Research Article: Investigation of Pseudomonas aeruginosa strain PcyII-10 variants resisting infection by N4-like phage Ab09 in search for genes involved in phage adsorption

Date Published: April 16, 2019

Publisher: Public Library of Science

Author(s): Libera Latino, Cédric Midoux, Gilles Vergnaud, Christine Pourcel, William M. Shafer.


Bacteria and their bacteriophages coexist and coevolve for the benefit of both in a mutualistic association. Multiple mechanisms are used by bacteria to resist phages in a trade-off between survival and maintenance of fitness. In vitro studies allow inquiring into the fate of virus and host in different conditions aimed at mimicking natural environment. We analyse here the mutations emerging in a clinical Pseudomonas aeruginosa strain in response to infection by Ab09, a N4-like lytic podovirus and describe a variety of chromosomal deletions and mutations conferring resistance. Some deletions result from illegitimate recombination taking place during long-term maintenance of the phage genome. Phage variants with mutations in a tail fiber gene are selected during pseudolysogeny with the capacity to infect resistant cells and produce large plaques. These results highlight the complex host/phage association and suggest that phage Ab09 promotes bacterial chromosome rearrangements. Finally this study points to the possible role of two bacterial genes in Ab09 phage adhesion to the cell, rpsB encoding protein S2 of the 30S ribosomal subunit and ORF1587 encoding a Wzy-like membrane protein involved in LPS biosynthesis.

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Bacteriophages play an important role in shaping the bacterial genome by exerting a selective pressure [1]. Bacterial resistance to bacteriophage is principally mediated by modification of the phage receptors although other mechanisms can play a role by blocking the viral entry and multiplication [2]. Recently a large number of new antiphage defense systems present in multicomponent defense islands (among which restriction-modification and CRISPR-Cas systems) have been described [3,4]. In return phage counteracts the defense mechanisms by developing different strategies [5]. Long-term phage/bacteria arm-race has been extensively investigated in the laboratory, mostly using coevolution in chemostats, and a few studies have observed coevolution in natural environment [6,7]. In the environment, many factors influence the fate of bacteria and phage variants by affecting the fitness as observed with Pseudomonas fluorescens for example [8]. In P. aeruginosa the filamentous Pf4 prophage is essential for several stages of the biofilm life-cycle and contributes to the bacterial virulence [9]. Adaptation of a bacterial strain to stress or to a particular niche may be accompanied by important rearrangements of the genome. Reduction of the genomic repertoire is the result of large deletions caused by homologous and illegitimate recombination during permanent association of pathogenic bacteria with their host [10]. Recombination between IS elements or prophages have been shown to be the most frequent mechanism for inversions and deletions [11,12,13]. Another actor in phage/bacteria evolution is the RecA-dependent spontaneous prophage induction (SPI) that influence bacterial fitness [14]. This may be linked to SOS response but also results from other mechanisms.

N4-like phages are podoviruses distributed into four genera frequently isolated from different Gram-negative species and characterized by the presence of a giant virion-associated RNA polymerase [34,35,36]. In P. aeruginosa the corresponding genus “Lit1virus” includes Lit1, PA26 and Ab09 which display a rather large host spectrum, making them interesting tools for phage therapy. They form plaques with a halo, likely related to degradation of capsular exopolysaccharides by a depolymerase activity [37,38,39]. The interactions between E. coli and phage N4, and the basis for phage-resistance have been largely studied but little is known about the other members of this family. We confirm here using the clinical and minimally subcultured P. aeruginosa strain PcyII-10 that Ab09 adopts a pseudolysogenic behavior at a high rate in starving bacteria, and that this favors the emergence of mutants as previously shown using the PAO1 laboratory strain [16]. During maintenance of the phage a small proportion of bacteria can support a full multiplication cycle and die releasing new phages, whereas some cells maintain small amounts of viral genome and other are cured, becoming prey for the free phages. As opposed to the carrier state established by the levivirus LeviOr01 in which large quantities of virions could be seen inside enlarged cells [40], there is apparently no Ab09 virions accumulating in the pseudolysogens, but the bacteria appear to be deeply affected. Therefore the blockage might be at the level of phage multiplication and not lysis. This was observed with phage T4 in starved cells, a phenomenon called lysis inhibition (LIN) induced by superinfection of T4-infected cells at any time during the multiplication cycle [41,42]. Some core and variable genes in members of the N4-like family show striking similarities to those of the T4 superfamily [43]. Phage Ab09 possesses two genes related to phage T4 rIIA and rIIB which appear to play an indirect role in LIN, promoting delayed lysis but there is apparently no Ab09 equivalent of gene rI which is the real actor of LIN [44,45,46].




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