Project description:KaiC is the central cog of the circadian clock in Cyanobacteria. Close homologs of this protein are widespread among bacteria not known to have a circadian physiology. The function, interaction network, and mechanism of action of these KaiC homologs are still largely unknown. Here, we focus on KaiC homologs found in environmental Pseudomonas species. We characterize experimentally the only KaiC homolog present in Pseudomonas putida KT2440 and Pseudomonas protegens CHA0. Through phenotypic assays and transcriptomics, we show that KaiC is involved in osmotic and oxidative stress resistance in P. putida and in biofilm production in both P. putida and P. protegens.
Project description:Pseudomonas putida KT2440 is a metabolically versatile soil bacterium useful both as a model biodegradative organism and as a host of catalytic activities of biotechnological interest. In this report, we present the high-resolution transcriptome of P. putida grown in different carbon sources as revealed by deep sequencing of the corresponding RNA pools. Examination of the data from growth on glycolytic (glucose, fructose) and gluconeogenic (succinate or glycerol) substrates revealed that > 20% of the P. putida genome is differentially expressed depending on the ensuing metabolic regime. Changes affected not only metabolic genes but also a suite of global regulators, e.g. the rpoS sigma subunit of RNAP, various cold-shock proteins and the three HU histone-like proteins. Specifically, the genes encoding HU subunit variants hupA, hupB and hupN drastically altered their expression levels (and thus their ability to form heterodimeric combinations) under the different growth conditions. Furthermore, we found that the two small RNAs crcZ and crcY, known to inhibit the Crc protein that mediates catabolite repression in P. putida, were both down-regulated by glucose. cDNA libraries from Pseudomonas supplemented with different carbon sources (glucose, glycerol, fructose, succinate) were sequenced using HiSeq 2000 to yield 91 paired-end reads. Gene expression values were compared.
Project description:Pseudomonas putida KT2440 is a metabolically versatile soil bacterium useful both as a model biodegradative organism and as a host of catalytic activities of biotechnological interest. In this report, we present the high-resolution transcriptome of P. putida grown in different carbon sources as revealed by deep sequencing of the corresponding RNA pools. Examination of the data from growth on glycolytic (glucose, fructose) and gluconeogenic (succinate or glycerol) substrates revealed that > 20% of the P. putida genome is differentially expressed depending on the ensuing metabolic regime. Changes affected not only metabolic genes but also a suite of global regulators, e.g. the rpoS sigma subunit of RNAP, various cold-shock proteins and the three HU histone-like proteins. Specifically, the genes encoding HU subunit variants hupA, hupB and hupN drastically altered their expression levels (and thus their ability to form heterodimeric combinations) under the different growth conditions. Furthermore, we found that the two small RNAs crcZ and crcY, known to inhibit the Crc protein that mediates catabolite repression in P. putida, were both down-regulated by glucose.
Project description:Alginate, a major exopolysaccharide (EPS) produced by P. putida, is known to create hydrated environments and alleviate the effect of water limitation. In addition to alginate, P. putida is capable of producing cellulose (bcs), putida exopolysaccharide a (pea), and putida exopolysaccharide b (peb). However, unlike alginate, not much is known about their roles under water limitation. Hence, in this study we examined the role of different EPS under water stress. To create environmentally realistic water stress conditions as observed in soil, we used Pressurized Porous Surface Model (PPSM). Our main hypothesis was that under water stress, absence of alginate would be compensated by the other EPS. To test our hypothesis, we investigated colony morphologies and whole genome transcriptomes of P. putida KT2440 WT and its mutants deficient in either alginate or all known EPS A custom-made Nimblegen (WI, USA) whole genome one-color oligonucleotide expression array (12x135K with 45-60 mer probes) of P. putida KT2440 was used to investigate effect of water stress on the differential expression of the whole genome. In this study Pseudomonas putida KT2440 wild type (WT) and two of its mutants deficient either in alginate (Alg-), or all known EPS (EPS-) production were used and grown under dry (water stress) and wet (without water stress) conditions. (Deleted genes in Alg-: PP1277-PP128; in EPS-: PP1277-1288 (alg) + PP2634-2638 (bcs) + PP3132-3142 (pea) + PP1795-1788 (peb)) (Nilsson et al., 2011).39. Nilsson, M., Chiang, W.C., Fazli, M., Gjermansen, M., Givskov, M., and Tolker-Nielsen, T. (2011) Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability. Environ Microbiol. 13 (5):1 357-1369