Bacteria have an extensive adaptive ability to live in close association with eukaryotic hosts, exhibiting detrimental, neutral or beneficial effects on host growth and health. However, the genes involved in niche adaptation are mostly unknown and their functions poorly characterized. Here, we present bacLIFE ( https://github.com/Carrion-lab/bacLIFE ) a streamlined computational workflow for genome annotation, large-scale comparative genomics, and prediction of lifestyle-associated genes (LAGs). As a proof of concept, we analyzed 16,846 genomes from the Burkholderia/Paraburkholderia and Pseudomonas genera, which led to the identification of hundreds of genes potentially associated with a plant pathogenic lifestyle. Site-directed mutagenesis of 14 of these predicted LAGs of unknown function, followed by plant bioassays, showed that 6 predicted LAGs are indeed involved in the phytopathogenic lifestyle of Burkholderia plantarii and Pseudomonas syringae pv. phaseolicola. These 6 LAGs encompassed a glycosyltransferase, extracellular binding proteins, homoserine dehydrogenases and hypothetical proteins. Collectively, our results highlight bacLIFE as an effective computational tool for prediction of LAGs and the generation of hypotheses for a better understanding of bacteria-host interactions., The project was financially supported, in part, by the Spanish “Ministerio
de Ciencia, Innovación y Universidades” project RYC2020-029240-I, by
the “Ayuda G.1. a la Actividad Investigadora de beneficiarios de los
programas Ramon y Cajal y Beatriz Galindo del II Plan Propio de la Uni versidad de Malaga” project 15 and by internal funding from Instituto de
Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA CSIC) and the Institute of Biology Leiden. A.P. was supported by Mar garita Salas Grant and co-funded by European Union-Next Generation
EU and Ministerio de Universidades (Spain). The contribution of J.M.R.
was supported by the Microp Gravitation project funded by NWO (grant
number 024.004.14). The Contribution of F.M.C. was supported by
PROYEXCEL21_00012, Junta de Andalucia. C.R. was supported by
PID2020-115177RB-C21 from the MCIN/AEI/ERDF. This publication was
supported by Proyecto QUAL21 012 IHSM, Consejería de Universidad,
Investigación e Innovación, Junta de Andalucía. We thank people from
the Carrión laboratory for their valuable feedback during group meet ings. All analyses were performed in the computing cluster of the Netherlands Institute of Ecology (NIOO-KNAW)., Peer reviewed

GacS/GacA is a widely distributed two-component system playing an essential role as a key global regulator, although its characterization in phytopathogenic bacteria has been deeply biased, being intensively studied in pathogens of herbaceous plants but barely investigated in pathogens of woody hosts. P. savastanoi pv. savastanoi (Psv) is characterized by inducing tumours in the stem and branches of olive trees. In this work, the model strain Psv NCPPB 3335 and a mutant derivative with a complete deletion of gene gacA were subjected to RNA-Seq analyses in a minimum medium and a medium mimicking in planta conditions, accompanied by RT-qPCR analyses of selected genes and phenotypic assays. These experiments indicated that GacA participates in the regulation of at least 2152 genes in strain NCPPB 3335, representing 37.9 % of the annotated CDSs. GacA also controls the expression of diverse rsm genes, and modulates diverse phenotypes, including motility and resistance to oxidative stresses. As occurs with other P. syringae pathovars of herbaceous plants, GacA regulates the expression of the type III secretion system and cognate effectors. In addition, GacA also regulates the expression of WHOP genes, specifically encoded in P. syringe strains isolated from woody hosts, and genes for the biosynthesis of phytohormones. A gacA mutant of NCPPB 3335 showed increased virulence, producing large immature tumours with high bacterial populations, but showed a significantly reduced competitiveness in planta. Our results further extend the role of the global regulator GacA in the virulence and fitness of a P. syringae pathogen of woody hosts., This research was supported by project grants PID2020-115177RB-C21
and PID2020-115177RB-C22 financed by the Spanish Ministry of
Science and Innovation (MCIN)/Agencia Estatal de Investigacioń
(AEI)/10.13039/501100011033/ and by the European Regional Development Fund (ERDF) “A way to make Europe”. CL-B was
supported by the FPI2018-084276 predoctoral grant. Open access
was partially funded by grant QUAL21 012 IHSM, Consejería de
Universidad, Investigación e Innovación, Junta de Andalucıa., Peer reviewed

