BACKGROUND
zz <dig>  a novel sequenced lytic phage that can efficiently infect acinetobacter baumannii clinical strains, was identified from fishpond water in zhengzhou, china  <cit> .
as is typical for t4-like virion morphology, the zz <dig> viral particle contains an isometric head and a contractile tail, which are visible with transmission electron microscopy.
the phage was reported to have different antibacterial activity against three a. baumannii clinical strains .
of the host bacteria, ab09v is the most sensitive, and zz <dig> causes large and distinguishable plaque formation on a lawn of ab09v and is highly infectious with a short latent period  and a large burst size   <cit> .
to date, the genomes of  <dig> acinetobacter phages have been completely sequenced and published in the ncbi genome database.
five belong to the myoviridae phage group, including  <dig>  acj <dig>  acj <dig>  and ac <dig>  and four belong to the siphoviridae phage group, including abp <dig> , bphi-b <dig> , ab <dig>  and ymc/09/02/b1251_aba_bp .
of the remaining  <dig> phages, ab <dig>  belongs to the podoviridae phage group; ap <dig> , an ssrna virus, belongs to the levivirus group; and the phage ime-ab <dig>  is unclassified.
an initial ncbi nucleotide blast analysis  of the complete genome sequence indicated that zz <dig> shares limited similarity with other sequenced phages.
the sequences from  <dig> acinetobacter phages, acj <dig> , acj <dig> , ac <dig> , and  <dig> , were the most similar to that of zz <dig>  <cit> .
the four acinetobacter phages were recently deposited in genbank and were previously annotated as t4-like phages  <cit> .
the t <dig> phage superfamily is one of the best-characterised groups of escherichia coli phages  <cit> .
most of the known t4-like phages specifically infect certain strains of e. coli or other enterobacteria, but several t4-like phages can propagate in bacteria that are more phylogenetically distant, such as vibrio, aeromonas, cyanobacteria, and acinetobacter .
although many t4-like phages have been sequenced, only a limited number of these phage genomes have been analysed in as much detail as the t4-like acinetobacter phages.
previous cross-genome comparisons of t <dig> and other t4-like phages  revealed that this family of phages shares a common core genome from an ancestral sequence encoding the dna replication modules, virion structural proteins, and some conserved predicted proteins.
furthermore, some of the proteins with unknown functions are conserved in t4-like phage genomes, and some of the minor differences between these phages may be related to their adaptations to different host ranges  <cit> .
a previous study found that each of the 4 t4-like acinetobacter phages, acj <dig>  acj <dig>  ac <dig>  and  <dig>  has a unique set of orfs that occupy ~35% of the genome.
in other words, each represents a different type of t4-related phage genome  <cit> .
however, to the best of our knowledge, no detailed bioinformatics analysis and comparative genomic analyses of zz <dig> compared to t <dig> and the other acinetobacter phages have been reported.
although detailed information about the relationship between the hosts of zz <dig> and the hosts of the other  <dig> acinetobacter phages is not available from the ncbi database and the current literature, it has been confirmed that the hosts of the  <dig> phages belong to acinetobacter .
these phages may have evolved from a common ancestor and switched hosts during their evolution.
the similarities and the differences between them can improve our understanding of their evolutionary strategy.
more importantly, it would be useful to identify regions that are variable between the genomes of different t4-like phages that might underlie host-specific adaptations.
here, we describe the genome organisation of the phage zz <dig>  which infects a. baumannii yet shares a common ancestor with the e. coli phage t <dig>  the comparative genomic analyses of zz <dig>  t <dig>  and the other 4 t4-like acinetobacter phages not only provides insights into viral diversity and evolution but also offers an exciting opportunity to understand the host-specific adaptation mechanism of these phages.table  <dig>
general genome features of zz <dig>  t <dig> and the other completely sequenced t4-like
acinetobacter
phages
RESULTS
annotation of the zz <dig> genome
sequence assembly yielded a closed, circular sequence with 114 bp inverted terminal repeats, indicating that zz <dig> phage particles contain linear, circularly permuted genomic dna, similar to phage t <dig> particles  <cit> .
the single copy genome is  <dig>  bp.
bioinformatics methods identified  <dig> putative protein-coding sequences.
the lengths of these cdss range from  <dig> to  <dig>  amino acid residues  and average  <dig> aa.
there are approximately  <dig>  genes per kbp, and  <dig> % of the zz <dig> genome is predicted to encode proteins .
atg  was the predominant initiation codon.
only  <dig> cdss began with gtg, and  <dig> began with ttg.
the predominant termination codons were taa  and tga .
we noted that most of the significant blastp hits  were proteins from phages , and the remaining hits were unknown proteins encoded by bacteria and other organisms.
of the  <dig> cdss from zz <dig>   <dig> were assigned a functional annotation .
only  <dig>  have no blastp matches in the non-redundant protein sequences database, indicating that genes encoding new viral proteins were revealed by the characterisation of this phage.
however, further batch cd-search analyses suggested that  <dig> of the  <dig> genes with functional annotations lack protein domain information.
of the  <dig> cdss,  <dig> were conserved hypothetical proteins, and only  <dig> of the proteins  had protein domain information .
further functional analysis revealed that the  <dig> cdss assigned putative functions could be classified into  <dig> functional categories according to previous descriptions of phage t <dig>  <cit>  .
most of the named functional proteins are highly conserved among t4-like phages and are either structural  or involved in dna replication, recombination, repair, packaging, and processing  .figure  <dig>
annotated genome map of phage zz <dig>  cdss are labelled with the name of the closest homolog, and thus t <dig> nomenclature is primarily indicated .
the numbers below the cds arrows are the cds ids used in the text, tables, and genbank.
following convention, the map starts at the bottom left with the riia gene and ends at the bottom right with the riib gene.
the individual cdss are depicted as arrows, with the orientation of the arrows indicating whether the genes are on the watson or crick strand.
the colour of the arrow identifies the functional category into which the homologous t <dig> gene was classified  <cit> .
the colour code for gene function is provided in the bottom of the figure.
summary of the  <dig> zz <dig> genes annotated by blastp and batch cd-search analyses.
pie chart showing the relative abundance of genes with and without named functions, including  <dig> kfp ,  <dig> chp ,  <dig> chp-d , and  <dig> hp  genes.
relative abundance of zz <dig> proteins in  <dig> functional categories: a, transcription; b, translation; c, nucleotide metabolism; d, dna replication, recombination, repair, packaging, and processing; e, virion proteins; f, chaperonins/assembly catalysts; g, lysis; h, host or phage interactions; i, host alteration/shutoff; j, homing endonucleases and homologs; k, predicted membrane spanning proteins in unknown function proteins; l, unknown function.
comparative genomics of t <dig> and zz1
the determination of phage relatedness is not based exclusively on sequence similarity but also takes genome organisation into consideration  <cit> .
the phage zz <dig> has similar virion morphology  <cit> , genome size and number of cdss compared to the coliphage t <dig>  .
to further investigate the genomic similarities of t <dig> and zz <dig>  we constructed comparative genome maps of zz <dig> and t <dig>  the putative gene riia from zz <dig> was positioned on the minus strand  and at the start of the map, following the t <dig> convention.
homologous genes are projected onto the t <dig> map, and the projections are colour-coded based on protein sequence similarity .
blastp analysis indicated that the zz <dig> cds <dig> shows significant similarity to the putative vs gene  from the other 4 t4-like acinetobacter phages, acj <dig>  acj <dig>   <dig>  and ac <dig> .
however, when we compared the zz <dig> cds <dig> to the set of all t <dig> proteins by aligning two or more sequences from blastp using the default parameters, no counterpart was found in the results.
further blastp analysis revealed that, of the  <dig> acinetobacter phages, only phage acj <dig> vs showed a slight similarity to the t <dig> vs .
