTPase superfamily domain, top hits bacterial, less sig- nificant hits -phage

A06ORF4 Partial PP2Ac su-

perfamily domain

- bis(5’-nucleosyl)- tetraphosphatase PrpE, bacterial

A06ORF5 -

A06ORF6 eukaryota hits including methyl-CpG binding do- main proteins, no pu- tative conserved domain detected

A06ORF7 -

A06ORF8 -

A06ORF9 -

A06ORF10 DNA polymerase

gamma (complete DNA pol A domain, possibly complete gene)

A07 1096627182099 Rangirora Atoll

(Polynesia Archipelagos, Fr. Polynesia, Coral Reef Atoll)

A07ORF1 - Unknown

A07ORF2 -

A07ORF3 -

A07ORF4 -

A07ORF5 DNA polymerase

gamma (partial DNA pol A domain)

A08 1096627376200 A08ORF1 DNA polymerase

gamma (complete DNA pol A domain, possibly incomplete gene)

Unknown

A09 1096628021027 A09ORF1 DNA polymerase

gamma (partial DNA pol A domain)

Unknown

A09ORF2 -

A09ORF3 -

A10 1096628023999 A10ORF1 Fungal DNA polymerase

gamma hits containing DnaQ like exonuclease superfamily domain

Unknown

A10ORF2 DNA polymerase

gamma (partial DNA pol A domain) A11 1101668141779 Chesapeake Bay, MD (North American East Coast, USA, estuary)

A11ORF1 DNA polymerase

gamma (partial DNA pol A domain)

Unknown

A12 1101668711231 Cabo Marshall,

Isabella Island (Galapagos Is- lands, Ecuador, coastal)

A12ORF1 DNA polymerase

gamma (partial DNA pol A domain)

Chapter 4. Phage genomic and proteomic characterisation 96

tree in Fig. 4.8 shows that they do not fall within the bacterial clade and therefore represent a new phylum of bacteria according to this gene.