Competition for nutrients and niches (CNN) is known to be one of the mechanisms for biocontrol mostly exhibited by Pseudomonas strains. Phenotypic and full genome analysis revealed Pseudomonas putida PCL1760 controlling tomato foot and root rot (TFRR) solely through CNN mechanism. Although the availability of nutrients and motility are the known conditions for CNN, persistence of bacteria through dormancy by ribosomal hibernation is a key phenomenon to evade both biotic and abiotic stress. To confirm this hypothesis, rsfS gene knockout mutant of PCL1760 (SB9) was first obtained through genetic constructions and compared with the wild type PCL1760. Primarily, relative expression of rsfS in PCL1760 was conducted on tomato seedlings which showed a higher expression at the apical part (1.02 ± 0.18) of the plant roots than the basal (0.41 ± 0.13). The growth curve and persistence in ceftriaxone after the induction of starvation with rifampicin were performed on both strains. Colonization on the tomato root by CFU and qPCR, including biocontrol ability against Fusarium, was also tested. The growth dynamics of both PCL1760 and SB9 in basal and rich medium statistically did not differ (p ≤ 0.05). There was a significant difference observed in persistence showing PCL1760 to be more persistent than its mutant SB9, while SB9 (pJeM2:rsfS) was 221.07 folds more than PCL1760. In colonization and biocontrol ability tests, PCL1760 was dominant over SB9 colonizing and controlling TFRR (in total, 3.044 × 10⁴ to 6.95 × 10³ fg/µL and 55.28% to 30.24%, respectively). The deletion of the rsfS gene in PCL1760 reduced the persistence and effectiveness of the strain, suggesting persistence as one important characteristic of the CNN.

Pseudomonasputida strain PCL1760 is a biocontrol agent protecting plants from pathogens via the mechanism of competition for nutrients and niches (CNN). To confirm this mechanism as well as to adapt the strain for biotechnological applications, full genome analysis was compared with the known biotechnological model, P. putida S12, and other related species, which were analyzed on different genomic databases. Moreover, the antibacterial activity of PCL1760 was tested against Staphylococcus aureus, Pseudomonas aeruginosa, and Pseudomonas syringae. No genetic systems involved in antibiosis were revealed among the secondary metabolite clusters of the strain of PCL1760. The only antagonistic effect was observed against P. syringae, which might be because of siderophore (yellow-greenish fluorescence), although less than 19% pyoverdin biosynthesis clusters were predicted using the AntiSMASH server. P. putida PCL1760 in comparison with the Pseudomonas simiae strain PCL1751, another biocontrol agent acting solely via CNN, which lost its ‘luxury’ genes necessary for antibiosis or parasitism/predation mechanisms, but carries genetic systems providing motility. Interestingly, immunity genes (CRISPR/Cas and prophages) showed PCL1760 to be robust in comparison with S12, while annotation on OrthoVenn2 showed PCL1760 to be amenable for genetic manipulations. It is tempting to state that rhizobacteria using the mechanism of CNN are distinguishable from biocontrol agents acting via antibiosis or parasitism/predation at the genomic level. This confirms the CNN of PCL1760 as the sole mechanism for biocontrol and we suggest the strain as a new model for genetic engineering.

We characterized the cryptic plasmid pM27181 (15,416 bp) isolated from the Stutzerimonas stutzeri strain DIA-8. Sequencing and bioinformatic analysis allowed identification of 20 open reading frames, including genes involved in replication (rep), mobilization (relaxase/mobilization protein), segregation (parA, parG), and site-specific recombination (XerC-like protein), as well as the putative Csr/Rsm regulatory module. Deletion analysis and random ligation yielded three minireplicons: pEBNT-1-K, pEBNT-2, and pEBNT-3. Each contains a replication module comprising a rep gene and an iteron-containing region. All three variants replicated autonomously in both Escherichia coli and Pseudomonas putida. Loss of one of the two iterons in variant pEBNT-3 relative to pEBNT-2 has a host-dependent effect. In P. putida, this deletion increased plasmid copy number, possibly due to attenuation of negative feedback, but reduced segregational stability. In E. coli cells the deletion decreased copy number without a corresponding decrease in segregational stability. pEBNT-1-K retained mobilization activity, whereas no mobilization was detected for pEBNT-2 and pEBNT-3 under the conditions tested, suggesting a reduced risk of horizontal dissemination. These findings suggest that replication control is host-dependent and may involve interaction among the Rep protein, iteron repeats, and intracellular factors. The minireplicons provide a basis for developing genetic tools for pseudomonads and a model for studying plasmid replication in different bacterial hosts.

Ribosome hibernation is a key bacterial survival strategy under stress conditions. While most eubacteria utilize ribosome silencing factor (RsfS) and hibernation promoting factor (HPF), the functional interplay of ribosome-associated factors, including ribosome modulation factor (RMF), short HPF, and YfiA in γ-proteobacteria remains poorly understood. Here, we systematically investigated the role of these four ribosome-associated factors in Pseudomonas putida PCL1760 by examining their gene expression patterns, effects of overexpression, and contribution to long-term survival. Transcriptional analysis revealed that rmf, hpf, and yfiA are strongly induced upon entry into stationary phase with distinct temporal expression patterns, whereas rsfS expression remained constitutive. Overexpression of RMF, YfiA, and RsfS caused strong growth inhibition, while HPF overexpression resulted only in moderate growth suppression. Long-term survival assays of knockout mutants revealed HPF as the most critical factor, with the Δhpf mutant showing a 75% loss of viability. The ΔhpfΔrmf double mutant exhibited severely reduced survival (94% loss), whereas Δrmf and ΔyfiA single mutants showed only moderate reduction, while the ΔrsfS mutant displayed no survival defects. Notably, deletion of yfiA against severely impaired ΔhpfΔrmf background partially restored viability, suggesting a compensatory interaction among ribosome-associated factors. These findings reveal a synergistic relationship between HPF and RMF in long-term cellular preservation and uncover a complex regulatory network, in which distinct temporal roles of involved proteins and their cross-compensation regulate ribosome hibernation. This work advances understanding of bacterial persistence mechanisms, with potential implications for bioremediation and agricultural biotechnology.

Monitoring individual strains in complex microbial communities remains a fundamental challenge in microbial ecology and biotechnology. Here, we present an integrated pipeline for identifying and validating strain-specific loci (SSL) in four biotechnologically relevant plant growth promoting strains from three genera (Stenotrophomonas, Bacillus, and Pseudomonas). The pipeline applies a two-round specificity-filtering strategy combining whole-genome comparison and high-sensitivity BLASTn validation of revealed strain-specific loci (SSL) against the NCBI nucleotide database. SSL count decreased with increasing Average nucleotide identity (ANIb) of the strains used for the analysis, ranging from one locus in B. halotolerans (ANIb = 98.91%) to 15 loci in S. rhizophila (ANIb = 86.49%). All 25 SSL were universally AT-rich, mainly accessory-genome-associated, with flanking regions enriched in genes of unknown function (34.6%) and mobile genetic elements (19.2%). TaqMan qPCR assays targeting SSL demonstrated high specificity—no target sequences were detected across ten geographically distinct soil samples, nor in a native rhizosphere metagenome—and sensitivity, with limits of detection of 0.01–0.1 pg of genomic DNA. Spike-in experiments in soil yielded method detection limits (MDL) of 850–15,000 CFU/g. All four strains were detected in the wheat rhizosphere seven days after consortium application in a field experiment, validating the pipeline for multi-strain field monitoring.