Additional conjugation systems for inter-domain plasmid transfer to the diatom Phaeodactylum tricornutum

Bacterial conjugation utilizes a type IV secretion system and a DNA transfer mechanism to deliver DNA from one cell to another. Conjugative partners are conventionally confined to the prokaryotic domain. In a prominent exception, Agrobacterium tumefaciens type IV secretion-mediated transfer of DNA to plant cells can result in subsequent chromosomal integration. Recently, we demonstrated interdomain conjugation from Escherichia coli to the diatom Phaeodactylum tricornutum with the subsequent maintenance of an episome at chromosomal copy numbers if it contains diatom centromeres or centromere-like elements. The genes involved in the conjugation process can be separated into those encoding the type IV secretion system, also called the mating pair formation (MPF) genes, and genes involved in DNA processing called the mobilization (MOB) genes. Various protein families compose each class of conjugation genes, including common MOB types F, P, and Q and MPF types F, P, and T. The conjugative transfer from E. coli to P. tricornutum was demonstrated with a vector expressing MOBP and MTFP. Here we show that the MOBPsystem can be deleted and complemented with a MOBQ system in E.coli-diatom conjugations with subsequent episomal maintenaince. Utilization of both MOBP and MOBQ systems results in substantially higher efficiencies in E. coli-diatom conjugation. Finally, we demonstrate conjugative gene transfer between P. tricornutum and A. tumefaciens expressing a MPFT, the first demonstration of this system in diatoms,resulting in episomal maintainance or chromosomal integration, depending on the ex-conjugant. The promiscuity of MOB and MTF systems permitting prokaryote to diatom conjugative DNA transfer suggest major environmental and evolutionary importance of this process. The increased efficiency of dual MOB systems immediately improves genetic engineering in diatoms and has interesting basic cellular biology implications.

Additional conjugation systems for inter-domain plasmid transfer to the diatom Phaeodactylum tricornutum

Bacterial conjugation utilizes a type IV secretion system and a DNA transfer mechanism to deliver DNA from one cell to another. Conjugative partners are conventionally confined to the prokaryotic domain. In a prominent exception, Agrobacterium tumefaciens type IV secretion-mediated transfer of DNA to plant cells can result in subsequent chromosomal integration. Recently, we demonstrated interdomain conjugation from Escherichia coli to the diatom Phaeodactylum tricornutum with the subsequent maintenance of an episome at chromosomal copy numbers if it contains diatom centromeres or centromere-like elements. The genes involved in the conjugation process can be separated into those encoding the type IV secretion system, also called the mating pair formation (MPF) genes, and genes involved in DNA processing called the mobilization (MOB) genes. Various protein families compose each class of conjugation genes, including common MOB types F, P, and Q and MPF types F, P, and T. The conjugative transfer from E. coli to P. tricornutum was demonstrated with a vector expressing MOBP and MTFP. Here we show that the MOBPsystem can be deleted and complemented with a MOBQ system in E.coli-diatom conjugations with subsequent episomal maintenaince. Utilization of both MOBP and MOBQ systems results in substantially higher efficiencies in E. coli-diatom conjugation. Finally, we demonstrate conjugative gene transfer between P. tricornutum and A. tumefaciens expressing a MPFT, the first demonstration of this system in diatoms,resulting in episomal maintainance or chromosomal integration, depending on the ex-conjugant. The promiscuity of MOB and MTF systems permitting prokaryote to diatom conjugative DNA transfer suggest major environmental and evolutionary importance of this process. The increased efficiency of dual MOB systems immediately improves genetic engineering in diatoms and has interesting basic cellular biology implications.

Heat Shock-Enhanced Conjugation Efficiency in Standard Campylobacter jejuni Strains

ABSTRACTCampylobacter jejuni, the leading bacterial cause of human gastroenteritis in the United States, displays significant strain diversity due to horizontal gene transfer. Conjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance. It has been observed that heat shock could increase transformation efficiency in some bacteria. In this study, the effect of heat shock onC. jejuniconjugation efficiency and the underlying mechanisms were examined. With a modifiedEscherichia colidonor strain, differentC. jejunirecipient strains displayed significant variation in conjugation efficiency ranging from 6.2 × 10−8to 6.0 × 10−3CFU per recipient cell. Despite reduced viability, heat shock of standardC. jejuniNCTC 11168 and 81-176 strains (e.g., 48 to 54°C for 30 to 60 min) could dramatically enhanceC. jejuniconjugation efficiency up to 1,000-fold. The phenotype of the heat shock-enhanced conjugation inC. jejunirecipient cells could be sustained for at least 9 h. Filtered supernatant from the heat shock-treatedC. jejunicells could not enhance conjugation efficiency, which suggests that the enhanced conjugation efficiency is independent of secreted substances. Mutagenesis analysis indicated that the clustered regularly interspaced short palindromic repeats system and the selected restriction-modification systems (Cj0030/Cj0031, Cj0139/Cj0140, Cj0690c, and HsdR) were dispensable for heat shock-enhanced conjugation inC. jejuni. Taking all results together, this study demonstrated a heat shock-enhanced conjugation efficiency in standardC. jejunistrains, leading to an optimized conjugation protocol for molecular manipulation of this organism. The findings from this study also represent a significant step toward elucidation of the molecular mechanism of conjugative gene transfer inC. jejuni.

Characterization ofndvD, the third gene involved in the synthesis of cyclic β-(13),(16)-D-glucans inBradyrhizobium japonicum

Previously, we identified two genes in Bradyrhizobium japonicum (ndvB, ndvC) that are required for cyclic β-(1[Formula: see text]3),(1[Formula: see text]6)-D-glucan synthesis and successful symbiotic interaction with soybean (Glycine max). In this study, we report a new open reading frame (ORF1) located in the intergenic region between ndvB and ndvC, which is essential for β-glucan synthesis and effective nodulation of G. max. This new gene is designated ndvD (nodule development). The ndvD translation product has a predicted molecular mass of 26.4 kDa with one transmembrane domain. Genetic experiments involving gene deletion, Tn5 insertion, and gene complementation revealed that the mutation of ndvD generated pleiotropic phenotypes, including hypoosmotic sensitivity, reduced motility, and defects in conjugative gene transfer, in addition to symbiotic ineffectiveness. Although deficient in in vivo β-glucan synthesis, membrane preparations from the ndvD mutant synthesized neutral β-glucans in vitro. Therefore, ndvD does not appear to be a structural gene for β-glucan synthesis. Our hypothesis for the mechanism of β-(1[Formula: see text]3),(1[Formula: see text]6)-D-glucan synthesis is presented. Key Words: β-glucans,Bradyrhizobium, soybean, nitrogen fixation.

High Rates of Conjugation in Bacterial Biofilms as Determined by Quantitative In Situ Analysis

ABSTRACT Quantitative in situ determination of conjugative gene transfer in defined bacterial biofilms using automated confocal laser scanning microscopy followed by three-dimensional analysis of cellular biovolumes revealed conjugation rates 1,000-fold higher than those determined by classical plating techniques. Conjugation events were not affected by nutrient concentration alone but were influenced by time and biofilm structure.