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Cyclic AMP receptor protein (CRP), also known as catabolite gene activator protein (CAP), is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed.
We have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution.
The CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The loss of CRPs in these species leads to the rapid loss of their binding sites in the genomes.
Cyclic AMP receptor protein (CRP), also known as catabolite gene activator protein (CAP), is an important transcriptional regulator widely distributed in a variety of bacterial groups [1,2]. The biological processes under the regulation of CRP are highly diverse, including energy metabolism [3,4], cell division and development [5], toxin production [1], competence development [6], quorum sensing [7] and cellular motility [8,9]. CRP belongs to the CRP/FNR transcription factor (TF) superfamily [10], which are generally believed to function as global regulators throughout the eubacteria [11]. Each member of the CRP/FNR superfamily contains an N-terminal effector binding domain and a C-terminal helix-turn-helix (HTH) DNA binding domain (DBD) [12]. The TFs of this superfamily form a homodimer in vivo, and are activated by the binding of specific small effector molecules to their effector binding domains [12]. The CRP dimer is activated by the binding of two cAMP molecules to the effector binding domain of each subunit, which causes a conformational change in the DBDs, allowing each to bind to half of a specific pseudo-palindromic DNA sequence in the promoters of the genes that are under CRP regulation [13]. Upon the binding, CRP interacts with the C-terminal domain of the alpha subunit of the RNA polymerase, affecting the RNA polymerase binding to the promoter, and thus leads to the change of the transcription initiation rate of the target gene [14-18].
The functions of CRP as well as its target genes (the CRP regulons) have been well studied in E. coli and other heterotrophic bacteria [19], and it seems that all the sequenced E. coli genomes encode one copy of the crp gene. CRP in E. coli is characterized as a global regulator, which controls the expression of more than 200 transcriptional units involved in various important biological processes of this organism [20,21]. Through decades of research, 269 CRP binding sites (RegulonDB release 5.8 [21]) in this species have been experimentally identified, which show a pseudo-palindromic consensus in the form of TGTGAN6TCACA. More recently, slightly different CRP binding sites with the consensus TGCGAN6TCGCA were also identified in E. coli and other γ-proteobacteria [22]. One of the major functions of CRP in E. coli involves the transcriptional regulation of genes related to organic carbon assimilation and energy metabolism [3,4].
As the life of E. coli and other heterotrophic organisms relies on the assimilation of organic carbon sources from the environment, it is not surprising that CRP works as an important global regulator to coordinate a variety of biological processes in these organisms. Cyanobacteria, on the other hand, are a group of autotrophic organisms capable of oxygenic photosynthesis; therefore, they do not rely on organic carbon source from the environment. Intriguingly, at least half of the sequenced cyanobacterial genomes encode at least one copy of the crp gene (see below). CRP proteins have been experimentally studied in two cyanobacterial strains, i.e. Synechocystis sp. PCC 6803 (PCC6803) [8,9,23-26] and Anabaena sp. PCC 7120 (PCC7120) [27,28]. In the PCC6803 genome, the open reading frame (ORF) sll1371 encodes a homologue to the E. coli crp gene [23], and has been named sycrp1. It has been shown that the product of this gene, SyCRP1 forms a homodimer, which can bind cAMP with high affinity in vitro [24]. Furthermore, in the presence of cAMP, SyCRP1 could form a complex with DNA that contains the consensus CRP binding site similar to that in E. coli (TGTGAN6TCACA) [24]. Further studies have revealed that SyCRP1 was essential for type IV pilus biogenesis and was involved in cell motility in PCC6803 [8,9,25,29,30]. On the other hand, in the PCC7120 genome two ORFs, alr0295 and alr2325, were found to encode putative CRPs, and were named ancrpA and ancrpB, respectively [27]. Equilibrium dialysis measurements showed that both AnCRPA and AnCRPB (the gene products of ancrpA and ancrpB respectively) could bind cAMP. Electrophoresis mobility shift assay (EMSA) further demonstrated that AnCrpA could bind to the consensus CRP binding site in E. coli [27]. It has also been reported that both AnCrpA and AnCrpB are functional in PCC7120, the former regulates the expression of several genes involved in nitrogen fixation [28], and the latter controls the genes induced by nitrogen depletion [31]. A few CRP binding sites in these two genomes have also been experimentally determined, which were found to form a palindromic motif with the consensus sequence TGTGAN6TCACA similar to that in E.coli [25,28]. In addition, the promoter regions of most of these identified CRP-activated genes in these two cyanobacterial genomes also contain an E. coli -10 σ70-like box (TAN3T), located ~22 bp downstream the CRP binding site. These studies also suggested that CRPs in cyanobacteria might regulate a very different set of genes than those in E. coli. However, a systematic genome-scale study of the potential CRP target genes as well as CRP binding sites in cyanobacteria is hitherto lacking. In this paper, we have predicted the CRP regulons as well as CRP binding sites in 12 sequenced cyanobacterial genomes that encode at least one copy of the crp gene using a highly effective motif scanning algorithm [32,33]. We have also investigated the degradation of the CRP binding sites in the rest of sequenced cyanobacterial genomes in which the crp genes were lost during the course of evolution.
