Growth phase-dependent expression profiles of three vital H-NS family proteins encoded on the chromosome of Pseudomonas putida KT2440 and on the pCAR1 plasmid

Abstract

BackgroundH-NS family proteins are nucleoid-associated proteins that form oligomers on DNA and function as global regulators. They are found in both bacterial chromosomes and plasmids, and were suggested to be candidate effectors of the interaction between them. TurA and TurB are the predominantly expressed H-NS family proteins encoded on the chromosome of Pseudomonas putida KT2440, while Pmr is encoded on the carbazole-degradative incompatibility group P-7 plasmid pCAR1. Previous transcriptome analyses suggested that they function cooperatively, but play different roles in the global transcriptional network. In addition to differences in protein interaction and DNA-binding functions, cell expression levels are important in clarifying the detailed underlying mechanisms. Here, we determined the precise protein amounts of TurA, TurB, and Pmr in KT2440 in the presence and absence of pCAR1.ResultsThe intracellular amounts of TurA and TurB in KT2440 and KT2440(pCAR1) were determined by quantitative western blot analysis using specific antibodies. The amount of TurA decreased from the log phase (~80,000 monomers per cell) to the stationary phase (~20,000 monomers per cell), while TurB was only detectable upon entry into the stationary phase (maximum 6000 monomers per cell). Protein amounts were not affected by pCAR1 carriage. KT2440(pCAR1pmrHis), where histidine-tagged Pmr is expressed under its original promotor, was used to determine the intracellular amount of Pmr, which was constant (~30,000 monomers per cell) during cell growth. Quantitative reverse transcription PCR demonstrated that the transcriptional levels of turA and turB were consistent with protein expression, though the transcriptional and translational profiles of Pmr differed.ConclusionThe amount of TurB increases as TurA decreases, and the amount of Pmr does not affect the amounts of TurA and TurB. This is consistent with our previous observation that TurA and TurB play complementary roles, whereas Pmr works relatively independently. This study provides insight into the molecular mechanisms underlying reconstitution of the transcriptional network in KT2440 by pCAR1 carriage.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-017-1091-6) contains supplementary material, which is available to authorized users.

Horizontally Acquired Homologs of Xenogeneic Silencers: Modulators of Gene Expression Encoded by Plasmids, Phages and Genomic Islands

“…Moreover, the effect of an hnsB mutation on the generation time, capsule expression, and bgl expression is stronger at 25 °C compared with 37 and 42 °C [53]. Contrary to the hfp pattern, the expression of sfh and pmr is high during the log phase, although it was observed that the amount of the Sfh protein increases during the stationary phase and Pmr remains relatively constant along the growth curve [55,124,125]. Further research is required to address the different conditions and contexts that might be modulating the expression of the horizontally acquired XS homologs.…”

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

“…Second, TurA and TurB may compensate for each other functionally during the stationary phase. The number of TurB molecules during the stationary phase was approximately 3,000 molecules per cell, and the number of TurA molecules decreased to 25,000 molecules per cell (Sun et al, 2017). Since the difference in the number of TurA and TurB molecules was smaller during the stationary phase than in the log phase, TurA and TurB may complement each other during the stationary phase if one of them is lost from the cell, resulting in a small number of differentially transcribed genes in KT2440 turA and KT2440 turB.…”

“…This difference in the numbers of the differentially transcribed genes after the deletion of turA or turB likely stems from differing numbers of TurA and TurB molecules in the cell. The number of TurA molecules per cell in KT2440 during the log phase was approximately 60,000 molecules, in contrast to the number of TurB molecules per cell, which was less than 1,000 molecules (Sun et al, 2017). On the other hand, during the stationary phase, only 18 and 13 genes were differentially transcribed in KT2440 turA and KT2440 turB, respectively (Figure 1).…”

“…As a result, Pmr may function as a molecular backup to TurA and TurB. Our previous study showed that Pmr numbers approximately 30,000 molecules per cell in both the log and stationary phases, which is almost the same expression level as that of TurA (Sun et al, 2017). Thus, Pmr might work to enlarge the pool of MvaT homologs in the cell.…”

“…From transcriptome analyses using Pseudomonas putida KT2440 as a host of pCAR1 and pCAR1 pmr, we previously found that Pmr has a "stealth" function resembling that of Sfh, while transcription levels of some genes on the chromosome and pCAR1 seemed to be specifically regulated by Pmr (Yun et al, 2010). Among the five genes encoding MvaT homologs in KT2440, turA and turB are considered to be transcribed predominantly, and their protein expression level was previously determined (Yuste et al, 2006;Renzi et al, 2010;Yun et al, 2010;Sun et al, 2017). TurA and TurB are homologs of MvaT and MvaU of Pseudomonas aeruginosa PAO1, which bind to almost the same DNA regions and function coordinately (Castang et al, 2008).…”