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1. Transcription Factor PecS Mediates Agrobacterium fabrum Fitness and Survival

   https://doi.org/10.1128/jb.00478-22  

   Nwokocha, George C. ; Adhikari, Prava ; Iqbal, Asif ; Elkholy, Hannah ; Doerrler, William T. ; Larkin, John C. ; Grove, Anne ( July 2023 , Journal of Bacteriology)

   Becker, Anke (Ed.)

   ABSTRACT The transcriptional regulator PecS is encoded by select bacterial pathogens. For instance, in the plant pathogen Dickeya dadantii , PecS controls a range of virulence genes, including pectinase genes and the divergently oriented gene pecM , which encodes an efflux pump through which the antioxidant indigoidine is exported. In the plant pathogen Agrobacterium fabrum (formerly named Agrobacterium tumefaciens ), the pecS-pecM locus is conserved. Using a strain of A. fabrum in which pecS has been disrupted, we show here that PecS controls a range of phenotypes that are associated with bacterial fitness. PecS represses flagellar motility and chemotaxis, which are processes that are important for A. fabrum to reach plant wound sites. Biofilm formation and microaerobic survival are reduced in the pecS disruption strain, whereas the production of acyl homoserine lactone (AHL) and resistance to reactive oxygen species (ROS) are increased when pecS is disrupted. AHL production and resistance to ROS are expected to be particularly relevant in the host environment. We also show that PecS does not participate in the induction of vir genes. The inducing ligands for PecS, urate, and xanthine, may be found in the rhizosphere, and they accumulate within the plant host upon infection. Therefore, our data suggest that PecS mediates A. fabrum fitness during its transition from the rhizosphere to the host plant. IMPORTANCE PecS is a transcription factor that is conserved in several pathogenic bacteria, where it regulates virulence genes. The plant pathogen Agrobacterium fabrum is important not only for its induction of crown galls in susceptible plants but also for its role as a tool in the genetic manipulation of host plants. We show here that A. fabrum PecS controls a range of phenotypes, which would confer the bacteria an advantage while transitioning from the rhizosphere to the host plant. This includes the production of signaling molecules, which are critical for the propagation of the tumor-inducing plasmid. A more complete understanding of the infection process may inform approaches by which to treat infections as well as to facilitate the transformation of recalcitrant plant species. 

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2. Pathways of DNA transfer to plants from Agrobacterium tumefaciens and related bacterial species

   Lacroix, B ; Citovsky, V. ( July 2019 , Annual review of phytopathology)

   Genetic transformation of host plants by Agrobacterium tumefaciens and related species represents a unique model for natural horizontal gene transfer. Almost five decades of studying the molecular interactions between Agrobacterium and its host cells have yielded countless fundamental insights into bacterial and plant biology, even though several steps of the DNA transfer process remain poorly understood. Agrobacterium spp. may utilizs different pathways for transfer of its DNA, which likely reflects the very wide host range of Agrobacterium. Moreover, closely related bacterial species, such as rhizobia, become able transfer DNA to host plant cells when they are provided with Agrobacterium DNA transfer machinery and T-DNA. Homologs of Agrobacterium virulence genes are found in many bacterial genomes, but only one non-Agrobacterium bacterial strain, Rhizobium etli CFN42, harbors a complete set of virulence genes and can mediate plant genetic transformation when carrying a T-DNA-containing plasmid. 

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3. Plant DNA repair and Agrobacterium T-DNA integration.

   https://doi.org/doi.org/10.3390/ijms22168458  

   Gelvin, S.B. ( July 2021 , International journal of molecular sciences)

   Agrobacterium species transfer DNA (T-DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T-DNA often contains, at its junctions with plant DNA, deletions of T-DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T-DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T-DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T-DNA integration into the plant genome. 

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4. Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR

   https://doi.org/10.7554/eLife.66834  

   Diebold, Peter J ; New, Felicia N ; Hovan, Michael ; Satlin, Michael J ; Brito, Ilana L ( July 2021 , eLife)

   null (Ed.)

   The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive, or prone to contamination. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links plasmid-encoded genes with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases within the gut microbiomes of neutropenic patients, as they are particularly vulnerable multidrug-resistant infections. We successfully detect the known association of a multi-drug resistant plasmid with Klebsiella pneumoniae , as well as the novel associations of two low-abundance genera, Romboutsia and Agathobacter . Further investigation with OIL-PCR confirmed that our detection of Romboutsia is due to its physical association with Klebsiella as opposed to directly harboring the beta-lactamase genes. Here we put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes, as well as detecting physical bacterial associations such as those occurring within biofilms and complex microbial communities. 

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5. Transformation and regeneration of DNA polymerase Θ mutant rice plants

   https://doi.org/10.1002/pld3.526  

   Nishizawa‐Yokoi, Ayako ; Gelvin, Stanton B. ( September 2023 , Plant Direct)

   AgrobacteriumT‐DNA integration into the plant genome is essential for the process of transgenesis and is widely used for genome engineering. The importance of the non‐homologous end‐joining (NHEJ) protein DNA polymerase Θ, encoded by thePolQgene, for T‐DNA integration is controversial, with some groups claiming it is essential whereas others claim T‐DNA integration inArabidopsisand ricepolQmutant plant tissue. Because of pleiotropic effects of PolQ loss on plant development, scientists have previously had difficulty regenerating transgenicpolQmutant plants. We describe a protocol for regenerating transgenicpolQmutant rice plants using a sequential transformation method. This protocol may be applicable to other plant species.

    

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