Indole-3-acetic acid (IAA) production is a pathogenicity/virulence factor in the Pseudomonas syringae complex, including Pseudomonas savastanoi. P. savastanoi pathovars (pvs.) genomes contain the iaaL gene, encoding an enzyme that catalyzes the biosynthesis of the less biologically active compound 3-indole-acetyl-ϵ-L-lysine (IAA-Lys). Previous studies have reported the identification of IAA-Lys in culture filtrates of P. savastanoi strains isolated from oleander (pv. nerii), but the conversion of IAA into a conjugate was not detectable in olive strains (pv. savastanoi). In this paper, we show the distribution of iaaL alleles in all available P. savastanoi genomes of strains isolated from woody hosts. Most strains encode two different paralogs, except for those isolated from broom (pv. retacarpa), which contain a single allele. In addition to the three previously reported iaaL alleles (iaaL Psv, iaaL Psn and iaaL Pto), we identified iaaL Psf, an exclusive allele of strains isolated from ash (pv. fraxini). We also found that the production of IAA-Lys in P. savastanoi pv. savastanoi and pv. nerii depends on a functional iaaL Psn allele, whereas in pv. fraxini depends on iaaL Psf. The production of IAA-Lys was detected in cultures of an olive strain heterologously expressing IaaLPsn-1, IaaLPsf-1 and IaaLPsf-3, but not when expressing IaaLPsv-1. In addition, Arabidopsis seedlings treated with the strains overproducing the conjugate, and thus reducing the free IAA content, alleviated the root elongation inhibitory effect of IAA. IAA-Lys synthase activity assays with purified allozymes confirmed the functionality and specificity of lysine as a substrate of IaaLPsn-1 and IaaLPsf-3, with IaaLPsf-3 showing the highest catalytic efficiency for both substrates. The IAA-Lys synthase activity of IaaLPsn-1 was abolished by the insertion of two additional tyrosine residues encoded in the inactive allozyme IaaLPsv-1. These results highlight the relevance of allelic variation in a phytohormone-related gene for the modulation of auxin production in a bacterial phytopathogen., AP and HD-C were supported by the FPU14/05551 and PRE2021-099113 predoctoral grants, respectively. CR was supported by the AGL2017-82492-C2-1-R and PID2020-115177RB-C21 project grants from the Spanish Ministry of Science, Innovation and Universities (Spain), co-financed by the European Regional Development Fund (ERDF). VP and VF were supported by the GV/2018/115 and CIACO/2021/092 grant projects from Generalitat Valenciana and the RTI2018-094350-B-C33 grant from the Spanish Ministry of Science, Innovation and Universities. MV and SL were supported by project grant A20-0079 from The North Carolina Biotechnology Center (NCBC)., Peer reviewed

Phytopathogenic bacteria secrete diverse virulence factors to manipulate host defenses and establish infection. Characterization of the type III secretion system (T3SS)- and HrpL-independent secretome (T3-IS) in Pseudomonas savastanoi pv. savastanoi (Psv), the causal agent of olive knot disease, identified five secreted LysM-containing proteins (LysM1-LysM5) associated with distinct physiological processes critical for infection. Functional predictions from network analyses suggest that LysM1, LysM2, and LysM4 may participate in type IV pilus-related functions, while LysM3 and LysM5 are likely to possess peptidoglycan hydrolase domains critical for cell division. Supporting these predictions, loss of LysM1 function resulted in impaired twitching and swimming motility, highlighting a role in pilus-mediated movement and early host colonization. In contrast, mutants lacking LysM3 or LysM5 exhibited pronounced filamentation and defective bacterial division, underscoring their essential role in septation, a process crucial for both in planta fitness and tumor formation. Structural modeling and protein stability assays demonstrate that LysM3 interacts with peptidoglycan fragments such as tetra-N-acetylglucosamine and meso-diaminopimelic acid, as well as with zinc ions, through conserved LysM and M23 domains. LysM3 also displayed selective bacteriostatic activity against co-inhabiting Gram-negative bacterial competitors, such as Pantoea agglomerans and Erwinia toletana. Our findings highlight the relevance of LysM proteins in maintaining bacterial integrity, motility, and competitive fitness, which are crucial for successful host infection. This study expands the functional repertoire of LysM-containing proteins and reveals their broader impact on bacterial virulence and adaptation to the plant-associated niche., This research was supported by project grants PID2020-115177RB-C21 (to CR and LR-M) and PID2020-115177RB-C22 (to JM) financed by the Ministerio de Ciencia, Innovación y Universidades (MICIU)/ Agencia Estatal de Investigación (AEI)/10.13039/501100011033/ and by the European Regional Development Fund (ERDF) “A way to make Europe”, and by grant 2024ICT153 (to IP-D) from the Consejo Superior de Investigaciones Científicas (CSIC). HD-C was supported by predoctoral grant PRE2021-099113 (MICIU). LB-M was supported by the A4 Program of the Universidad de Málaga, Peer reviewed

