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cAMP receptor protein (CRP) downregulatesKlebsiella pneumoniae nif promoters inEscherichia coli
Zhiting Li,Yicheng Sun,Xianjun Mao,Yiping Wang
Chinese Science Bulletin , 2002, DOI: 10.1007/BF03184111
Abstract: In enteric bacteria, in response to the PTS system, the cAMP receptor protein (CRP) mediates the glucose effect,via regulating σ70-dependent catabolic genes at transcriptional level. In this study, it is observed that the nitrogen fixation capacity ofKlebsiella pneumoniae varies strongly when cells are grown on different carbohydrates, and this carbon effect occurs at the level ofnif gene expression. Here we show that CRP can repress σ54-dependentnif promoters (nifB, nifE, nifF, nifH, nifJ, nifLA andnifU), in a cAMP dependent fashion, in closed relatedK coli background. Sequence analysis of thesenif promoters indicates that there is no direct correlation between the fold of CRP-cAMP-mediated inhibition and the upstreamcis elements at the promoters. In addition, thecrp gene ofK. pneumoniae has been isolated and sequenced, which is structural and functional highly homologous to that ofE. coli. This suggests that CRP-cAMP-mediated inhibition on thenif promoters could be the reason for carbon effect on nitrogen fixation and thus has its physiological significance. A novel regulatory linkage between carbon metabolism and nitrogen fixation is proposed.
Novel Roles of cAMP Receptor Protein (CRP) in Regulation of Transport and Metabolism of Carbon Sources  [PDF]
Tomohiro Shimada, Nobuyuki Fujita, Kaneyoshi Yamamoto, Akira Ishihama
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0020081
Abstract: CRP (cAMP receptor protein), the global regulator of genes for carbon source utilization in the absence of glucose, is the best-studied prokaryotic transcription factor. A total of 195 target promoters on the Escherichia coli genome have been proposed to be under the control of cAMP-bound CRP. Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites. Based on their location on the E. coli genome, we predict a total of at least 183 novel regulation target operons, altogether with the 195 hitherto known targets, reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP. All the promoters selected from the newly identified targets and examined by using the lacZ reporter assay were found to be under the control of CRP, indicating that the Genomic SELEX screening allowed to identify the CRP targets with high accuracy. Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.
Comparative Analysis of Fatty Acid Desaturases in Cyanobacterial Genomes  [PDF]
Xiaoyuan Chi,Qingli Yang,Fangqing Zhao,Song Qin,Yu Yang,Junjun Shen,Hanzhi Lin
Comparative and Functional Genomics , 2008, DOI: 10.1155/2008/284508
Abstract: Fatty acid desaturases are enzymes that introduce double bonds into the hydrocarbon chains of fatty acids. The fatty acid desaturases from 37 cyanobacterial genomes were identified and classified based upon their conserved histidine-rich motifs and phylogenetic analysis, which help to determine the amounts and distributions of desaturases in cyanobacterial species. The filamentous or N2-fixing cyanobacteria usually possess more types of fatty acid desaturases than that of unicellular species. The pathway of acyl-lipid desaturation for unicellular marine cyanobacteria Synechococcus and Prochlorococcus differs from that of other cyanobacteria, indicating different phylogenetic histories of the two genera from other cyanobacteria isolated from freshwater, soil, or symbiont. Strain Gloeobacter violaceus PCC 7421 was isolated from calcareous rock and lacks thylakoid membranes. The types and amounts of desaturases of this strain are distinct to those of other cyanobacteria, reflecting the earliest divergence of it from the cyanobacterial line. Three thermophilic unicellular strains, Thermosynechococcus elongatus BP-1 and two Synechococcus Yellowstone species, lack highly unsaturated fatty acids in lipids and contain only one Δ9 desaturase in contrast with mesophilic strains, which is probably due to their thermic habitats. Thus, the amounts and types of fatty acid desaturases are various among different cyanobacterial species, which may result from the adaption to environments in evolution.
