Three of a Kind: Genetically Similar Tsukamurella Phages TIN2, TIN3, and TIN4

Abstract

bThree Tsukamurella phages, TIN2, TIN3, and TIN4, were isolated from activated sludge treatment plants located in Victoria, Australia, using conventional enrichment techniques. Illumina and 454 whole-genome sequencing of these Siphoviridae viruses revealed that they had similar genome sequences, ranging in size between 76,268 bp and 76,964 bp. All three phages shared 74% nucleotide sequence identity to the previously described Gordonia phage GTE7. Genome sequencing suggested that phage TIN3 had suffered a mutation in one of its lysis genes compared to the sequence of phage TIN4, to which it is genetically very similar. Mass spectroscopy data showed the unusual presence of a virion structural gene in the DNA replication module of phage TIN4, disrupting the characteristic modular genome architecture of Siphoviridae phages. All three phages appeared highly virulent on strains of Tsukamurella inchonensis and Tsukamurella paurometabola. The genus Tsukamurella contains Gram-positive organisms in the Corynebacteriales (1) and currently includes 11 species (2). Members of this genus are strictly aerobic and weakly/variably acid-fast, nonmotile, non-spore-forming organisms whose cell envelopes contain long-chain unsaturated mycolic acids (3-8).Together with other mycolic acid-producing genera, including Mycobacterium, Millisia, Nocardia, Rhodococcus, Williamsia, Rhodococcus, and Gordonia, they are often referred to collectively as the mycolata, a group of organisms whose mycolic acids render their cells highly hydrophobic (9, 10).The high hydrophobicity and the biosurfactants produced by the mycolata implicate them as stabilizing agents of foams in activated sludge plants (9, 11). Several Tsukamurella species including T. spumae (12), T. sunchonensis (13), and T. pseudospumae (12) have been isolated from these foams. Stable foams complicate sludge management, have a negative impact on plant esthetics, and increase maintenance costs (9).Several opportunistic Tsukamurella pathogens are known to exist, thus posing a potential health hazard to plant operators from aerosol foam dispersal (9). Thomas et al. (14) proposed that a phage therapy biocontrol approach might be an attractive option for treating this global operational problem and successfully isolated 17 phages infective for foaming mycolata from Australian wastewater treatment plants. More mycolata phages have been isolated and described since then (15-21). Among them is the TPA2 phage, infective for members of the genus Tsukamurella (15).As of March 2015, genomes of 228 phages infective for members of the closely related genus Mycobacterium have been sequenced. To better understand their genomics, Hatfull et al. (22) devised a clustering system that claimed to reveal insights into their evolutionary interrelationships. In this system, TPA2 phage would be placed into their cluster B as its genome organization is similar to that of the Mycobacterium phage Rosebush (15). Despite this similarity between TPA2 and Mycobacterium phages, little else is known about the exte...

Bacteriophages in Natural and Artificial Environments

“…Despite an understanding of the activated sludge process and the viruses involved, little research into phage communities had been conducted prior to 2011 [94]. With the advent of next-generation DNA sequencing (NGS) of isolated phages and metagenomics studies, this situation has improved [95,96,97,98,99,100,101,102,103,104,105,106]. It is now clear that the genomes of many bacteria present in activated sludge systems contain CRISPR-Cas regions, suggesting that these have in the past been infected by phage, and therefore their presence makes the problem of determining host/phage relationships in the absence of conventional ability to culture host cells, and consequently phage recovery [107].…”

Application of Biological Nanopore Sequencing Technology in the Detection of Microorganisms†

“…This study indicated that all strains of pond species were considered multidrug resistant and could lead to the emergence of ESBL bacteria, posing a threat to human health upon exposure. Furthermore, nanopore sequencing technology has also been applied to industrial wastewater, [ 93 ] activated sludge, [ 94 ] animal husbandry, [ 95 ] medical waste, [ 96 ] and other environments.…”

Biological control of problematic bacterial populations causing foaming in activated sludge wastewater treatment plants—phage therapy and beyond

“…It is clear that phages have evolved to overcome these powerful mechanisms as they can be isolated readily from activated sludge plants, and are capable of lysing many Mycolata members including the important foam stabilizing Gordonia (Petrovski et al 2011d(Petrovski et al , 2012bLiu et al 2015;Dyson et al 2015a;Pope et al 2017;Franco et al 2021), Rhodococcus (Shibayama and Dabbs 2011;Summer et al 2011;Petrovski et al 2011bPetrovski et al , 2012aPetrovski et al , 2013aPetrovski et al , 2013b, Tsukamurella (Petrovski et al 2011c;Dyson et al 2015b) and Nocardia (Pulverer et al 1975;Stratton et al 1996;Taylor et al 2019). Phages isolated and characterized to date are all double-stranded DNA phages that belong mainly to the Siphoviridae family, which all share similar shaped capsids with long non-contractile tails, although differing markedly in their size (Fig.…”