The type III secretion system in Pseudomonas syringae complex pathogens delivers type III effectors (T3Es) into plant cells to manipulate host processes, enhance survival, and promote disease. While substantial research has focused on herbaceous pathogens, T3Es in strains infecting woody hosts are less understood. This study investigates the HopBL family of effectors in Pseudomonas savastanoi, a pathogen of woody plants. HopBL1 and HopBL2, core effectors in P. savastanoi, are restricted to phylogroup 3 strains of the P. syringae complex, all isolated from woody hosts. Phylogenetic analysis suggests recent horizontal acquisition of these effectors across multiple P. syringae pathovars, integrated into genomic islands flanked by mobile genetic elements. Structural analysis shows that both HopBL effectors contain SUMO protease and DNA-binding domains, with HopBL1 also possessing an ethylene-responsive motif, all characteristic of XopD from Xanthomonas spp. Despite low sequence identity, HopBL effectors exhibit structural similarity to XopD, with HopBL1 showing greater resemblance, particularly in the arrangement of these domains. Functional assays in olive and oleander revealed strain-specific contributions of HopBL1 and HopBL2 to virulence. In oleander, the natural host of P. savastanoi pv. nerii, mutation of either effector gene resulted in reduced symptom development. We show that HopBL2 localised predominantly to subnuclear foci and associated with plasmodesmata, with partial overlap observed along microtubules, suggesting a potential role in cytoskeleton manipulation. These findings underscore the importance of T3Es unique to P. syringae strains infecting woody hosts and their adaptation to modulate host cellular structures to promote disease., This research was supported by project grant PID2020-115177RB-C21 from the Spanish Ministry of Science and Innovation (MCIN)/Agencia Estatal de Investigación (AEI)/10.13039/501100011033/, the European Regional Development Fund (ERDF)–‘A way to make Europe’, and the National Institutes of Health (NIH) under (Grant 2R35GM136402), Peer reviewed

[Data Availability Statement]
RNA-seq data have been deposited in the Sequence Read Archive (SRA) at NCBI under accession number PRJNA1141921. All other data supporting the findings of this study are included in the Supporting Information or are available from the corresponding author upon reasonable request., The Pseudomonas syringae complex WHOP genomic island underpins virulence in woody hosts by mediating the catabolism of aromatic compounds. However, the biochemical functions of the ipoABC and dhoAB operons and the regulatory gene whpR remain unknown. Comparative genomics revealed WHOP-like clusters beyond P. syringae, found in diverse plant-associated, environmental and clinical bacteria, including indole degraders. We propose that ipoABC and dhoAB mediate indole degradation via anthranilate, linking indole detoxification to central metabolism through the β-ketoadipate pathway. In the olive pathogen P. savastanoi pv. savastanoi, ipoABC promotes indole degradation, indigo production, cell aggregation and biofilm formation. WhpR, an AraC-family regulator structurally related to CuxR and ToxT, defines a regulon comprising repression of most WHOP operons along with genes outside this region, including trpAB, reflecting integrated regulation of indole catabolism and tryptophan biosynthesis. In line with the observed transcriptional repression of WHOP genes, deletion of whpR led to hypervirulence and significantly altered bacterial fitness in woody olive plants. These findings define the WHOP region as a regulatory hub linking indole detoxification, multicellular behaviour and virulence, emerging as a target for novel control strategies against woody plant diseases., This research was supported by grant PID2020-115177RB-C21 from the Spanish Ministerio de Ciencia e Innovación (MCIN)/Agencia Estatal de Investigación (AEI)/10.13039/501100011033/, co-financed by the European Regional Development Fund (ERDF) – ‘A way to make Europe, Peer reviewed