similar results were observed in the annotation of zz <dig> cds <dig>  which showed significant similarity to the putative 57a  from phages ac <dig> and acj <dig> although no counterpart was found in the blastp results when we compared zz <dig> cds <dig> to the set of all t <dig> proteins.
thus, we annotated zz <dig> cds <dig> and  <dig> as hypothetical proteins , and of the  <dig> cdss from zz <dig>  only  <dig>  could be assigned a functional annotation, and  <dig>  were conserved hypothetical proteins .
overall, such gradual evolution of the t <dig> genes between these closely and distantly related phages may eventually provide some interesting insights into the particular steps of genome divergence.figure  <dig>
t4-zz <dig> comparison genomic mapping and zz <dig> orthologous genes from the other 4
acinetobacter
phages.
the genomes and coding regions of t <dig> and zz <dig> are drawn in parallel approximately to scale.
alignment of different genes is from t <dig>  and from zz <dig> .
following convention, the map starts at the top left with the riia gene and ends at the bottom right with the riib gene.
horizontal arrows indicate transcription direction.
the likely function of the genes is indicated by the colour of the arrows as described in figure  <dig>  genes sharing protein sequence identity are linked according to the colour key provided at the middle left.
zz <dig> genes are annotated randomly with their gene order in the genbank database.
the map was artificially split into upper halves and lower halves.
zz <dig> genes that are orthologous to the  <dig> other acinetobacter phages are indicated by coloured boxes under the corresponding zz <dig> genes.
the amino acid sequence identity of zz <dig> genes to these orthologous genes was also indicated by colour as described in the key provided at the upper right.
the % coverage of zz <dig> genes to these orthologous genes is indicated by the grey boxes under each coloured shade or box according to the grey scale key at the upper right.
the genes indicated by the grey vertical arrows share significant similarity with proteins from the  <dig> other acinetobacter phages, but share no significant similarity with t <dig> proteins.
notably,  <dig> of the  <dig> zz <dig> cdss  shared up to 73% amino acid sequence identity with t <dig> proteins  .
long blocks of synteny containing homologous genes are interspersed with stretches that lack homology.
the lower halves of the genome, which are closely related, include three separate clusters of rightward-transcribed genes, specifically:  a cluster of base plate wedge/head/tail genes ,  a smaller base plate hub gene cluster in its vicinity , and  further away, a tail fibre gene cluster consisting of a few large genes .
two clusters of leftward-oriented non-structural genes separate the  <dig> rightward-oriented clusters and mainly encode proteins involved in nucleotide metabolism.
the degree of sequence identity varied substantially.
the degree of sequence conservation did not follow structural/non-structural gene divisions.
the region that displays the most significant difference between zz <dig> and t <dig> is located in the upper halves of the genome that exclusively encode leftward-oriented, non-structural genes , including a large dna replication module.
large regions of nonaligned genome segments were thought to have resulted from genetic gains, losses, or replacements, i.e., events similar to those that have shaped the evolution of all microbial genomes in nature.
for example, zz <dig> riia.
<dig> ,  <dig>  and moba are flanked by  <dig> unknown genes and  <dig> genes were deleted between zz <dig> vs. <dig>  and segb .
further alignment and comparison of t <dig> genes that are homologous to zz <dig> revealed that the order and transcriptional direction of t <dig> genes are broadly maintained in the zz <dig> genome, which helped provide the phylogenetic distance between zz <dig> and t <dig>  there were three major differences: 1) the rnlb gene was at the junction of  <dig> in the lower genome half in t <dig> but was adjacent to the cds <dig> in the upper genome half of zz <dig> ; 2) the topoisomerase gene  and the dna polymerase gene  were split and intact, respectively, in t <dig> but intact and split, respectively, in phage zz1; and 3) a cluster of alt genes including three putative alt-like genes differs between t <dig> and zz <dig>  as shown in figure  <dig>  the transcriptional direction of the three zz <dig> alt-like genes was different from the t <dig> alt, revealing an unusual genomic organisation for the acinetobacter phage zz <dig>  moreover, the first alt counterpart  is  <dig> aa in length and shares 98% coverage and 28% amino acid identity with the t <dig> alt .
the second alt-like gene  is  <dig> aa in length and shares 35% amino acid identity with the t <dig> alt segment that extends from  <dig> to  <dig> amino acids, and the third alt analogue  is  <dig> aa in length and shares 33% amino acid identity with the t <dig> alt segment that extends from  <dig> to  <dig> aa.
apparently, the last two zz <dig> alt-like genes resulted from an even splitting event from the ancestral alt gene .
the duplication of the alt gene was also present in the genomes of the acinetobacter phage ac <dig> , the coliphage js <dig> , and the enterobacteria phage bp <dig> , but the splitting of the alt gene was not observed in other t4-like phages.figure  <dig>
divergence of the core gene organisation between t <dig> and t4-like
acinetobacter
phages.
the map was artificially split into upper and lower halves.
the bars indicate identified orfs and are drawn to scale.
genes transcribed in the forward direction  are displayed above those genes transcribed in the reverse direction .
the functional category of t <dig> genes is indicated by the color-coded bars described in figure  <dig>  the numbers and acronyms shown directly above the color-coded bars refer to the gene names.
core and quasicore genes described by petrov et al.
<cit>  are shown in red and blue font, respectively.
homologous signature genes, shared by t <dig> and the 5 t4-related acinetobacter phages, are connected with light blue shading.
conversely, homologous unknown genes, which shared significant similarity with all 5 t4-related acinetobacter phages but no significant similarity with t <dig> proteins, are linked by dark and light purple shading.
genes linked by dark purple shading are specific to the 5 t4-related acinetobacter phages in the genbank database.
in addition,  <dig> signature t <dig> genes, which are linked by green shading, could reflect a dominant feature in the evolution of all 5 t4-related acinetobacter phages.
t4-like phages were often shaped by gene duplications.
for example, in t <dig>  genes  <dig> and  <dig> share 29% amino acid identity  <cit> , and  <dig> proteins  were identified as likely to have been duplicated in the kvp <dig> lineage  <cit> .
to further examine duplications, we aligned genes from zz <dig> with other zz <dig> genes using blastp at the amino acid level and blastn at the dna level.
those with better matches  were considered candidates for lineage-specific duplications.
using this method,  <dig> proteins were identified as likely to have been duplicated in the zz <dig> lineage .
we further reasoned that if the homologous genes are the result of duplication of an ancestral gene, and if the different degrees of sequence diversification of these duplicated genes resulted from the accumulation of nucleotide point mutations resulting from differential selection pressure, then the genes with lineage-specific duplications should have significant nucleotide sequence identities.
however, although the duplicated genes have significant amino acid identities, no significant nucleotide sequence identities were observed.
thus, the similarity of the genes is more likely to be the result of convergent evolution than the result of duplication of an ancestral gene.
for example, zz <dig>  <dig> and  <dig> share 25% amino acid identity but negligible nucleotide sequence identity .
the  <dig> protein plays an interesting role in t <dig> head maturation.
the t <dig> prohead consists of an outer shell  and an inner scaffold .
when  <dig> is added to the prohead, maturation cleavage occurs:  <dig> loses its n-terminal portion, whereas  <dig> is degraded completely  <cit> .
in addition, in phage kvp <dig>  the duplication of three genes encoding proteins associated with the phage tail or the tail fibre  suggests added flexibility in the range of host adaptation and the infection process  <cit> .
in our study, duplication was observed in three putatively duplicated gp <dig>  homologues , which encode tail fibre-associated proteins and share 29-30% amino acid identity with zz <dig> gp <dig>  overall, splitting and/or duplication followed by sequence diversification and new gene insertion might be a mode of t4-like phage evolution.
similarities and distinctions in the genome features of the e. coliphage t <dig> and the t4-like acinetobacterphages
alignment of the zz <dig> genome with the t4-like phages in the tulane database showed that zz <dig> was closely related to  <dig> acinetobacter phages.
