Yurika Takahashi

Response of the Pseudomonas host chromosomal transcriptome to carriage of the IncP-7 plasmid pCAR1

Plasmid carriage requires appropriate expression of the genes on the plasmid or host chromosome through cooperative transcriptional regulation. To clarify the impact of plasmid carriage on the host chromosome, we compared the chromosomal RNA maps of plasmid-free and plasmid-containing host strains using the incompatibility group P-7 archetype plasmid pCAR1, which is involved in carbazole degradation, and three distinct Pseudomonas strains. The possession of pCAR1 altered gene expression related to the iron acquisition systems in each host. Expression of the major siderophore pyoverdine was greater in plasmid-containing P. putida KT2440 and P. aeruginosa PAO1 than in the plasmid-free host strains, in part due to the expression of carbazole-degradative genes on pCAR1. The mexEFoprN operon encoding an efflux pump of the resistance-nodulation-cell division family was specifically upregulated by the carriage of pCAR1 in P. putida KT2440, whereas the expression of orthologous genes in the other species remained unaltered. Induction of the mexEFoprN genes increased the resistance of pCAR1-containing KT2440 to chloramphenicol compared with pCAR1-free KT2440. Our findings indicate that the possession of pCAR1 altered the growth rate of the host via the expression of genes on pCAR1 and the host chromosomes.

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

Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins (NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is transmissible among various Pseudomonas strains and carries a gene encoding the H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not affect the transcription of these five genes and that only pmr, turA, and turB were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed that Pmr strongly interacted with itself and with TurA, TurB, and TurE. Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1) strains indicated that pmr disruption had greater effects on the host transcriptome than did pCAR1 carriage. The transcriptional levels of some genes that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and parI, reverted with pmr disruption to levels in pCAR1-free KT2440. Transcriptional levels of putative horizontally acquired host genes were not altered by pCAR1 carriage but were altered by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification coupled with high-density tiling chip) analysis demonstrated that Pmr preferentially binds to horizontally acquired DNA regions. The Pmr binding sites overlapped well with the location of the genes differentially transcribed following pmr disruption on both the plasmid and the chromosome. Our findings indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and the host chromosome.

The Complete Nucleotide Sequence of pCAR2: pCAR2 and pCAR1 Were Structurally Identical IncP-7 Carbazole Degradative Plasmids

pCAR1 and pCAR2 are IncP-7 self-transmissible carbazole degradative plasmids. Their respective hosts showed clearly different conjugative host ranges. Their complete nucleotide sequences were virtually the same, and can be regarded as structurally the same plasmid, indicating that the difference in the conjugative host range was caused by host cell backgrounds.

Carbazole-degradative IncP-7 plasmid pCAR1.2 is structurally unstable in Pseudomonas fluorescens Pf0-1, which accumulates catechol, the intermediate of the carbazole degradation pathway.

ABSTRACT We determined the effect of the host on the function and structure of the nearly identical IncP-7 carbazole-degradative plasmids pCAR1.1 and pCAR1.2. We constructed Pseudomonas aeruginosa PAO1(pCAR1.2) and P. fluorescens Pf0-1Km(pCAR1.2) and compared their growth on carbazole- and succinate-containing media with that of P. putida KT2440(pCAR1.1). We also assessed the stability of the genetic structures of the plasmids in each of the three hosts. Pf0-1Km(pCAR1.2) showed dramatically delayed growth when carbazole was supplied as the sole carbon source, while the three strains grew at nearly the same rate on succinate. Among the carbazole-grown Pf0-1Km(pCAR1.2) cells, two types of deficient strains appeared and dominated the population; such dominance was not observed in the other two strains or for succinate-grown Pf0-1Km(pCAR1.2). Genetic analysis showed that the two deficient strains possessed pCAR1.2 derivatives in which the carbazole-degradative car operon was deleted or its regulatory gene, antR, was deleted by homologous recombination between insertion sequences. From genomic information and quantitative reverse transcription-PCR analyses of the genes involved in carbazole mineralization by Pf0-1Km(pCAR1.2), we found that the cat genes on the chromosome of Pf0-1Km, which are necessary for the degradation of catechol (a toxic intermediate in the carbazole catabolic pathway), were not induced in the presence of carbazole. The resulting accumulation of catechol may have enabled the strain that lost its carbazole-degrading ability to have overall higher fitness than the wild-type strain. These results suggest that the functions of the chromosomal genes contributed to the selection of plasmid derivatives with altered structures.

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

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.

MvaT Family Proteins Encoded on IncP-7 Plasmid pCAR1 and the Host Chromosome Regulate the Host Transcriptome Cooperatively but Differently

ABSTRACT MvaT proteins are members of the H-NS family of proteins in pseudomonads. The IncP-7 conjugative plasmid pCAR1 carries an mvaT-homologous gene, pmr. In Pseudomonas putida KT2440 bearing pCAR1, pmr and the chromosomally carried homologous genes, turA and turB, are transcribed at high levels, and Pmr interacts with TurA and TurB in vitro. In the present study, we clarified how the three MvaT proteins regulate the transcriptome of P. putida KT2440(pCAR1). Analyses performed by a modified chromatin immunoprecipitation assay with microarray technology (ChIP-chip) suggested that the binding regions of Pmr, TurA, and TurB in the P. putida KT2440(pCAR1) genome are almost identical; nevertheless, transcriptomic analyses using mutants with deletions of the genes encoding the MvaT proteins during the log and early stationary growth phases clearly suggested that their regulons were different. Indeed, significant regulon dissimilarity was found between Pmr and the other two proteins. Transcription of a larger number of genes was affected by Pmr deletion during early stationary phase than during log phase, suggesting that Pmr ameliorates the effects of pCAR1 on host fitness more effectively during the early stationary phase. Alternatively, the similarity of the TurA and TurB regulons implied that they might play complementary roles as global transcriptional regulators in response to plasmid carriage.