Pseudomonas syringae pv. savastanoi NCPPB 3335 is the causal agent of olive knot disease and contains three virulence plasmids: pPsv48A (pA), 80 kb; pPsv48B (pB), 45 kb, and pPsv48C (pC), 42 kb. Here we show that pB contains a complete MPFT (previously type IVA secretion system) and a functional origin of conjugational transfer adjacent to a relaxase of the MOBP family; pC also contains a functional oriT-MOBP array, whereas pA contains an incomplete MPFI (previously type IVB secretion system), but not a recognizable oriT. Plasmid transfer occurred on solid and in liquid media, and on leaf surfaces of a non-host plant (Phaseolus vulgaris) with high (pB) or moderate frequency (pC); pA was transferred only occasionally after cointegration with pB. We found three plasmid-borne and three chromosomal relaxase genes, although the chromosomal relaxases did not contribute to plasmid dissemination. The MOBP relaxase genes of pB and pC were functionally interchangeable, although with differing efficiencies. We also identified a functional MOBQ mobilization region in pC, which could only mobilize this plasmid. Plasmid pB could be efficiently transferred to strains of six phylogroups of P. syringae sensu lato, whereas pC could only be mobilized to two strains of phylogroup 3 (genomospecies 2). In two of the recipient strains, pB was stably maintained after 21 subcultures in liquid medium. The carriage of several relaxases by the native plasmids of P. syringae impacts their transfer frequency and, by providing functional diversity and redundancy, adds robustness to the conjugation system., This work was funded by the Spanish Plan Nacional I+D+i grants PID2020-115177RB-C21 and PID2020-115177RB-C22 financed by the Ministerio de Ciencia e Innovación and the Agencia Estatal de Investigación, MCIN/AEI/10.13039/501100011033/, Spain. The funders had no role in study design, data collection, and interpretation or the decision to submit the work for publication., Peer reviewed

Phytopathogenic bacteria secrete diverse virulence factors to manipulate host defenses and establish infection. Characterization of the type III secretion system (T3SS)- and HrpL-independent secretome (T3-IS) in Pseudomonas savastanoi pv. savastanoi (Psv), the causal agent of olive knot disease, identified five secreted LysM-containing proteins (LysM1–LysM5) associated with distinct physiological processes critical for infection. Functional predictions from network analyses suggest that LysM1, LysM2, and LysM4 may participate in type IV pilus-related functions, while LysM3 and LysM5 are likely to possess peptidoglycan hydrolase domains critical for cell division. Supporting these predictions, loss of LysM1 function resulted in impaired twitching and swimming motility, highlighting a role in pilus-mediated movement and early host colonization. In contrast, mutants lacking LysM3 or LysM5 exhibited pronounced filamentation and defective bacterial division, underscoring their essential role in septation, a process crucial for both in planta fitness and tumor formation. Structural modeling and protein stability assays demonstrate that LysM3 interacts with peptidoglycan fragments such as tetra-N-acetylglucosamine and meso-diaminopimelic acid, as well as with zinc ions, through conserved LysM and M23 domains. LysM3 also displayed selective bacteriostatic activity against co-inhabiting Gram-negative bacterial competitors, such as Pantoea agglomerans and Erwinia toletana. Our findings highlight the relevance of LysM proteins in maintaining bacterial integrity, motility, and competitive fitness, which are crucial for successful host infection. This study expands the functional repertoire of LysM-containing proteins and reveals their broader impact on bacterial virulence and adaptation to the plant-associated niche., This research was supported by project grants PID2020-115177RB-C21 (to CR and LR-M) and PID2020-115177RB-C22 (to JM) financed by the Ministerio de Ciencia, Innovación y Universidades (MICIU)/ Agencia Estatal de Investigación (AEI)/10.13039/501100011033/ and by the European Regional Development Fund (ERDF) 'A way to make Europe', and by grant 2024ICT153 (to IP-D) from the Consejo Superior de Investigaciones Científicas (CSIC). HD-C was supported by predoctoral grant PRE2021-099113 (MICIU). LB-M was supported by the A4 Program of the Universidad de Málaga.