based on the total search score, the closest was acj <dig>  followed by acj <dig>  ac <dig>  and  <dig>  no other acinetobacter phages were uncovered.
in dna sequence dot plots, we observed a frequently interrupted but straight diagonal line between zz <dig> and the  <dig> phages .
overall, their genomes are colinear but are frequently interrupted by replacements with unrelated genome segments of comparable lengths, especially in the first halves of their genome.
further comparison of these genome sequences  at the dna level using mauve showed remarkable synteny in five large conserved regions  .
the most conserved syntenic region covered the morphogenesis module as well as the dna replication and metabolism module, which are consistent with previous analyses  <cit> .figure  <dig>
multiple genome alignment and the genomes of zz <dig> and 5 t4-like phages.
six genomes were compared using mauve software, and similarity profiles were generated.
boxes with identical colours represent local colinear blocks  indicating homologous dna regions shared by two or more genomes without sequence rearrangements.
the relative locations of orthologous genes, shown by open boxes, reveal a high degree of synteny.
white regions within lcbs represent nonidentical sequences.
protein-by-protein comparison of the  <dig> phages using blastp and coregenes revealed that zz <dig> shares  <dig>  protein homologues with acinetobacter phage acj <dig>   <dig>  with acj <dig>   <dig>  with  <dig>  and  <dig>  with ac <dig>  although zz <dig> shares only  <dig> protein homologues with coliphage t <dig>  in contrast,  <dig> protein homologues are present in t <dig> and in all  <dig> acinetobacter phages, whereas  <dig> homologous genes are present in all  <dig> of the acinetobacter phages , which is further evidence that zz <dig> and the  <dig> other acinetobacter phages belong to the same genus .
the full portrait of the t <dig> phage superfamily that has emerged from the many reported genomic comparisons  is that the t <dig> superfamily can be distilled down to core signature genes, which are known as the “core genome” of the t4-related phages or t4-like viruses  <cit> .
the core genome primarily includes homologues of essential t <dig> genes, such as the virion structure and dna replication genes.
in contrast, the hyper plastic regions  contain mostly novel genes of unknown function and origin  <cit> .
the core genome of the t4-related phages has been considered to consist of two genetic components: one highly resistant component was termed the core genes, which are essential for all known conditions; and the other component, which is somewhat permissive to attrition in evolution, was termed the quasicore genes and can be substituted or circumvented in certain genetic backgrounds of phage and/or bacterial hosts  <cit> .
the genetic background for the core genome can vary considerably between t <dig> relatives, and thus, the number of the core genes that constitute the core genome depends on the precise subset of phages considered, which constitutes an important criterion for distinguishing between close and distant relatives  <cit> .
a closer look at the components and organization of the core genome in the 6 t4-like phages  shows that all  <dig> acinetobacter phages share a core genome with t <dig> interrupted by several hprs, which are where most of their divergence occurs.
this finding is consistent with the previous observations of t4-like phages  <cit> .
all of the core genes described by petrov et al.
exist in all 5 t4-related acinetobacter phages and are indicated by the red font directly above the coloured genes bars in figure  <dig>  the topology of the set of t <dig> phage core genes is also shared by all 5 t4-related acinetobacter phages.
moreover, we observed that almost all the quasicore genes described by petrov et al.
were shared by all 5 t4-related acinetobacter phages, except for uvsx , uvsy , nrdc , nrdg , tk ,  <dig> , cd , and dena .
these genes are permissive to attrition in evolution of all 5 t4-related acinetobacter phages .
in addition, nearly half of the other t <dig> signature genes  are shared by all 5 t4-related acinetobacter phages, including the gene topology.
thus, all 5 t4-related acinetobacter phages use similar mechanisms to control propagation in their hosts.
these phages all shared a common ancestral genome but, during the course of evolution, they have modified it in numerous ways.
these conserved t <dig> genes allowed the 5 t4-related acinetobacter phages to conserve their highly successful virion design and mode of replication.
notably, several major distinctive genome features in all 5 t4-related acinetobacter phages  could reflect a dominant feature in the evolution of all 5 t4-related acinetobacter phages.
the most significant difference between the 5 t4-related acinetobacter phages and t <dig> is in a series of hprs, which are interspersed between the conserved t <dig> core sequences and especially enriched in the upper genome halves of all 5 t4-related acinetobacter phages.
these regions vary greatly in gene number and content.
the hprs were primarily composed of genes of unknown origin, but they do contain some identifiable sequences from bacteria and unrelated phages.
thus, the hprs have been predicted to be the result of an evolutionary history of isolation within distinct hosts and extensive lateral gene transfer , i.e., importing genes or exchanges with diverse biological entities in nature  <cit> .
our observations showed that all of the genes lack t <dig> homologues in the 5 t4-related acinetobacter phage genomes and cannot be assigned a putative function, most of which likely appear to be homologous genes from other phages, bacteria or other organisms.
notably, of the  <dig> zz <dig> genes that have significant homology with genes present in  <dig> of the acinetobacter phages,  <dig> have unknown functions.
of these  <dig> genes,  <dig>  which are indicated by the vertical grey arrows in figure  <dig> , share no significant similarity to t <dig> proteins.
moreover, most of these homologous genes maintain a consistent order and content in the 5 t4-related acinetobacter phages .
further, blastp analyses suggested that  <dig> of the  <dig> zz <dig> genes , whose counterparts are linked with dark purple shading in figure  <dig>  lack significant matches to any of the phage sequences in genbank except for the  <dig> other acinetobacter phages  and a few other organisms .
in particular,  <dig>  share high similarities  with the other  <dig> acinetobacter phages.
the diversity among phage “pangenome”  is a reflection of the adaptations of a phage ancestor to a variety of evolutionary challenges, including encountering new host environments.
lgt most certainly has played a role in such adaptations, and these adaptations may have been facilitated in part by certain particularities of the t <dig> phage recombination and gene expression systems  <cit> .
however, to date, there are few clues about the agents that might mediate such a transfer.
t <dig> recombines most efficiently early in the infection before the host genome is degraded, and this process could facilitate the acquisition of host genes by the phage  <cit> .
as little as 50 bp of homology  is sufficient for t <dig> recombination system to recombine at reasonable frequencies  <cit> .
thus, “semilegitimate” recombination that relies on the small but generally conserved regulatory signals in the intergenic regions  could perhaps have mediated the acquisition of such foreign genes  <cit> .
once the foreign genes are acquired, the t <dig> expression system could exploit their endogenous bacterial promoters because these sequences are very similar to the early promoter sequences of the t4-type phages  <cit> .
overall, different phages sharing a common ancestor undergo a specific and similar adaptation process for different bacterial strains that belong to the same genera or species.
the existence of these highly conserved unknown homologous genes specific to acinetobacter phages in hprs could reflect the complex interactions of acinetobacter phages with conserved cell components that are specific to acinetobacter bacterial hosts and are distantly related to e. coli or other bacterial hosts.
theoretically, the uniqueness of homologous genes or certain sequences in specific phage genomes or lineages might help distinguish between the different clusters or types of phage and help predict the bacterial host range when treating the corresponding bacterial genera clinical isolates.
additional research is required to elucidate the highly conserved genes that are specific to acinetobacter phages.
gc skews in the zz <dig> genome
phage zz <dig> dna contains only  <dig> % gc, which is slightly lower than the value  observed in sequenced a. baumannii strains .
to date, it is the lowest reported gc content for sequenced t4-like acinetobacter phages .
the gc content of zz <dig> is comparable with that of the enterobacteria phage t <dig> .
the mol% gc of the t <dig> genome is also substantially lower than that of its host   <cit> .
the difference is in disagreement with previous studies suggesting that the gc content of phage genomes such as the mycobacteriophages  <cit>  and staphylococcus aureus phages  <cit>  have a gc content similar to their respective hosts.
