Deciphering stringent response proteins and toxin-antitoxin systems in the arms race between bacteria and phages

Project Information

PhaBacArms

Grant agreement ID: 101116205

DOI

10.3030/101116205

EC signature date 11 October 2023

Start date 1 January 2024

End date 31 December 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 499 250,00

EU contribution

€ 1 499 250,00

Coordinated by

TARTU ULIKOOL
Estonia

Periodic Reporting for period 1 - PhaBacArms (Deciphering stringent response proteins and toxin-antitoxin systems in the arms race between bacteria and phages)

Reporting period: 2024-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

Bacteriophages, viruses of bacteria, are the most abundant life form on Earth. The prospect of using them in the fight against pathogenic bacteria has long been recognised, but their high specificity and unpredictable reproduction limits their use in medicine. The mechanisms phages use to fight bacteria are attractive candidates for the development of novel antimicrobial applications as they are more general, but are still very poorly understood. Indirect evidence exists of toxin antitoxin systems (TAS) and stringent response involvement in phage defense. Stringent response is a stress response of bacteria that reprograms them from growth (when they are sensitive to different antimicrobial applications) to stress survival (when bacteria are highly tolerant). Although the constant co-evolution of phages and bacteria suggests that phages have a way of overcoming all of the wide variety of bacterial defense systems, yet, no direct phage mechanisms targeting bacterial stringent response have been identified.
We hypothesize that as the bacterial toxin-antitoxin systems and stringent response play a part in the arms race between bacteria and phages, we can provide direct molecular evidence and identify phage proteins involved in these interactions. We will create and characterise, morphologically and molecularly, a novel library of phages infecting a biotechnologically important bacterium - Pseudomonas putida PaW85, and uncover the stringent response- and TAS-mediated phenotypic effects and their molecular mechanisms on P. putida phage tolerance. By functionally and structurally characterizing the anti-stringent response phage effectors, we will bridge several gaps in the phage-bacteria interaction research. By knowing better the phage defense determinants of P. putida, we will advance the phage research of this biosynthesis and biodegradation strain. We will also provide crucial basic knowledge that can lead to the development of novel, efficient phage therapy solutions that directly target bacterial stress-mechanisms.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We isolated bacteriophages against P. putida PaW85. We isolated a collection of environmental phages infecting our host strain by optimizing different isolation methodologies to suit the host bacterium. To describe the phages, we screened the isolated phages for their sensitivity to chromosomal toxin-antitoxin systems (TAS) of P. putida PaW85. We discovered that the chromosomal TA systems do not provide phage protections. On the other hand the cryptic prophages in the genome of the becterium protect aganist many different phages. We also show that the infection of P. putida phages is temperature sensitive and is usually most efficient at 20 C. Determining the host-range showed that the phages are very specific to a certain host, most of them being able to only infect the specific PaW85 strain of P. putida. We discovered that a large TOL plasmid (pWWO) carried by the environmental ancestor strain of PaW85 protects bacteria from specific phages. These results were published in Environmental Microbiology. (Brauer et al 2024).
The TOL plasmid that conferred protection against one genera of phages carries a TAS. The phage defence factor from TOL plasmid is being determined.

We created a strain of P. putida lacking relA and spoT genes to create a stringent response deficient (ppGpp0) strain. The stringent response deficient strain is prototrophic, tolerates different chemical stressors well, but experiences membrane damage in late stationary phase. This results in increased membrane permeability, lowered membrane potential and even cell death. The problems probably originate from the inability of the cells to switch from growth to stress tolerance state as exemplified by the low expression of anti-sigma70 factors, which was determined by full cell proteome analysis comparing ppGpp0 strain to the wild type P. putida PaW85. We screened the environmental phages for their sensitivity to stringent response and discovered that the lack of stringent response can affect phage infection both positively and negatively. More precisely, determining the phage infection steps of two of affected phages showed that the lack of ppGp may result in a prolonged infection of the phage, thus resulting in a lowered infection efficiency. On the other hand, ppGpp present in wild type cells my protect the bacteria from attack from other phages as it seems that not all cells that phages bind to can carry out an efficient infection. The stringent response results were published in January 2026 (Lewanczyk et al 2026).

The prophage P1 in P. putida genome protects against several phages. This prophage also carries a HicAB TAS. We hypothesized that this system could be involved in P1-provided phage defense, but instead, a RexAB-like system proved to be the defense factor encoded by P1. This research was carried out in collaboration with R. Hõrak and the results were accepted for publication in Scientific Reports in January 2026.
The phage isolation was continued with a weakened strain of P. putida (lacking several potential phage defense systems from the genome), to create a more varied phage collection and increase the test pool for finding phages that interact with the bacterial TAS or stringent response. The increased collection will be published in the coming year.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

1. CEPEST phage collection - a collection of environmental phages infecting P. putida PaW85.
This collection of phages infecting a biotechnologically relevant bacterium has gained a lot of attention from scientists from different fields (molecular biology, biotechnology and even researcher studying environmental changes like global warming). Almost no phages infecting this bacterium was available before.