Cyanobacterial contribution to the genomes of the plastid-lacking protists
Shinichiro Maruyama, Motomichi Matsuzaki, Kazuharu Misawa, Hisayoshi Nozaki
BMC Evolutionary Biology , 2009, DOI: 10.1186/1471-2148-9-197
Abstract: We identified 12 gene families with cyanobacterial ancestry in the genomes of a taxonomically wide range of plastid-lacking eukaryotes (Phytophthora [Chromalveolata], Naegleria [Excavata], Dictyostelium [Amoebozoa], Saccharomyces and Monosiga [Opisthokonta]) using a novel phylogenetic pipeline. The eukaryotic gene clades with cyanobacterial ancestry were mostly composed of genes from bikonts (Archaeplastida, Chromalveolata, Rhizaria and Excavata). We failed to find genes with cyanobacterial ancestry in Saccharomyces and Dictyostelium, except for a photorespiratory enzyme conserved among fungi. Meanwhile, we found several Monosiga genes with cyanobacterial ancestry, which were unrelated to other Opisthokonta genes.Our data demonstrate that a considerable number of genes with cyanobacterial ancestry have contributed to the genome composition of the plastid-lacking protists, especially bikonts. The origins of those genes might be due to lateral gene transfer events, or an ancient primary or secondary endosymbiosis before the diversification of bikonts. Our data also show that all genes identified in this study constitute multi-gene families with punctate distribution among eukaryotes, suggesting that the transferred genes could have survived through rounds of gene family expansion and differential reduction.Cyanobacterial ancestors gave rise to plastids (chloroplasts) in the ancestor of a eukaryotic lineage. The birth of the plastid had an impact on eukaryotic genome evolution, by way of endosymbiotic gene transfer (EGT), a particular form of lateral gene transfer (LGT) from endosymbionts into the phylogenetically discontiguous host genome [1]. Subsequently, an algal ancestor gave rise to secondary plastids in several punctate lineages of eukaryotes. A number of these secondarily phototrophic lineages lost their photosynthetic ability and further diverged into secondarily heterotrophic, plastid-lacking protists [2,3].Although the position of the root of eukaryotes is s
Phycobilisomes linker family in cyanobacterial genomes: divergence and evolution
Xiangyu Guan, Song Qin, Fangqing Zhao, Xiaowen Zhang, Xuexi Tang
International Journal of Biological Sciences , 2007,
Abstract: Cyanobacteria are the oldest life form making important contributions to global CO2 fixation on the Earth. Phycobilisomes (PBSs) are the major light harvesting systems of most cyanobacteria species. Recent availability of the whole genome database of cyanobacteria provides us a global and further view on the complex structural PBSs. A PBSs linker family is crucial in structure and function of major light-harvesting PBSs complexes. Linker polypeptides are considered to have the same ancestor with other phycobiliproteins (PBPs), and might have been diverged and evolved under particularly selective forces together. In this paper, a total of 192 putative linkers including 167 putative PBSs-associated linker genes and 25 Ferredoxin-NADP oxidoreductase (FNR) genes were detected through whole genome analysis of all 25 cyanobacterial genomes (20 finished and 5 in draft state). We compared the PBSs linker family of cyanobacteria in terms of gene structure, chromosome location, conservation domain, and polymorphic variants, and discussed the features and functions of the PBSs linker family. Most of PBSs-associated linkers in PBSs linker family are assembled into gene clusters with PBPs. A phylogenetic analysis based on protein data demonstrates a possibility of six classes of the linker family in cyanobacteria. Emergence, divergence, and disappearance of PBSs linkers among cyanobacterial species were due to speciation, gene duplication, gene transfer, or gene loss, and acclimation to various environmental selective pressures especially light.
Enhancing E. coli Tolerance towards Oxidative Stress via Engineering Its Global Regulator cAMP Receptor Protein (CRP)  [PDF]
Souvik Basak, Rongrong Jiang
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0051179
Abstract: Oxidative damage to microbial hosts often occurs under stressful conditions during bioprocessing. Classical strain engineering approaches are usually both time-consuming and labor intensive. Here, we aim to improve E. coli performance under oxidative stress via engineering its global regulator cAMP receptor protein (CRP), which can directly or indirectly regulate redox-sensing regulators SoxR and OxyR, and other ~400 genes in E. coli. Error-prone PCR technique was employed to introduce modifications to CRP, and three mutants (OM1~OM3) were identified with improved tolerance via H2O2 enrichment selection. The best mutant OM3 could grow in 12 mM H2O2 with the growth rate of 0.6 h?1, whereas the growth of wild type was completely inhibited at this H2O2 concentration. OM3 also elicited enhanced thermotolerance at 48°C as well as resistance against cumene hydroperoxide. The investigation about intracellular reactive oxygen species (ROS), which determines cell viability, indicated that the accumulation of ROS in OM3 was always lower than in WT with or without H2O2 treatment. Genome-wide DNA microarray analysis has shown not only CRP-regulated genes have demonstrated great transcriptional level changes (up to 8.9-fold), but also RpoS- and OxyR-regulated genes (up to 7.7-fold). qRT-PCR data and enzyme activity assay suggested that catalase (katE) could be a major antioxidant enzyme in OM3 instead of alkyl hydroperoxide reductase or superoxide dismutase. To our knowledge, this is the first work on improving E. coli oxidative stress resistance by reframing its transcription machinery through its native global regulator. The positive outcome of this approach may suggest that engineering CRP can be successfully implemented as an efficient strain engineering alternative for E. coli.
Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)  [PDF]
Huiqing Chong, Jianwei Yeow, Ivy Wang, Hao Song, Rongrong Jiang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0077422
Abstract: The presence of acetate exceeding 5 g/L is a major concern during E. coli fermentation due to its inhibitory effect on cell growth, thereby limiting high-density cell culture and recombinant protein production. Hence, engineered E. coli strains with enhanced acetate tolerance would be valuable for these bioprocesses. In this work, the acetate tolerance of E. coli was much improved by rewiring its global regulator cAMP receptor protein (CRP), which is reported to regulate 444 genes. Error-prone PCR method was employed to modify crp and the mutagenesis libraries (~3×106) were subjected to M9 minimal medium supplemented with 5–10 g/L sodium acetate for selection. Mutant A2 (D138Y) was isolated and its growth rate in 15 g/L sodium acetate was found to be 0.083 h-1, much higher than that of the control (0.016 h-1). Real-time PCR analysis via OpenArray? system revealed that over 400 CRP-regulated genes were differentially expressed in A2 with or without acetate stress, including those involved in the TCA cycle, phosphotransferase system, etc. Eight genes were chosen for overexpression and the overexpression of uxaB was found to lead to E. coli acetate sensitivity.
Improving Ethanol Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)  [PDF]
Huiqing Chong, Lei Huang, Jianwei Yeow, Ivy Wang, Hongfang Zhang, Hao Song, Rongrong Jiang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0057628
Abstract: A major challenge in bioethanol fermentation is the low tolerance of the microbial host towards the end product bioethanol. Here we report to improve the ethanol tolerance of E. coli from the transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP), which is known to regulate over 400 genes in E. coli. Three ethanol tolerant CRP mutants (E1– E3) were identified from error-prone PCR libraries. The best ethanol-tolerant strain E2 (M59T) had the growth rate of 0.08 h?1 in 62 g/L ethanol, higher than that of the control at 0.06 h?1. The M59T mutation was then integrated into the genome to create variant iE2. When exposed to 150 g/l ethanol, the survival of iE2 after 15 min was about 12%, while that of BW25113 was <0.01%. Quantitative real-time reverse transcription PCR analysis (RT-PCR) on 444 CRP-regulated genes using OpenArray? technology revealed that 203 genes were differentially expressed in iE2 in the absence of ethanol, whereas 92 displayed differential expression when facing ethanol stress. These genes belong to various functional groups, including central intermediary metabolism (aceE, acnA, sdhD, sucA), iron ion transport (entH, entD, fecA, fecB), and general stress response (osmY, rpoS). Six up-regulated and twelve down-regulated common genes were found in both iE2 and E2 under ethanol stress, whereas over one hundred common genes showed differential expression in the absence of ethanol. Based on the RT-PCR results, entA, marA or bhsA was knocked out in iE2 and the resulting strains became more sensitive towards ethanol.
Type 1 Fimbriae, a Colonization Factor of Uropathogenic Escherichia coli, Are Controlled by the Metabolic Sensor CRP-cAMP  [PDF]
Claudia M. Müller,Anna ?berg,Jurate Strasevi?iene,Levente Em?dy,Bernt Eric Uhlin ,Carlos Balsalobre
PLOS Pathogens , 2009, DOI: 10.1371/journal.ppat.1000303
Abstract: Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues.
Characterization of ligand response properties of the CRP protein from Pseudomonas putida
Feng Jiang,ZheXian Tian,YiPing Wang
Chinese Science Bulletin , 2012, DOI: 10.1007/s11434-012-5360-3
Abstract: cAMP receptor protein (CRP) plays profound roles in many bacteria as a global regulator. In Escherichia coli, CRP E. coli modulates the expression of many operons involved in carbon catabolism, in response to the fluctuation of intracellular cAMP level caused by carbon catabolism. A crp homologue gene has been identified in the genome of Pseudomonas putida, however, little is known about its cellular function. In this work, we investigated ligand response properties of this CRP protein (CRP P. putida ). The results showed that in the presence of exogenous cAMP or cGMP, CRP P. putida can activate the lac promoter in E. coli cya crp mutant. In vitro isothermal titration calorimetry (ITC) assays indicated that CRP P. putida could bind cAMP as well as cGMP. Its affinity to cAMP is much higher than CRP E. coli . Sequence alignment of the CRP proteins suggested that the Thr132 of CRP P. putida (analogous to Ser128 of CRP E. coli ) could be the key determinant for all ligand responsive properties observed above. When Thr132 of CRP P. putida is mutated to Serine, two phenomena were observed: (i) its affinity to cAMP or cGMP was reduced to a level similar to CRP E. coli ; (ii) its transcriptional activation activity on E. coli lac promoter was diminished. The potential physiological implications of these ligand binding properties are discussed.
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