although zz <dig> has a lower gc content,  <dig> of the predicted phage zz <dig> genes contain more than 40% gc, including one major head gene , three tail fibre genes , one nucleotide metabolism gene , and seven unknown genes.
notably, the gc content of the zz <dig>  <dig> gene is  <dig> %, which is significant lower than the local gc content of t <dig>  <dig>  and the t4-like phage js <dig>  <dig>   <cit> .
gene  <dig> has the highest proportion of codons that are translationally optimal for the host  in keeping with its very high level of expression; approximately  <dig>  copies of the protein are needed per synthesised phage particle  <cit> .
interestingly, the gc content of the zz <dig> host, a. baumannii strains, is approximately 39% and is lower than the gc content of the host strains for t <dig> and js <dig>  which is  <dig> %.
if the gc content of phages and their hosts become more similar during extended co-evolution, the difference between the gc content of zz <dig> gene  <dig> and the gc content of gene  <dig> in the phage t <dig> and js <dig> presumably reflects the differences in host range, gc content, and life history of the respective hosts.
thus, the low gc content of zz <dig>  <dig> suggests that phage zz <dig> might have acquired the ability to infect a. baumannii strains over a long period of time.
one region was found to be devoid of cdss, extending from  <dig>  to  <dig> .
in an effort to determine whether this region served as the replication origin, we used gc-skew analysis  <cit> .
a putative origin of replication in the region of nt  <dig> and a putative terminus of replication in the region of nt  <dig> were revealed .
the putative origin of replication is close to but not within the cds-devoid region.
however, trna detection showed that the trna genes from phage zz <dig> were encoded between cds <dig> and  <dig>  and  <dig> clusters of trnas  were located near the three adjacent genes, cds <dig>   <dig>  and  <dig>  the two regions were in this cds-devoid region .
a previous study revealed that the phage zz <dig> had a very short latent period of 9 min  <cit> .
the large number of trna genes and their strategic locations might enable the phage to translate its sequence more efficiently, reducing its latency time and increasing its reproduction rate and thus its infectivity.figure  <dig>
cumulative gc skew analysis of the genome sequence.
the global minimum and maximum are displayed in the cumulative graph.
the minimum and maximum of a gc-skew can be used to predict the origin of replication  and the terminus location .
codon usage and trnas
eight trna genes were predicted in the zz <dig> genome .
the t <dig> genome also has  <dig> trna genes, whereas ac <dig> has slightly fewer , and others have substantially more including phage  <dig> , acj <dig> , and acj <dig> , as shown in figure  <dig>  the different numbers and types of trnas present in the  <dig> acinetobacter phages are consistent with a previous study of trnas in t4-like phages  <cit> .
in addition, although zz <dig> has a low mol% gc content ,  <dig>  out of the  <dig> trnas recognise codons with a in the third position .table  <dig>
trna genes from zz <dig> and homologous trnas from  <dig> other
acinetobacter
phages and t4
a
b
atwo trna scan tools, trnascan-se and aragorn, were used for prediction.
all of the predicted trna genes are located on the minus strand of the zz <dig> genome.
btrnas homologous to zz <dig> in other phages revealed by nucleotide comparison  are also located on the minus strand of the phage genomes.
codon usage and trna availability in
acinetobacter
phages.
the total number of codons is  <dig>  for phage zz <dig> ,  <dig>  for phage acj <dig> ,  <dig>  for phage acj <dig> ,  <dig>  for phage ac <dig> , and  <dig>  for phage  <dig> .
optimal codons that have relative phage-encoded trna are indicated with black boxes.
phage-encoded trnas related to the highly used codons are shaded grey, as is the corresponding codon.
the exact function of phage-encoded trnas in the phage-infected cell is still not clear.
they might be involved in the adaptation of the host translation apparatus to the demands of the phage codon usage pattern.
in other words, the trnas overcome the phage codon usage problem, and the presence of trnas in a phage genome has been suggested to compensate for differences in codon usage between the phage and the host, corresponding to codons that are expected to be poorly translated by the host machinery  <cit> .
previous studies indicated that t4-encoded trnas are related to codons that are highly used in t <dig> genes but rarely used in the host and that the phage trnas can enhance the low expression of t <dig> late-stage protein genes through optimal codon usage in translation  <cit> .
similarly, analysis of the zz1-encoded trnas indicated that over half of them  might be related to the optimal codon usage of zz <dig> proteins .
however, the codon usage frequencies of the  <dig> other acinetobacter phages suggested that phage-encoded trnas might be unrelated to decoding the relatively more frequent codons in all of the genes .
thus, the functional role of the trna genes for these phages remains unclear.
although the types and numbers of phage-encoded trnas differ between the  <dig> phages, some consistency was observed.
for example, all  <dig> phages encode met-trna and trp-trna, which are related to optimal codon usage, but arg-trna, which was not related to optimal codon usage, was also common to all  <dig> phages .
of the  <dig> trna genes in zz <dig>   <dig>  shared up to  <dig> and 90% nucleotide sequence similarity to trnas from phage acj <dig> and phage  <dig>  respectively;  <dig>  shared up to 89% nucleotide sequence similarity to the trnas from phage acj61; and only  <dig> trna showed 83% sequence similarity with the trna-thr from phage ac <dig> .
in addition, the zz1-encoded trna-thr gene shared 83% identity with the t4-encoded trna-pro gene .
the high degree of conservation of some of the trnas suggests an important functional role.
the number and type of homologous trna appears to confirm the relationship between zz <dig> and the other phages, suggesting some elements of vertical evolution in these trnas.
mobile elements
the homing endonuclease genes are not genuine phage dna; rather, they belong to intron-associated selfish dna elements  <cit> .
there are  <dig> seg,  <dig> mob, and  <dig> intron nuclease  genes in the t <dig> genome  <cit> , which have efficiently invaded the t <dig> genome and are also frequently found in the dna transaction module opposite the tail fibre cluster  and the dna replication module  .
inconsistent distribution of these elements has been described for t <dig>  t <dig> and t <dig> phages  <cit> .
one notable difference between the t <dig> and zz <dig> genomes is that zz <dig> appears to lack counterparts in the corresponding regions, except for two mobile genes, a seg-like gene and a mob-like gene.
the zz <dig> seg-like mobile endonuclease , which is located in the dna replication module, has  <dig> to 54% similarity to  <dig> seg genes in t <dig> .
however, the zz <dig> seg-like gene has higher similarity with the seg counterparts of the other  <dig> acinetobacter phages: 73% amino acid identity with the seg counterpart from acj <dig>  and  <dig> to 58% identity with seg counterparts from acj <dig>   <dig> and ac <dig>  the zz <dig> mob-like homing endonuclease , which was found in the cluster of neck genes, lacks sequence similarity with the corresponding t <dig> genes.
nevertheless it has 52% sequence similarity with the mob counterparts from acj <dig> and  <dig> to 39% sequence similarity with the mob counterparts from  <dig> and ac <dig>  the other  <dig> acinetobacter phages also lack mobile elements in the corresponding locations.
although each of them has two to four seg genes, no mob-like genes were found in phage acj <dig>  only one mob-like gene was found in phage  <dig> and ac <dig>  and only two mob-like genes were found in acj <dig> .
t <dig> has three self-splicing group i introns, one each in td, nrdb, and nrdd  <cit>  .
a few group i-like introns have been found in other phages, such as t <dig>  the s. aureus phage twort  <cit> , the bacillus phages i-basi and i-hmui  <cit> , the lactobacillus phage ll-h, and the lactococcus phage r1t  <cit> .
however, none of the three t <dig> intron nuclease genes were found in phage zz <dig>  acj <dig>  or acj <dig>  and only one i-tevi-like homing endonuclease from the giy-yig family was found in the  <dig> and ac <dig> genome .
these mobile element genes do not commonly exist in the t4-like phages, and the differences in mobile elements between the  <dig> acinetobacter phages and t <dig> phage genomes are consistent with the results for another t4-like phage  <cit>  apparently, there are ill-defined barriers to intron promiscuity in bacteriophages  <cit> .