Effects of three different nucleoid-associated proteins encoded on IncP-7 plasmid pCAR1 on host Pseudomonas putida KT2440.

ABSTRACT Nucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption of pmr, pnd, and phu were assessed in host Pseudomonas putida KT2440. When pmr and pnd or pmr and phu were simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation of pmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype.

Inhibition of Pseudomonas aeruginosa swarming motility by 1-naphthol and other bicyclic compounds bearing hydroxyl groups.

ABSTRACT Many bacteria convert bicyclic compounds, such as indole and naphthalene, to oxidized compounds, including hydroxyindoles and naphthols. Pseudomonas aeruginosa, a ubiquitous bacterium that inhabits diverse environments, shows pathogenicity against animals, plants, and other microorganisms, and increasing evidence has shown that several bicyclic compounds alter the virulence-related phenotypes of P. aeruginosa. Here, we revealed that hydroxyindoles (4- and 5-hydroxyindoles) and naphthalene derivatives bearing hydroxyl groups specifically inhibit swarming motility but have minor effects on other motilities, including swimming and twitching, in P. aeruginosa. Further analyses using 1-naphthol showed that this effect is also associated with clinically isolated hyperswarming P. aeruginosa cells. Swarming motility is associated with the dispersion of cells from biofilms, and the addition of 1-naphthol maintained biofilm biomass without cell dispersion. We showed that this 1-naphthol-dependent swarming inhibition is independent of changes of rhamnolipid production and the intracellular level of signaling molecule cyclic-di-GMP (c-di-GMP). Transcriptome analyses revealed that 1-naphthol increases gene expression associated with multidrug efflux and represses gene expression associated with aerotaxis and with pyochelin, flagellar, and pilus synthesis. In the present study, we showed that several bicyclic compounds bearing hydroxyl groups inhibit the swarming motility of P. aeruginosa, and these results provide new insight into the chemical structures that inhibit the specific phenotypes of P. aeruginosa.

Effects of carbazole‐degradative plasmid pCAR1 on biofilm morphology in Pseudomonas putida KT2440

Bacteria typically form biofilms under natural conditions. To elucidate the effect of the carriage of carbazole-degradative plasmid pCAR1 on biofilm formation by host bacteria, we compared the biofilm morphology, using confocal laser scanning microscopy, of three pCAR1-free and pCAR1-carrying Pseudomonas hosts: P. putida KT2440, P. aeruginosa PAO1 and P. fluorescens Pf0-1. Although pCAR1 did not significantly affect biofilm formation by PAO1 or Pf0-1, pCAR1-carrying KT2440 became filamentous and formed flat biofilms, whereas pCAR1-free KT2440 formed mushroom-like biofilms. pCAR1 contains three genes encoding nucleoid-associated proteins (NAPs), namely, Pmr, Pnd and Phu. The enhanced filamentous morphology was observed in two double mutants [KT2440(pCAR1ΔpmrΔpnd) and KT2440(pCAR1ΔpmrΔphu)], suggesting that these NAPs are involved in modulating the filamentous phenotype. Transcriptome analyses of the double mutants identified 32 candidate genes that may be involved in filamentation of KT2440. Overexpression of PP_2193 in KT2440 induced filamentation and overexpression of PP_0308 or PP_0309 in KT2440(pCAR1) enhanced filamentation of cells over time. This suggests that pCAR1 induces development of an abnormal filamentous morphology by KT2440 via a process involving overexpression of several genes, such as PP_2193. In addition, pCAR1-encoded NAPs partly suppress too much filamentation of KT2440(pCAR1) by repressing transcription of some genes, such as PP_0308 and PP_0309.

Conjugative Elements: Host Chromosome Function Modifiers

Conjugative transfer of plasmids and integrative and conjugative elements (ICE s) play a key role in rapid bacterial evolution and adaptation. These elements give a host several new phenotypes that can be advantageous for host survival in some cases, but disadvantageous in others. Comparisons of growth rate and biomass yield as well as competition assays have shown that carriage of plasmids frequently imposes a fitness cost on the host cells. New phenotypes are caused by cross talk between plasmid- and chromosome-encoded factors during implantation of regulatory circuits into the host native transcriptional network. Nucleoid-associated proteins (NAPs ) are important factors for adaptation of the ICEs to the new host cell environment. Plasmids and ICEs, containing genes involved in the biodegradation of various compounds, are excellent models to study the impact on the host. A chlorocatechol-degradative ICE, ICE clc , can express its degradative genes without changing the fitness of the new host. The host of toluene/xylene-degradative plasmid pWW0 builds cooperative regulatory systems to express metabolic activity. The influence of naphthalene-degradative plasmid NAH7 on the host cells is modest, and the carriage of NAH7 alleviates host stress when exposed to naphthalene. The effects of carbazole-degradative pCAR1 carriage on the host cells depend on the type of host, and the NAPs encoded on pCAR1 are important for controlling the transcriptional network of the host. The extent of the impact on the host cells changes the genetic stability of these elements in the host, which is important for spreading these genetic elements among different bacteria.