Pseudomonas syringae pv. savastanoi NCPPB 3335 is the causal agent of
olive knot disease and contains three virulence plasmids: pPsv48A (pA), 80 kb;
pPsv48B (pB), 45 kb, and pPsv48C (pC), 42 kb. Here we show that pB contains a
complete MPFT (previously type IVA secretion system) and a functional origin
of conjugational transfer adjacent to a relaxase of the MOBP family; pC also
contains a functional oriT-MOBP array, whereas pA contains an incomplete
MPFI (previously type IVB secretion system), but not a recognizable oriT.
Plasmid transfer occurred on solid and in liquid media, and on leaf surfaces
of a non-host plant (Phaseolus vulgaris) with high (pB) or moderate frequency
(pC); pA was transferred only occasionally after cointegration with pB. We
found three plasmid-borne and three chromosomal relaxase genes, although
the chromosomal relaxases did not contribute to plasmid dissemination.
The MOBP relaxase genes of pB and pC were functionally interchangeable,
although with di ering eciencies. We also identified a functional MOBQ
mobilization region in pC, which could only mobilize this plasmid. Plasmid
pB could be eciently transferred to strains of six phylogroups of P. syringae
sensu lato, whereas pC could only be mobilized to two strains of phylogroup 3
(genomospecies 2). In two of the recipient strains, pB was stably maintained
after 21 subcultures in liquid medium. The carriage of several relaxases
by the native plasmids of P. syringae impacts their transfer frequency and,
by providing functional diversity and redundancy, adds robustness to the
conjugation system., This work was funded by the Spanish Plan Nacional
I+D+i grants PID2020-115177RB-C21 and PID2020-
115177RB-C22 financed by the Ministerio de Ciencia
e Innovación and the Agencia Estatal de Investigación,
MCIN/AEI/10.13039/501100011033/, Spain.

GacS/GacA is a widely distributed two-component system playing an essential role as a key global regulator, although its characterization in phytopathogenic bacteria has been deeply biased, being intensively studied in pathogens of herbaceous plants but barely investigated in pathogens of woody hosts. P. savastanoi pv. savastanoi (Psv) is characterized by inducing tumours in the stem and branches of olive trees. In this work, the model strain Psv NCPPB 3335 and a mutant derivative with a complete deletion of gene gacA were subjected to RNA-Seq analyses in a minimum medium and a medium mimicking in planta conditions, accompanied by RT-qPCR analyses of selected genes and phenotypic assays. These experiments indicated that GacA participates in the regulation of at least 2152 genes in strain NCPPB 3335, representing 37.9 % of the annotated CDSs. GacA also controls the expression of diverse rsm genes, and modulates diverse phenotypes, including motility and resistance to oxidative stresses. As occurs with other P. syringae pathovars of herbaceous plants, GacA regulates the expression of the type III secretion system and cognate effectors. In addition, GacA also regulates the expression of WHOP genes, specifically encoded in P. syringe strains isolated from woody hosts, and genes for the biosynthesis of phytohormones. A gacA mutant of NCPPB 3335 showed increased virulence, producing large immature tumours with high bacterial populations, but showed a significantly reduced competitiveness in planta. Our results further extend the role of the global regulator GacA in the virulence and fitness of a P. syringae pathogen of woody hosts., This research was supported by project grants PID2020-115177RB-C21 and PID2020-115177RB-C22 financed by the Spanish Ministry of Science and Innovation (MCIN)/Agencia Estatal de Investigación (AEI)/10.13039/501100011033/ and by the European Regional Development Fund (ERDF) “A way to make Europe”. CL-B was supported by the FPI2018-084276 predoctoral grant. Open access was partially funded by grant QUAL21 012 IHSM, Consejería de Universidad, Investigación e Innovación, Junta de Andalucía.