CONCLUSIONS
we determined and revised the complete genome sequence of the phage zz <dig> that infects pathogenic a. baumannii strains.
a total of  <dig> potential proteins and  <dig> trnas were predicted.
the blastp analysis reveals that only a small portion of its proteins  are clearly related to coliphage t <dig> proteins and share up to 73% protein sequence identity with the corresponding t <dig> proteins.
further analysis revealed that  <dig>   <dig>   <dig>  and  <dig> proteins from zz <dig> share up to  <dig>   <dig>   <dig>  and 83% amino acid identity, respectively, with acinetobacter phage acj <dig>  acj <dig>   <dig>  and ac <dig> proteins, respectively.
nine zz <dig> genes lack significant matches to any of the phage sequences in the genbank database except for the  <dig> other acinetobacter phages.
in addition, the high degree of conservation of some trnas between zz <dig> and the other t4-like phages suggests that they may have an important functional role in addition to overcoming the phage codon usage problem.
the number and identity of the homologous trnas supports the evolutionary relationship between zz <dig> and the other phages, suggesting some elements of vertical evolution in these trnas.
overall, although more than 200 t4-like phages have been examined  <cit> , only a very limited number of the t4-like acinetobacter phage genomes have been explored or exploited.
as additional genome sequencing of acinetobacter phage species are completed, more acinetobacter phages similar to t <dig> might be discovered.
host-specific adaptation mechanisms might be revealed by a more comprehensive understanding of the genomic diversity within the acinetobacter bacteriophage population in the future.

 zz a novel sequenced lytic phage that can efficiently infect acinetobacter baumannii clinical strains, was identified from fishpond water in zhengzhou, china. as is typical for t4-like virion morphology, the zz viral particle contains an isometric head and a contractile tail, which are visible with transmission electron microscopy. the phage was reported to have different antibacterial activity against three a. baumannii clinical strains. of the host bacteria, ab09v is the most sensitive, and zz causes large and distinguishable plaque formation on a lawn of ab09v and is highly infectious with a short latent period and a large burst size. to date, the genomes of acinetobacter phages have been completely sequenced and published in the ncbi genome database. five belong to the myoviridae phage group, including acj acj and ac and four belong to the siphoviridae phage group, including abp, bphi-b, ab and ymc/09/02/b1251_aba_bp. of the remaining phages, ab belongs to the podoviridae phage group; ap, an ssrna virus, belongs to the levivirus group; and the phage ime-ab is unclassified. an initial ncbi nucleotide blast analysis of the complete genome sequence indicated that zz shares limited similarity with other sequenced phages. the sequences from acinetobacter phages, acj, acj, ac, and, were the most similar to that of zz. the four acinetobacter phages were recently deposited in genbank and were previously annotated as t4-like phages. the t phage superfamily is one of the best-characterised groups of escherichia coli phages. most of the known t4-like phages specifically infect certain strains of e. coli or other enterobacteria, but several t4-like phages can propagate in bacteria that are more phylogenetically distant, such as vibrio, aeromonas, cyanobacteria, and acinetobacter. although many t4-like phages have been sequenced, only a limited number of these phage genomes have been analysed in as much detail as the t4-like acinetobacter phages. previous cross-genome comparisons of t and other t4-like phages revealed that this family of phages shares a common core genome from an ancestral sequence encoding the dna replication modules, virion structural proteins, and some conserved predicted proteins. furthermore, some of the proteins with unknown functions are conserved in t4-like phage genomes, and some of the minor differences between these phages may be related to their adaptations to different host ranges. a previous study found that each of the 4 t4-like acinetobacter phages, acj acj ac and has a unique set of orfs that occupy ~35% of the genome. in other words, each represents a different type of t4-related phage genome. however, to the best of our knowledge, no detailed bioinformatics analysis and comparative genomic analyses of zz compared to t and the other acinetobacter phages have been reported. although detailed information about the relationship between the hosts of zz and the hosts of the other acinetobacter phages is not available from the ncbi database and the current literature, it has been confirmed that the hosts of the phages belong to acinetobacter. these phages may have evolved from a common ancestor and switched hosts during their evolution. the similarities and the differences between them can improve our understanding of their evolutionary strategy. more importantly, it would be useful to identify regions that are variable between the genomes of different t4-like phages that might underlie host-specific adaptations. here, we describe the genome organisation of the phage zz which infects a. baumannii yet shares a common ancestor with the e. coli phage t the comparative genomic analyses of zz t and the other 4 t4-like acinetobacter phages not only provides insights into viral diversity and evolution but also offers an exciting opportunity to understand the host-specific adaptation mechanism of these phages.table general genome features of zz t and the other completely sequenced t4-like acinetobacter phages  annotation of the zz genome sequence assembly yielded a closed, circular sequence with 114 bp inverted terminal repeats, indicating that zz phage particles contain linear, circularly permuted genomic dna, similar to phage t particles. the single copy genome is  bp. bioinformatics methods identified putative protein-coding sequences. the lengths of these cdss range from to amino acid residues and average aa. there are approximately genes per kbp, and % of the zz genome is predicted to encode proteins. atg was the predominant initiation codon. only cdss began with gtg, and began with ttg. the predominant termination codons were taa and tga. we noted that most of the significant blastp hits were proteins from phages, and the remaining hits were unknown proteins encoded by bacteria and other organisms. of the cdss from zz were assigned a functional annotation. only have no blastp matches in the non-redundant protein sequences database, indicating that genes encoding new viral proteins were revealed by the characterisation of this phage. however, further batch cd-search analyses suggested that of the genes with functional annotations lack protein domain information. of the cdss, were conserved hypothetical proteins, and only of the proteins had protein domain information. further functional analysis revealed that the cdss assigned putative functions could be classified into functional categories according to previous descriptions of phage t. most of the named functional proteins are highly conserved among t4-like phages and are either structural or involved in dna replication, recombination, repair, packaging, and processing.figure annotated genome map of phage zz cdss are labelled with the name of the closest homolog, and thus t nomenclature is primarily indicated. the numbers below the cds arrows are the cds ids used in the text, tables, and genbank. following convention, the map starts at the bottom left with the riia gene and ends at the bottom right with the riib gene. the individual cdss are depicted as arrows, with the orientation of the arrows indicating whether the genes are on the watson or crick strand. the colour of the arrow identifies the functional category into which the homologous t gene was classified. the colour code for gene function is provided in the bottom of the figure. summary of the zz genes annotated by blastp and batch cd-search analyses. pie chart showing the relative abundance of genes with and without named functions, including kfp, chp, chp-d, and hp genes. relative abundance of zz proteins in functional categories: a, transcription; b, translation; c, nucleotide metabolism; d, dna replication, recombination, repair, packaging, and processing; e, virion proteins; f, chaperonins/assembly catalysts; g, lysis; h, host or phage interactions; i, host alteration/shutoff; j, homing endonucleases and homologs; k, predicted membrane spanning proteins in unknown function proteins; l, unknown function. comparative genomics of t and zz1 the determination of phage relatedness is not based exclusively on sequence similarity but also takes genome organisation into consideration. the phage zz has similar virion morphology, genome size and number of cdss compared to the coliphage t. to further investigate the genomic similarities of t and zz we constructed comparative genome maps of zz and t the putative gene riia from zz was positioned on the minus strand and at the start of the map, following the t convention. homologous genes are projected onto the t map, and the projections are colour-coded based on protein sequence similarity. blastp analysis indicated that the zz cds shows significant similarity to the putative vs gene from the other 4 t4-like acinetobacter phages, acj acj and ac. however, when we compared the zz cds to the set of all t proteins by aligning two or more sequences from blastp using the default parameters, no counterpart was found in the results. further blastp analysis revealed that, of the acinetobacter phages, only phage acj vs showed a slight similarity to the t vs. similar results were observed in the annotation of zz cds which showed significant similarity to the putative 57a from phages ac and acj although no counterpart was found in the blastp results when we compared zz cds to the set of all t proteins. thus, we