Transcriptome Analysis ofPseudomonas putidaKT2440 Harboring the Completely Sequenced IncP-7 Plasmid pCAR1

Abstract

The IncP-7 plasmid pCAR1 of Pseudomonas resinovorans CA10 confers the ability to degrade carbazole upon transfer to the recipient strain P. putida KT2440. We designed a customized whole-genome oligonucleotide microarray to study the coordinated expression of pCAR1 and the chromosome in the transconjugant strain KT2440(pCAR1). First, the transcriptome of KT2440(pCAR1) during growth with carbazole as the sole carbon source was compared to that during growth with succinate. The carbazole catabolic car and ant operons were induced, along with the chromosomal cat and pca genes involved in the catechol branch of the ␤-ketoadipate pathway. Additionally, the regulatory gene antR encoding the AraC/XylS family transcriptional activator specific for car and ant operons was upregulated. The characterization of the antR promoter revealed that antR is transcribed from an RpoN-dependent promoter, suggesting that the successful expression of the carbazole catabolic operons depends on whether the chromosome contains the specific RpoN-dependent activator. Next, to analyze whether the horizontal transfer of a plasmid alters the transcription network of its host chromosome, we compared the chromosomal transcriptomes of KT2440(pCAR1) and KT2440 under the same growth conditions. Only subtle changes were caused by the transfer of pCAR1, except for the significant induction of the hypothetical gene PP3700, designated parI, which encodes a putative ParA-like ATPase with an N-terminal Xre-type DNA-binding motif. Further transcriptional analyses showed that the parI promoter was positively regulated by ParI itself and the pCAR1-encoded protein ParA.Many catabolic plasmids can be transferred horizontally between different bacteria and play an important role in the distribution of the ability to degrade and utilize recalcitrant chemical compounds (15,90). Several catabolic plasmids within members of the genus Pseudomonas have been identified and have been classified mostly into incompatibility groups IncP-1, IncP-2, IncP-7, and IncP-9. Recently, the complete genome sequences of several IncP-1, IncP-7, and IncP-9 catabolic plasmids were determined (16,28,44,45,47,76,77,83,86,93). The 199,035-bp catabolic plasmid pCAR1 was originally discovered in Pseudomonas resinovorans CA10, which is able to utilize carbazole as its sole source of carbon, nitrogen, and energy (53, 56), and was the first IncP-7 plasmid to be completely sequenced (45). pCAR1 carries the car and ant operons, which encode the upper and meta pathway enzymes and the anthranilate 1,2-dioxygenase, respectively (see Fig. 1A). The operons are transcribed from two identical anthranilate-inducible promoters, P ant , under the control of the AraC/XylS family activator AntR (85), and the constitutive promoter P carAa also originates the transcription of the car operon (50). Carbazole catabolism begins with the upper pathway to yield anthranilate and 2-hydroxypenta-2,4-dienoate, and then 2-hydroxypenta-2,4-dienoate is mineralized into pyruvate and acetyl coenzyme A (acetylCoA) by the met...

Pmr, a Histone-Like Protein H1 (H-NS) Family Protein Encoded by the IncP-7 Plasmid pCAR1, Is a Key Global Regulator That Alters Host Function

“…Following inactivation of the DNase by the addition of the stop reagent and subsequent incubation at 65°C for 10 min, RNA samples were repurified with the Primer extension and pmr disruption. We identified the transcription start point (tsp) of pmr by primer extension analysis, performed as described previously (25). We used the IRD800-labeled primer PMR-R (Aloka Co., Ltd., Tokyo, Japan) (see Table S1 in the supplemental material), which anneals to the coding region of pmr from ϩ218 to ϩ237 (77782 to 77763 on pCAR1; see Table S1).…”

“…pCAR1 carries the pmr gene, encoding the H-NS family protein designated Pmr (plasmid-encoded MvaT-like regulator) (25) and belonging to the above-mentioned group III. The effect of plasmid carriage on host strains may change in different hosts, and therefore, we performed transcriptome comparisons between pCAR1-free and pCAR1-containing KT2440 strains (25,35).…”

“…pCAR1 carries the pmr gene, encoding the H-NS family protein designated Pmr (plasmid-encoded MvaT-like regulator) (25) and belonging to the above-mentioned group III. The effect of plasmid carriage on host strains may change in different hosts, and therefore, we performed transcriptome comparisons between pCAR1-free and pCAR1-containing KT2440 strains (25,35). Based on the comparisons, pCAR1 carriage affected the iron acquisition system of the host KT2440 strain, enhanced resistance to chloramphenicol by inducing the mexEF-oprN operon, and induced the transcription of PP_3700 (parI) (35).…”

The Behavior and Significance of Degradative Plasmids Belonging to Inc Groups in <i>Pseudomonas</i> within Natural Environments and Microcosms

“…are the principal host of pCAR1; indeed, bacteria of other genera are unable to replicate the plasmid (91), indicating a narrow host range (108). In the past 5 years, the effect of pCAR1 on host bacteria has been investigated through comparisons of chromosomal and plasmid transcriptomes (59,60,95). Its transfer frequency varies across bacterial species (90).…”

The Presence of Conjugative Plasmid pLS20 Affects Global Transcription of Its Bacillus subtilis Host and Confers Beneficial Stress Resistance to Cells

“…The transcriptional analysis of host and plasmid gene expression in different Escherichia coli or Pseudomonas putida strains containing large conjugative plasmids revealed host genotypespecific differences in gene expression on both the plasmid and the chromosome (9,10). These studies did not address whether the conjugative plasmid decreases fitness of host cells.…”