The environmental bacterium, Pseudomonas putida , possesses a broad spectrum of metabolic pathways. This makes it highly promising for use in biotechnological production as a cell factory, as well as in bioremediation strategies to degrade various aromatic pollutants. For P. putida to flourish in its environment, it must withstand the continuous threats posed by bacteriophages. Interestingly, until now, only a handful of phages have been isolated for the commonly used laboratory strain, P. putida KT2440, and no phage defence mechanisms have been characterized. In this study, we present a new Collection of Environmental P. putida Phages from Estonia, or CEPEST. This collection comprises 67 double‐stranded DNA phages, which belong to 22 phage species and 9 phage genera. Our findings reveal that most phages in the CEPEST collection are more infectious at lower temperatures, have a narrow host range, and require an intact lipopolysaccharide for P. putida infection. Furthermore, we show that cryptic prophages present in the P. putida chromosome provide strong protection against the infection of many phages. However, the chromosomal toxin–antitoxin systems do not play a role in the phage defence of P. putida . This research provides valuable insights into the interactions between P. putida and bacteriophages, which could have significant implications for biotechnological and environmental applications.

Type II toxin-antitoxin (TA) systems are genetic elements usually encoding two proteins: a stable toxin and an antitoxin, which binds the toxin and neutralizes its toxic effect. The disturbance in the intracellular toxin and antitoxin ratio typically leads to inhibition of bacterial growth or bacterial cell death. Despite the fact that TA modules are widespread in bacteria and archaea, the biological role of these systems is ambiguous. Nevertheless, a number of studies suggests that the TA modules are engaged in such important processes as biofilm formation, stress response or virulence and maintenance of mobile genetic elements. The Dickeya dadantii 3937 strain serves as a model for pathogens causing the soft-rot disease in a wide range of angiosperm plants. Until now, several chromosome-encoded type II TA systems were identified in silico in the genome of this economically important bacterium, however so far only one of them was experimentally validated. In this study, we investigated three putative type II TA systems in D. dadantii 3937: ccdAB_2Dda, phd-doc_Dda and dhiTA, which represents a novel toxin/antitoxin superfamily. We provide an experimental proof for their functionality in vivo both in D. dadantii and Escherichia coli. Finally, we examined the prevalence of those systems across the Pectobacteriaceae family by a phylogenetic analysis.

Guanosine tetra- and pentaphosphate ((p)ppGpp) are one of the key players in the stress response of bacteria. Accumulation of these alarmones activates the stringent response, usually triggered by different nutritional stresses. For Pseudomonas putida, there is only limited data available on the importance of the stringent response in stress situations. Also, in recent years, different specific phage defence systems have received much attention, but little is known about the involvement of stringent response in phage infection. Here, we show that P. putida PaW85 (p)ppGpp0 is prototrophic and tolerates chemical stress well. However, in the stationary phase P. putida cells deprived of (p)ppGpp have impaired membrane integrity. In addition, we conducted a large-scale screening of stringent response effects on phage infections using the CEPEST phage collection. We tested 67 phages of 22 different species and revealed that the lack of (p)ppGpp has opposing effects on phage infection with nearly half of the tested phages showing higher infection efficiency on the (p)ppGpp0 cells, whereas the other half shows reduced infection. We show that the differences in phage infection efficiency for phages Aura and Amme-3 are not caused by adsorption rate differences, but alterations in downstream steps of the infection cycle—prolonged latent period in the absence of (p)ppGpp or unproductive infection in the presence of (p)ppGpp. Altogether, results indicate that the role of stringent response in phage infection is highly diverse, and over half of the times the presence of (p)ppGpp facilitates phage infections rather than protects the cells.