annotated zz cds and as hypothetical proteins, and of the cdss from zz only could be assigned a functional annotation, and were conserved hypothetical proteins. overall, such gradual evolution of the t genes between these closely and distantly related phages may eventually provide some interesting insights into the particular steps of genome divergence.figure t4-zz comparison genomic mapping and zz orthologous genes from the other 4 acinetobacter phages. the genomes and coding regions of t and zz are drawn in parallel approximately to scale. alignment of different genes is from t and from zz. following convention, the map starts at the top left with the riia gene and ends at the bottom right with the riib gene. horizontal arrows indicate transcription direction. the likely function of the genes is indicated by the colour of the arrows as described in figure  genes sharing protein sequence identity are linked according to the colour key provided at the middle left. zz genes are annotated randomly with their gene order in the genbank database. the map was artificially split into upper halves and lower halves. zz genes that are orthologous to the other acinetobacter phages are indicated by coloured boxes under the corresponding zz genes. the amino acid sequence identity of zz genes to these orthologous genes was also indicated by colour as described in the key provided at the upper right. the % coverage of zz genes to these orthologous genes is indicated by the grey boxes under each coloured shade or box according to the grey scale key at the upper right. the genes indicated by the grey vertical arrows share significant similarity with proteins from the other acinetobacter phages, but share no significant similarity with t proteins. notably, of the zz cdss shared up to 73% amino acid sequence identity with t proteins. long blocks of synteny containing homologous genes are interspersed with stretches that lack homology. the lower halves of the genome, which are closely related, include three separate clusters of rightward-transcribed genes, specifically: a cluster of base plate wedge/head/tail genes, a smaller base plate hub gene cluster in its vicinity, and further away, a tail fibre gene cluster consisting of a few large genes. two clusters of leftward-oriented non-structural genes separate the rightward-oriented clusters and mainly encode proteins involved in nucleotide metabolism. the degree of sequence identity varied substantially. the degree of sequence conservation did not follow structural/non-structural gene divisions. the region that displays the most significant difference between zz and t is located in the upper halves of the genome that exclusively encode leftward-oriented, non-structural genes, including a large dna replication module. large regions of nonaligned genome segments were thought to have resulted from genetic gains, losses, or replacements, i.e., events similar to those that have shaped the evolution of all microbial genomes in nature. for example, zz riia., and moba are flanked by unknown genes and genes were deleted between zz vs. and segb. further alignment and comparison of t genes that are homologous to zz revealed that the order and transcriptional direction of t genes are broadly maintained in the zz genome, which helped provide the phylogenetic distance between zz and t there were three major differences: 1) the rnlb gene was at the junction of in the lower genome half in t but was adjacent to the cds in the upper genome half of zz; 2) the topoisomerase gene and the dna polymerase gene were split and intact, respectively, in t but intact and split, respectively, in phage zz1; and 3) a cluster of alt genes including three putative alt-like genes differs between t and zz as shown in figure  the transcriptional direction of the three zz alt-like genes was different from the t alt, revealing an unusual genomic organisation for the acinetobacter phage zz moreover, the first alt counterpart is aa in length and shares 98% coverage and 28% amino acid identity with the t alt. the second alt-like gene is aa in length and shares 35% amino acid identity with the t alt segment that extends from to amino acids, and the third alt analogue is aa in length and shares 33% amino acid identity with the t alt segment that extends from to aa. apparently, the last two zz alt-like genes resulted from an even splitting event from the ancestral alt gene. the duplication of the alt gene was also present in the genomes of the acinetobacter phage ac, the coliphage js, and the enterobacteria phage bp, but the splitting of the alt gene was not observed in other t4-like phages.figure divergence of the core gene organisation between t and t4-like acinetobacter phages. the map was artificially split into upper and lower halves. the bars indicate identified orfs and are drawn to scale. genes transcribed in the forward direction are displayed above those genes transcribed in the reverse direction. the functional category of t genes is indicated by the color-coded bars described in figure  the numbers and acronyms shown directly above the color-coded bars refer to the gene names. core and quasicore genes described by petrov et al. are shown in red and blue font, respectively. homologous signature genes, shared by t and the 5 t4-related acinetobacter phages, are connected with light blue shading. conversely, homologous unknown genes, which shared significant similarity with all 5 t4-related acinetobacter phages but no significant similarity with t proteins, are linked by dark and light purple shading. genes linked by dark purple shading are specific to the 5 t4-related acinetobacter phages in the genbank database. in addition, signature t genes, which are linked by green shading, could reflect a dominant feature in the evolution of all 5 t4-related acinetobacter phages. t4-like phages were often shaped by gene duplications. for example, in t genes and share 29% amino acid identity, and proteins were identified as likely to have been duplicated in the kvp lineage. to further examine duplications, we aligned genes from zz with other zz genes using blastp at the amino acid level and blastn at the dna level. those with better matches were considered candidates for lineage-specific duplications. using this method, proteins were identified as likely to have been duplicated in the zz lineage. we further reasoned that if the homologous genes are the result of duplication of an ancestral gene, and if the different degrees of sequence diversification of these duplicated genes resulted from the accumulation of nucleotide point mutations resulting from differential selection pressure, then the genes with lineage-specific duplications should have significant nucleotide sequence identities. however, although the duplicated genes have significant amino acid identities, no significant nucleotide sequence identities were observed. thus, the similarity of the genes is more likely to be the result of convergent evolution than the result of duplication of an ancestral gene. for example, zz and share 25% amino acid identity but negligible nucleotide sequence identity. the protein plays an interesting role in t head maturation. the t prohead consists of an outer shell and an inner scaffold. when is added to the prohead, maturation cleavage occurs: loses its n-terminal portion, whereas is degraded completely. in addition, in phage kvp the duplication of three genes encoding proteins associated with the phage tail or the tail fibre suggests added flexibility in the range of host adaptation and the infection process. in our study, duplication was observed in three putatively duplicated gp homologues, which encode tail fibre-associated proteins and share 29-30% amino acid identity with zz gp overall, splitting and/or duplication followed by sequence diversification and new gene insertion might be a mode of t4-like phage evolution. similarities and distinctions in the genome features of the e. coliphage t and the t4-like acinetobacterphages alignment of the zz genome with the t4-like phages in the tulane database showed that zz was closely related to acinetobacter phages. based on the total search score, the closest was acj followed by acj ac and no other acinetobacter phages were uncovered. in dna sequence dot plots, we observed a frequently interrupted but straight diagonal line between zz and the phages. overall, their genomes are colinear but are frequently interrupted by replacements with unrelated genome segments of comparable lengths, especially in the first halves of their genome. further comparison of these genome sequences at the dna level using mauve showed remarkable synteny in five large conserved regions. the most conserved syntenic region covered the morphogenesis module as well as the dna replication and metabolism module, which are consistent with previous analyses.figure multiple genome alignment and the genomes of zz and 5 t4-like phages. six genomes were compared using mauve software, and similarity profiles were generated. boxes with identical colours represent local colinear blocks indicating homologous dna regions shared by two or more genomes without sequence rearrangements. the relative locations of orthologous genes, shown by open boxes, reveal a high degree of synteny. white regions within lcbs represent nonidentical sequences. protein-by-protein comparison of the phages using blastp and coregenes revealed that zz shares protein homologues with acinetobacter phage acj with acj with and with ac although zz shares only protein homologues with coliphage t in contrast, protein homologues are present in t and in all acinetobacter phages, whereas homologous genes are present in all of the acinetobacter phages, which is further evidence that zz and the other acinetobacter phages belong to the same genus. the full portrait of the t phage superfamily that has emerged from the many reported genomic comparisons is that the t superfamily can be distilled down to core signature genes, which