Modulation of primary cell function of host Pseudomonas bacteria by the conjugative plasmid pCAR1

Abstract

The impacts of plasmid carriage on the host cell were comprehensively analysed using the conjugative plasmid pCAR1 in three different Pseudomonas hosts, P. putida KT2440, P. aeruginosa PAO1 and P. fluorescens Pf0-1. Plasmid carriage reduced host fitness, swimming motility, and resistance to osmotic or pH stress. Plasmid carriage brought about alterations in primary metabolic capacities in the TCA cycle of the hosts. Differentially transcribed genes in the three hosts associated with plasmid carriage were identified by growth phase-dependent transcriptome analyses. Plasmid carriage commonly showed a greater effect on the host transcriptome at the transition and early stationary phases. The transcriptome alterations were similar between KT2440 and PAO1. Transcriptions of numbers of genes encoding ribosomal proteins, F-type ATPase, and RNAP core in both strains were not suppressed enough in the early stationary phase by plasmid carriage. These responses may have been responsible for the reduction in host fitness, motility and stress resistances. Host-specific responses to plasmid carriage were transcriptional changes of genes on putative prophage or foreign DNA regions. The extents of the impacts on host phenotypes and transcriptomes were similarly greatest in KT2440 and lowest in Pf0-1. These findings suggest that host cell function was actively regulated by plasmid carriage.

Effects of Three Different Nucleoid-Associated Proteins Encoded on IncP-7 Plasmid pCAR1 on Host Pseudomonas putida KT2440

“…We also demonstrated that carriage of the plasmid affects chromosomal gene expression and the host phenotype using three different hosts: P. putida KT2440, P. aeruginosa PAO1, and P. fluorescens Pf0-1 (8). Moreover, comprehensive analysis of the host phenotype revealed that pCAR1 carriage reduces host fitness, swimming motility, and resistance to osmotic or pH stress and alters the primary metabolic capacity of the host cells (9). These effects on host phenotypes were considered to be dependent on various factors from both the plasmid and the host chromosome.…”

“…Extraction of total RNA from P. putida KT2440 (pCAR1) at the log growth phase was performed as described previously (9). cDNA synthesis, 5=-rapid amplification of cDNA ends (5=-RACE) PCR, and nested PCR were performed using a SMARTer RACE cDNA amplification kit (Clontech, Mountain View, CA) in accordance with the manufacturer's instructions.…”

“…Extraction of total RNA from P. putida KT2440(pCAR1) was performed as described previously (9). The primers were designed using the Primer3 program (29), and the cDNA was quantified using an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA) as described previously (9).…”

“…Probes for colony hybridization were prepared from a 1.7-kb HindIII-XbaI fragment of pCAR1 containing the repA gene and a 1.0-kb BglII-XhoI fragment of pCAR1 containing the carAc gene. Hybridization and detection was performed as described previously (9). To assess the effects of RecT overexpression on the stability of pCAR1, KT2440(pCAR1) harboring pBBad18K (33) containing the recT gene of pCAR1 was used in similar medium supplemented with 0.2% (wt/vol) L-arabinose to induce the expression of recT.…”

“…Transcriptome analyses were performed with custom-made tiling arrays, which were constructed as described previously (8,35). Total RNA extraction and cDNA syntheses in the presence of actinomycin D (Sigma-Aldrich, St. Louis, MO) were performed as described previously (9). The fragmentation and labeling of cDNA, hybridization with the tiling arrays, a KT2440(pCAR1⌬pmr), which was used in our previous study (23), was reconstructed in this study as described in Materials and Methods.…”