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H-NS Family Proteins Drastically Change Their Targets in Response to the Horizontal Transfer of the Catabolic Plasmid pCAR1.

ABSTRACT: H-NS family proteins regulate the expression of many genes by preferably binding to AT-rich genomic regions and altering DNA topology. They are found in both bacterial chromosomes and plasmids, and plasmid-encoded H-NS family proteins have sometimes been suggested to act as a molecular backup of the chromosomally encoded ones. Pmr is an H-NS family protein encoded on the catabolic plasmid pCAR1, which belongs to incompatibility P-7 group. We have investigated the function of Pmr in Pseudomonas putida KT2440, where two H-NS family proteins (TurA and TurB) encoded on the chromosome are expressed predominantly. Previous transcriptome analyses suggested that TurA, TurB, and Pmr cooperatively regulate numerous genes, but the differentially transcribed genes in KT2440?turA(pCAR1), KT2440?turB(pCAR1), and KT2440(pCAR1?pmr) compared with those in KT2440(pCAR1) were somewhat different. Here, we performed RNA sequencing analyses to compare the differentially transcribed genes after the deletion of turA or turB in KT2440, and turA, turB or pmr in KT2440(pCAR1). Three pCAR1-free strains (KT2440, KT2440?turA, KT2440?turB) and four pCAR1-harboring strains [KT2440(pCAR1), KT2440?turA(pCAR1), KT2440?turB(pCAR1), KT2440(pCAR1?pmr)], grown until the log and stationary phases, were used. In KT2440, TurA was the major H-NS family protein regulating a large number and wide range of genes, and both TurA and TurB were suggested to functionally compensate each other, particularly during the stationary phase. In KT2440(pCAR1), the numbers of differentially transcribed genes after the deletion of turA or turB drastically increased compared to those in KT2440. Notably, more than half of the differentially transcribed genes in KT2440?turA and KT2440?turB did not overlap with those in KT2440?turA(pCAR1) and KT2440?turB(pCAR1). This dynamic change could be explained by the acquisition of pCAR1 itself and the expression of Pmr. After pCAR1 was transferred into the host, TurA and TurB could be detached from the chromosome of KT2440 and they could newly bind to pCAR1. Moreover, Pmr could reconstitute the chromosome-binding heteromeric oligomers which were formed by TurA and TurB. Our study revealed that horizontal transfer of a plasmid changes the transcriptional network of the chromosomally encoded H-NS family proteins.

SUBMITTER: Nakamura T

PROVIDER: S-EPMC7273181 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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H-NS Family Proteins Drastically Change Their Targets in Response to the Horizontal Transfer of the Catabolic Plasmid pCAR1.

Nakamura Taisuke T Suzuki-Minakuchi Chiho C Kawano Hibiki H Kanesaki Yu Y Kawasaki Shinji S Okada Kazunori K Nojiri Hideaki H

Frontiers in microbiology 20200529

H-NS family proteins regulate the expression of many genes by preferably binding to AT-rich genomic regions and altering DNA topology. They are found in both bacterial chromosomes and plasmids, and plasmid-encoded H-NS family proteins have sometimes been suggested to act as a molecular backup of the chromosomally encoded ones. Pmr is an H-NS family protein encoded on the catabolic plasmid pCAR1, which belongs to incompatibility P-7 group. We have investigated the function of Pmr in <i>Pseudomona ...[more]

PMID: 32547524

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Project description:To elucidate why plasmid-borne catabolic ability differs among host bacteria, we assessed the expression dynamics of the Pant promoter on the carbazole-degradative conjugative plasmid pCAR1 in Pseudomonas putida KT2440(pCAR1) (hereafter, KTPC) and Pseudomonas resinovorans CA10. The Pant promoter regulates the transcription of both the car and ant operons, which are responsible for converting carbazole into anthranilate and anthranilate into catechol, respectively. In the presence of anthranilate, transcription of the Pant promoter is induced by the AraC/XylS family regulator AntR, encoded on pCAR1. A reporter cassette containing the Pant promoter followed by gfp was inserted into the chromosomes of KTPC and CA10. After adding anthranilate, GFP expression in the population of CA10 showed an unimodal distribution, whereas a small population with low GFP fluorescence intensity appeared for KTPC. CA10 has a gene, antRCA, that encodes an iso-functional homolog of AntR on its chromosome. When antRCA was disrupted, a small population with low GFP fluorescence intensity appeared. In contrast, overexpression of pCAR1-encoded AntR in KTPC resulted in unimodal expression under the Pant promoter. These results suggest that the expression of pCAR1-encoded AntR is insufficient to ameliorate the stochastic expression of the Pant promoter. Raman spectra of single cells collected using deuterium-labeled carbazole showed that the C-D Raman signal exhibited greater variability for KTPC than CA10. These results indicate that heterogeneity at the transcriptional level of the Pant promoter due to insufficient AntR availability causes fluctuations in the pCAR1-borne carbazole-degrading capacity of host bacterial cells.IMPORTANCEHorizontally acquired genes increase the competitiveness of host bacteria under selective conditions, although unregulated expression of foreign genes may impose fitness costs. The "appropriate" host for a plasmid is empirically known to maximize the expression of plasmid-borne traits. In the case of pCAR1-harboring Pseudomonas strains, P. resinovorans CA10 exhibits strong carbazole-degrading capacity, whereas P. putida KT2440 harboring pCAR1 exhibits low degradation capacity. Our results suggest that a chromosomally encoded transcription factor affects transcriptional and metabolic fluctuations in host cells, resulting in different carbazole-degrading capacities as a population. This study may provide a clue for determining appropriate hosts for a plasmid and for regulating the expression of plasmid-borne traits, such as the degradation of xenobiotics and antibiotic resistance.

Project description:UNLABELLED:Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. IMPORTANCE:Diseases caused by multidrug-resistant bacteria are taking an important toll with respect to human morbidity and mortality. The most relevant antibiotic resistance genes come to human pathogens carried by plasmids, mainly using conjugation as a transmission mechanism. Here, we identified and characterized a series of compounds that were active against several plasmid groups of clinical relevance, in a wide variety of bacterial hosts. These inhibitors might be used for fighting antibiotic-resistance dissemination by inhibiting conjugation. Potential inhibitors could be used in specific settings (e.g., farm, fish factory, or even clinical settings) to investigate their effect in the eradication of undesired resistances.

Dataset Information

H-NS Family Proteins Drastically Change Their Targets in Response to the Horizontal Transfer of the Catabolic Plasmid pCAR1.

Publications

H-NS Family Proteins Drastically Change Their Targets in Response to the Horizontal Transfer of the Catabolic Plasmid pCAR1.

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