are known as the “core genome” of the t4-related phages or t4-like viruses. the core genome primarily includes homologues of essential t genes, such as the virion structure and dna replication genes. in contrast, the hyper plastic regions contain mostly novel genes of unknown function and origin. the core genome of the t4-related phages has been considered to consist of two genetic components: one highly resistant component was termed the core genes, which are essential for all known conditions; and the other component, which is somewhat permissive to attrition in evolution, was termed the quasicore genes and can be substituted or circumvented in certain genetic backgrounds of phage and/or bacterial hosts. the genetic background for the core genome can vary considerably between t relatives, and thus, the number of the core genes that constitute the core genome depends on the precise subset of phages considered, which constitutes an important criterion for distinguishing between close and distant relatives. a closer look at the components and organization of the core genome in the 6 t4-like phages shows that all acinetobacter phages share a core genome with t interrupted by several hprs, which are where most of their divergence occurs. this finding is consistent with the previous observations of t4-like phages. all of the core genes described by petrov et al. exist in all 5 t4-related acinetobacter phages and are indicated by the red font directly above the coloured genes bars in figure  the topology of the set of t phage core genes is also shared by all 5 t4-related acinetobacter phages. moreover, we observed that almost all the quasicore genes described by petrov et al. were shared by all 5 t4-related acinetobacter phages, except for uvsx, uvsy, nrdc, nrdg, tk,, cd, and dena. these genes are permissive to attrition in evolution of all 5 t4-related acinetobacter phages. in addition, nearly half of the other t signature genes are shared by all 5 t4-related acinetobacter phages, including the gene topology. thus, all 5 t4-related acinetobacter phages use similar mechanisms to control propagation in their hosts. these phages all shared a common ancestral genome but, during the course of evolution, they have modified it in numerous ways. these conserved t genes allowed the 5 t4-related acinetobacter phages to conserve their highly successful virion design and mode of replication. notably, several major distinctive genome features in all 5 t4-related acinetobacter phages could reflect a dominant feature in the evolution of all 5 t4-related acinetobacter phages. the most significant difference between the 5 t4-related acinetobacter phages and t is in a series of hprs, which are interspersed between the conserved t core sequences and especially enriched in the upper genome halves of all 5 t4-related acinetobacter phages. these regions vary greatly in gene number and content. the hprs were primarily composed of genes of unknown origin, but they do contain some identifiable sequences from bacteria and unrelated phages. thus, the hprs have been predicted to be the result of an evolutionary history of isolation within distinct hosts and extensive lateral gene transfer, i.e., importing genes or exchanges with diverse biological entities in nature. our observations showed that all of the genes lack t homologues in the 5 t4-related acinetobacter phage genomes and cannot be assigned a putative function, most of which likely appear to be homologous genes from other phages, bacteria or other organisms. notably, of the zz genes that have significant homology with genes present in of the acinetobacter phages, have unknown functions. of these genes, which are indicated by the vertical grey arrows in figure , share no significant similarity to t proteins. moreover, most of these homologous genes maintain a consistent order and content in the 5 t4-related acinetobacter phages. further, blastp analyses suggested that of the zz genes, whose counterparts are linked with dark purple shading in figure  lack significant matches to any of the phage sequences in genbank except for the other acinetobacter phages and a few other organisms. in particular, share high similarities with the other acinetobacter phages. the diversity among phage “pangenome” is a reflection of the adaptations of a phage ancestor to a variety of evolutionary challenges, including encountering new host environments. lgt most certainly has played a role in such adaptations, and these adaptations may have been facilitated in part by certain particularities of the t phage recombination and gene expression systems. however, to date, there are few clues about the agents that might mediate such a transfer. t recombines most efficiently early in the infection before the host genome is degraded, and this process could facilitate the acquisition of host genes by the phage. as little as 50 bp of homology is sufficient for t recombination system to recombine at reasonable frequencies. thus, “semilegitimate” recombination that relies on the small but generally conserved regulatory signals in the intergenic regions could perhaps have mediated the acquisition of such foreign genes. once the foreign genes are acquired, the t expression system could exploit their endogenous bacterial promoters because these sequences are very similar to the early promoter sequences of the t4-type phages. overall, different phages sharing a common ancestor undergo a specific and similar adaptation process for different bacterial strains that belong to the same genera or species. the existence of these highly conserved unknown homologous genes specific to acinetobacter phages in hprs could reflect the complex interactions of acinetobacter phages with conserved cell components that are specific to acinetobacter bacterial hosts and are distantly related to e. coli or other bacterial hosts. theoretically, the uniqueness of homologous genes or certain sequences in specific phage genomes or lineages might help distinguish between the different clusters or types of phage and help predict the bacterial host range when treating the corresponding bacterial genera clinical isolates. additional research is required to elucidate the highly conserved genes that are specific to acinetobacter phages. gc skews in the zz genome phage zz dna contains only % gc, which is slightly lower than the value observed in sequenced a. baumannii strains. to date, it is the lowest reported gc content for sequenced t4-like acinetobacter phages. the gc content of zz is comparable with that of the enterobacteria phage t. the mol% gc of the t genome is also substantially lower than that of its host. the difference is in disagreement with previous studies suggesting that the gc content of phage genomes such as the mycobacteriophages and staphylococcus aureus phages have a gc content similar to their respective hosts. although zz has a lower gc content, of the predicted phage zz genes contain more than 40% gc, including one major head gene, three tail fibre genes, one nucleotide metabolism gene, and seven unknown genes. notably, the gc content of the zz gene is %, which is significant lower than the local gc content of t and the t4-like phage js. gene has the highest proportion of codons that are translationally optimal for the host in keeping with its very high level of expression; approximately copies of the protein are needed per synthesised phage particle. interestingly, the gc content of the zz host, a. baumannii strains, is approximately 39% and is lower than the gc content of the host strains for t and js which is %. if the gc content of phages and their hosts become more similar during extended co-evolution, the difference between the gc content of zz gene and the gc content of gene in the phage t and js presumably reflects the differences in host range, gc content, and life history of the respective hosts. thus, the low gc content of zz suggests that phage zz might have acquired the ability to infect a. baumannii strains over a long period of time. one region was found to be devoid of cdss, extending from to. in an effort to determine whether this region served as the replication origin, we used gc-skew analysis. a putative origin of replication in the region of nt and a putative terminus of replication in the region of nt were revealed. the putative origin of replication is close to but not within the cds-devoid region. however, trna detection showed that the trna genes from phage zz were encoded between cds and and clusters of trnas were located near the three adjacent genes, cds and the two regions were in this cds-devoid region. a previous study revealed that the phage zz had a very short latent period of 9 min. the large number of trna genes and their strategic locations might enable the phage to translate its sequence more efficiently, reducing its latency time and increasing its reproduction rate and thus its infectivity.figure cumulative gc skew analysis of the genome sequence. the global minimum and maximum are displayed in the cumulative graph. the minimum and maximum of a gc-skew can be used to predict the origin of replication and the terminus location. codon usage and trnas eight trna genes were predicted in the zz genome. the t genome also has trna genes, whereas ac has slightly fewer, and others have substantially more including phage, acj, and acj, as shown in figure  the different numbers and types of trnas present in the acinetobacter phages are consistent with a previous study of trnas in t4-like phages. in addition, although zz has a low mol% gc content, out of the trnas recognise codons with a in the third position.table trna genes from zz and homologous trnas from other acinetobacter phages and t4 a b atwo trna scan tools, trnascan-se and aragorn, were used for prediction. all of the predicted trna genes are located on the minus strand of the zz genome. btrnas homologous to zz in other phages revealed by nucleotide comparison are also located on the minus strand