Morganella Phage Mecenats66 Utilizes an Evolutionarily Distinct Subtype of Headful Genome Packaging with a Preferred Packaging Initiation Site

Abstract

Both recognized species from the genus Morganella (M. morganii and M. psychrotolerans) are Gram-negative facultative anaerobic rod-shaped bacteria that have been documented as sometimes being implicated in human disease. Complete genomes of seven Morganella-infecting phages are publicly available today. Here, we report on the genomic characterization of an insect associated Morganella sp. phage, which we named Mecenats66, isolated from dead worker honeybees. Phage Mecenats66 was propagated, purified, and subjected to whole-genome sequencing with subsequent complete genome annotation. After the genome de novo assembly, it was noted that Mecenats66 might employ a headful packaging with a preferred packaging initiation site, although its terminase amino acid sequence did not fall within any of the currently recognized headful packaging subtype employing phage (that had their packaging strategy experimentally verified) with clusters on a terminase sequence phylogenetic tree. The in silico predicted packaging strategy was verified experimentally, validating the packaging initiation site and suggesting that Mecenats66 represents an evolutionarily distinct headful genome packaging with a preferred packaging initiation site strategy subtype. These findings can possibly be attributed to several of the phages already found within the public biological sequence repositories and could aid newly isolated phage packaging strategy predictions in the future.

Keywords: Morganella; bacteriophage; complete genome; genome termini; headful packaging; packaging initiation (pac) site; terminase.

Figure 1

The maximum-likelihood tree of the partial 16S rRNA gene sequences of the bacterial isolate B1-1 and closely-related bacterial species. The analysis involved 40 nucleotide sequences (16S rRNA gene sequences of isolate B1-1 and 39 other most closely related bacterial species identified). The tip labels correspond to the taxa and are in the format of “Species|Strain|Accession” and are colored based on the genus of the bacteria from which the sequence was derived. Isolate B1-1 is indicated by the black font, and the clade corresponding to the 16S rRNA sequences of bacterial genus Morganella representatives is highlighted in light blue. Input alignment had 1465 columns, 284 distinct patterns, 183 parsimony-informative, 41 singleton sites, and 1241 constant sites. The tree was built using TN + F + I + G4 as the best-fit substitution model. Near zero-length branches were collapsed into polytomies. The tree shown is midpoint rooted. The percentage of replicate trees in which the associated sequences clustered together in the ultrafast bootstrap (UFBoot; 1000 replicates) is shown next to the branches for branches with UFBoot support higher or equal to 95%. The tree is drawn to scale, branch lengths represent the number of nucleotide substitutions per site.

Figure 2

The transmission electron micrograph demonstrating the morphology of the Morganella bacteriophage Mecenats66 virion negatively stained with 0.5% uranyl acetate.

Figure 3

Pairwise genome organization and encoded protein comparison of Morganella phage Mecenats66 (middle) to Pseudomonas phage Dolphis (upper) and to Bordetella phage vB_BbrM_PHB04 (lower). Genomes are drawn to scale, and the genomes of Dolphis and PHB04 were reordered as indicated to ensure collinearity with Mecenats66; the scale bar indicates 2500 base pairs. Arrows representing open reading frames point in the direction of the transcription and are color-coded based on the function of their putative product according to the legend. Slanted labels above the arrows indicate the predicted function for the given ORF putative product in the case it had a function assigned (original annotations were retained for Dolphis and PHB04; predicted Mecenats66 ORF123 crossing the genome termini junction is not shown). Ribbons connect homologous proteins of phages and are colored according to the predicted functional group of the respective Mecenats66 ORF product. Ribbon labels indicate the pairwise similarity between the connected ORF products (for products having ≥30% identity). Comparisons were carried out using clinker (v.0.0.23.; [65]).

Figure 4

The maximum-likelihood tree of terminase/terminase large subunit (TerL) amino acid sequences for the prediction of the phage Mecenats66 packaging strategy. Input alignment had 48 sequences with 1539 columns, 1482 distinct patterns, 996 parsimony-informative, 346 singleton sites, and 197 constant sites. The tree was built using VT + F + R4 as the best-fit substitution model. Near zero-length branches were collapsed into polytomies. The tree shown is midpoint rooted. Tip labels are colored based on the distinct packaging strategies the phages seen in the tree employ (experimentally verified for most of the phages represented in the tree [34]). The percentage of replicate trees in which the associated sequences clustered together in the ultrafast bootstrap (UFBoot; 1000 replicates) is shown next to the branches for branches having a UFBoot support higher or equal to 95% (except for the MRCA node of the SPO1 type and Mecenats66 type TerL/terminase sequence clades for which a UFBoot of 90% is also shown). The tree is drawn to scale, and branch lengths represent the number of amino acid substitutions per site. Tip labels correspond to the phages from which the respective terminase/TerL amino acid sequences were derived and are in the format of “Protein accession|Phage”. Colored bars next to the labels indicate evolutionary distinct TerL clades, which correspond to different packaging strategies (LDTR stands for long direct terminal repeats, SDTR—short direct terminal repeats).

Figure 5

Restriction enzyme generated fragments from Morganella phage Mecenats66 DNA digestion by the selected restrictases. Tracks are named according to the FastDigest restrictase used. Track marked “M” indicates a marker/ladder track, and numeric values on the fragments indicate the ladder fragment sizes in base pairs. In the tracks named AanI, EcoRI, and SmiI, faint bands corresponding to the pac fragments are indicated by grey stars to the right of the respective fragment.

Figure 6

The Morganella phage Mecenats66 exact genome termini verification. The first track shows the rightward terminus nucleotide sequence of the Mecenats66 genome (first 22 bases). Tracks 2, 3, and 4 show Sanger-based sequencing read chromatograms corresponding to both termini, peaks are colored based on the base-call (red—adenine, green—thymine, yellow—guanine, blue—cytosine) and their heights represent the relative signal intensities. Cyan bars in the background represent the relative quality of the called base. In all of the tracks, the first base in the genome of Mecenats66 corresponding to an adenine is marked in red. In track 4, the thymine base with a cyan background (an adenine in the non-reverse-complemented original read) represents a non-template single base overhang added after the physical end of the most sequenced pac site-containing molecules.

Figure 7

The maximum-likelihood tree of representative terminase/terminase large subunit (TerL) amino acid sequence diversity. Input alignment had 793 sequences with 6470 columns, 4725 distinct patterns, 3011 parsimony-informative, 1119 singleton sites, and 2340 constant sites. The tree was built using Blosum62 + R10 as the best-fit substitution model. Near zero-length branches were collapsed into polytomies. The tree shown is midpoint rooted. Tip labels are provided for phages that had their packaging strategy/genome termini type verified experimentally and are colored based on the distinct packaging strategies phages within the presumed same packaging strategy clades employ (sequences from the extended “core” dataset have their respective packaging strategies indicated at the corresponding tips [34]; Figure 4) The clade consisting of the terminase sequence from Mecenats66, Dolphis, and PHB04 is highlighted in grey. LDTR stands for long direct terminal repeats, SDTR for short direct terminal repeats. Distal nodes of branches having UFBoot support higher or equal to 95% are indicated by green squares. The tree is drawn to scale, branch lengths represent the number of amino acid substitutions per site, and the radial circles are spaced 0.5 amino acid substitutions per site apart.