Iron is a nutrient of critical importance for the strict anaerobe Clostridium acetobutylicum, as it is involved in numerous basic cellular functions and metabolic pathways. A gene encoding a putative ferric uptake regulator (Fur) has been identified in the genome of C. acetobutylicum. In this work, we inactivated the fur gene by using insertional mutagenesis. The resultant mutant showed a slow-growing phenotype and enhanced sensitivity to oxidative stress, but essentially no dramatic change in its fermentation pattern. A unique feature of its physiology was the overflowing production of riboflavin. To gain further insights into the role of the Fur protein and the mechanisms for establishment of iron balance in C. acetobutylicum, we characterized and compared the gene-expression profile of the fur mutant and the iron-limitation stimulon of the parental strain. Not surprisingly, a repertoire of iron-transport systems was upregulated in both microarray datasets, suggesting that they are regulated by Fur according to the availability of iron. In addition, iron limitation and inactivation of fur affected the expression of several genes involved in energy metabolism. Among them, two genes, encoding a lactate dehydrogenase and a flavodoxin, were highly induced. In order to support the function of the latter, the ribDBAH operon responsible for riboflavin biosynthesis was also upregulated significantly. Furthermore, the iron-starvation response of C. acetobutylicum involved transcriptional modifications that were not detected in the fur mutant, suggesting that there exist additional mechanisms for adaptation to low-iron environments. Collectively, these results demonstrate that the strict anaerobe C. acetobutylicum senses and responds to availability of iron on multiple levels using a sophisticated system, and that Fur plays an important role in this process.

Understanding the interplay between a plasmid and its host system is a bottleneck towards prediction of the fate of plasmid-harbouring strains in the natural environments. Here, we studied the impact of the conjugative plasmid pCAR1, involved in carbazole degradation, on the proteome of Pseudomonas putida KT2440 using SILAC method. Furthermore, we investigated two acyl lysine modifications (acetylation and succinylation) that respond to the metabolic status of the cell and are implicated in regulation of various cellular processes. The total proteome analysis revealed that the abundance of key proteins involved in metabolism, signal transduction and motility was affected by pCAR1 carriage. In total, we identified 1359 unique acetylation sites on 637 proteins and 567 unique succinylation sites on 259 proteins. Changes in the acylation status of proteins involved in metabolism and translation by pCAR1 carriage were detected. Remarkably, acylation was identified on proteins involved in important plasmid functions, including partitioning and carbazole degradation, and on nucleoid-associated proteins that play a key role in the functional interaction with the chromosome. This study provides a novel insight on the functional consequences of plasmid carriage and improves our understanding of the plasmid-host cross-talk.

e 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.

H-NS family proteins play key roles in bacterial nucleoid compaction and global transcription. MvaT homologues in Pseudomonas have almost negligible amino acid sequence identity with H-NS, but can complement an hns-related phenotype of Escherichia coli. Here, we report the crystal structure of the N-terminal dimerization/oligomerization domain of TurB, an MvaT homologue in Pseudomonas putida KT2440. Our data identify two dimerization sites; the structure of the central dimerization site is almost the same as the corresponding region of H-NS, whereas the terminal dimerization sites are different. Our results reveal similarities and differences in dimerization and oligomerization mechanisms between H-NS and TurB.

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.