of the phage genomes. codon usage and trna availability in acinetobacter phages. the total number of codons is for phage zz, for phage acj, for phage acj, for phage ac, and for phage. optimal codons that have relative phage-encoded trna are indicated with black boxes. phage-encoded trnas related to the highly used codons are shaded grey, as is the corresponding codon. the exact function of phage-encoded trnas in the phage-infected cell is still not clear. they might be involved in the adaptation of the host translation apparatus to the demands of the phage codon usage pattern. in other words, the trnas overcome the phage codon usage problem, and the presence of trnas in a phage genome has been suggested to compensate for differences in codon usage between the phage and the host, corresponding to codons that are expected to be poorly translated by the host machinery. previous studies indicated that t4-encoded trnas are related to codons that are highly used in t genes but rarely used in the host and that the phage trnas can enhance the low expression of t late-stage protein genes through optimal codon usage in translation. similarly, analysis of the zz1-encoded trnas indicated that over half of them might be related to the optimal codon usage of zz proteins. however, the codon usage frequencies of the other acinetobacter phages suggested that phage-encoded trnas might be unrelated to decoding the relatively more frequent codons in all of the genes. thus, the functional role of the trna genes for these phages remains unclear. although the types and numbers of phage-encoded trnas differ between the phages, some consistency was observed. for example, all phages encode met-trna and trp-trna, which are related to optimal codon usage, but arg-trna, which was not related to optimal codon usage, was also common to all phages. of the trna genes in zz shared up to and 90% nucleotide sequence similarity to trnas from phage acj and phage respectively; shared up to 89% nucleotide sequence similarity to the trnas from phage acj61; and only trna showed 83% sequence similarity with the trna-thr from phage ac. in addition, the zz1-encoded trna-thr gene shared 83% identity with the t4-encoded trna-pro gene. the high degree of conservation of some of the trnas suggests an important functional role. the number and type of homologous trna appears to confirm the relationship between zz and the other phages, suggesting some elements of vertical evolution in these trnas. mobile elements the homing endonuclease genes are not genuine phage dna; rather, they belong to intron-associated selfish dna elements. there are seg, mob, and intron nuclease genes in the t genome, which have efficiently invaded the t genome and are also frequently found in the dna transaction module opposite the tail fibre cluster and the dna replication module. inconsistent distribution of these elements has been described for t t and t phages. one notable difference between the t and zz genomes is that zz appears to lack counterparts in the corresponding regions, except for two mobile genes, a seg-like gene and a mob-like gene. the zz seg-like mobile endonuclease, which is located in the dna replication module, has to 54% similarity to seg genes in t. however, the zz seg-like gene has higher similarity with the seg counterparts of the other acinetobacter phages: 73% amino acid identity with the seg counterpart from acj and to 58% identity with seg counterparts from acj and ac the zz mob-like homing endonuclease, which was found in the cluster of neck genes, lacks sequence similarity with the corresponding t genes. nevertheless it has 52% sequence similarity with the mob counterparts from acj and to 39% sequence similarity with the mob counterparts from and ac the other acinetobacter phages also lack mobile elements in the corresponding locations. although each of them has two to four seg genes, no mob-like genes were found in phage acj only one mob-like gene was found in phage and ac and only two mob-like genes were found in acj. t has three self-splicing group i introns, one each in td, nrdb, and nrdd. a few group i-like introns have been found in other phages, such as t the s. aureus phage twort, the bacillus phages i-basi and i-hmui, the lactobacillus phage ll-h, and the lactococcus phage r1t. however, none of the three t intron nuclease genes were found in phage zz acj or acj and only one i-tevi-like homing endonuclease from the giy-yig family was found in the and ac genome. these mobile element genes do not commonly exist in the t4-like phages, and the differences in mobile elements between the acinetobacter phages and t phage genomes are consistent with the results for another t4-like phage apparently, there are ill-defined barriers to intron promiscuity in bacteriophages.  we determined and revised the complete genome sequence of the phage zz that infects pathogenic a. baumannii strains. a total of potential proteins and trnas were predicted. the blastp analysis reveals that only a small portion of its proteins are clearly related to coliphage t proteins and share up to 73% protein sequence identity with the corresponding t proteins. further analysis revealed that and proteins from zz share up to and 83% amino acid identity, respectively, with acinetobacter phage acj acj and ac proteins, respectively. nine zz genes lack significant matches to any of the phage sequences in the genbank database except for the other acinetobacter phages. in addition, the high degree of conservation of some trnas between zz and the other t4-like phages suggests that they may have an important functional role in addition to overcoming the phage codon usage problem. the number and identity of the homologous trnas supports the evolutionary relationship between zz and the other phages, suggesting some elements of vertical evolution in these trnas. overall, although more than 200 t4-like phages have been examined, only a very limited number of the t4-like acinetobacter phage genomes have been explored or exploited. as additional genome sequencing of acinetobacter phage species are completed, more acinetobacter phages similar to t might be discovered. host-specific adaptation mechanisms might be revealed by a more comprehensive understanding of the genomic diversity within the acinetobacter bacteriophage population in the future. 
the -bp double-stranded dna genome of zz has the lowest gc content of the sequenced t4-like acinetobacter phages. forty-three percent of the predicted zz proteins share up to 73% amino acid identity with t proteins, and the homologous genes generally retained the same order and transcriptional direction. of the zz proteins, and 56% share up to and 83% amino acid identity with acj acj and ac proteins, respectively. zz has a different number and types of trnas than the other acinetobacter phages, although some of the zz1-encoded trnas share high sequence similarity with the trnas from these phages. however, this correlation was not present in any of the other acinetobacter phages. the comparative genomic analysis of these phages provided some new insights into the evolution and diversity of acinetobacter phages, which might elucidate the evolutionary origin and host-specific adaptation of these phages. beyond the conserved structural and dna replication modules, t and zz have diverged substantially by the acquisition and deletion of large blocks of unrelated genes, especially in the first halves of their genomes. to gain a better understanding of the genetic characteristics of zz bioinformatics and comparative genomic analyses of the t phages were performed. phage genome annotationphage genome organisationcomparative genomic analysest4-like phageissue-copyright-statement© the author 2014 in addition, zz and the four other t4-like acinetobacter phage genomes share a well-organised and highly conserved core genome, particularly in the regions encoding dna replication and virion structural proteins. over half of zz1-encoded trnas are related to optimal codon usage for zz proteins.
 phage zz which efficiently infects pathogenic acinetobacter baumannii strains, is the fifth completely sequenced t4-like acinetobacter phage to date. to gain a better understanding of the genetic characteristics of zz bioinformatics and comparative genomic analyses of the t phages were performed.  the -bp double-stranded dna genome of zz has the lowest gc content of the sequenced t4-like acinetobacter phages. a total of protein-coding genes and trna genes were predicted. forty-three percent of the predicted zz proteins share up to 73% amino acid identity with t proteins, and the homologous genes generally retained the same order and transcriptional direction. beyond the conserved structural and dna replication modules, t and zz have diverged substantially by the acquisition and deletion of large blocks of unrelated genes, especially in the first halves of their genomes. in addition, zz and the four other t4-like acinetobacter phage genomes share a well-organised and highly conserved core genome, particularly in the regions encoding dna replication and virion structural proteins. of the zz proteins, and 56% share up to and 83% amino acid identity with acj acj and ac proteins, respectively. zz has a different number and types of trnas than the other acinetobacter phages, although some of the zz1-encoded trnas share high sequence similarity with the trnas from these phages. over half of zz1-encoded trnas are related to optimal codon usage for zz proteins. however, this correlation was not present in any of the other acinetobacter phages.  the comparative genomic analysis of these phages provided some new insights into the evolution and diversity of acinetobacter phages, which might elucidate the evolutionary origin and host-specific adaptation of these phages. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords phage genome annotationphage genome organisationcomparative genomic analysest4-like phageissue-copyright-statement© the author 2014 