1. Materials
Genome sequences, predicted ORFs and annotation files of the following 29 cyanobacterial genomes were downloaded from the NCBI website at ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria webcite: Acaryochloris marina MBIC11017 (MBIC11017), Anabaena variabilis ATCC 29413 (ATCC29413), Anabaena sp. PCC 7120 (PCC7120), Gloeobacter violaceus PCC 7421 (PCC7421), Prochlorococcus marinus str. AS9601 (AS9601), Prochlorococcus marinus str. MIT 9211 (MIT9211), Prochlorococcus marinus str. MIT 9215 (MIT9215), Prochlorococcus marinus str. MIT 9301 (MIT9301), Prochlorococcus marinus MIT9303 (MIT9303), Prochlorococcus marinus MIT9312 (MIT9312), Prochlorococcus marinus str. MIT 9313 (MIT9313), Prochlorococcus marinus str. MIT 9515 (MIT9515), Prochlorococcus marinus str. NATL1A (NATL1A), Prochlorococcus marinus str. NATL2A (NATL2A), Prochlorococcus marinus CCMP1375 (CCMP1375), Prochlorococcus marinus MED4 (MED4), Synechococcus elongatus PCC 6301 (PCC6301), Synechococcus elongatus PCC 7942 (PCC7942), Synechococcus sp. CC9311 (CC9311), Synechococcus sp. CC9605 (CC9605), Synechococcus sp. JA-2-3B'a(2–13) (B-Prime), Synechococcus sp. JA-3-3Ab (A-Prime), Synechocystis sp. PCC 6803 (PCC6803), Synechococcus sp. CC9902 (CC9902), Synechococcus sp. RCC307 (RCC307), Synechococcus sp. WH 7803 (WH7803), Synechococcus sp. WH8102 (WH8102), Thermosynechococcus elongatus BP-1 (BP-1), and Trichodesmium erythraeum IMS101 (IMS101).
2. Prediction of operons
We predicted operon structures in each cyanobacterial genomes using the Operon Finder Software (OFS) developed by Westover et al [34]. OFS predicts operons based on three pieces of information, including the intergenic distance, functional relatedness of gene annotations, and conserved gene neighborhoods. In this study, both a multi-gene operon and a singleton operon containing only one gene are referred as a transcription unit (TU).
3. Prediction of orthologues
A simple bidirectional best hit (BDBH) approach using the BLASTP program with an E-value cutoff 10-10 was used for the prediction of orthologous genes between each pair of genomes.
4. Phylogenetic analysis
To construct the CRP tree, the full length amino acid sequences of cyanobacterial CRPs were identified by the criteria described above using SyCRP1 (sll1371) in PCC6803 as the query sequence. Multiple sequence alignments of the identified cyanobacterial CRP sequences and that of E. coli K12 were performed using ClustalW implemented in MEGA [35] with default settings. A neighbor-joining (NJ) tree with Poisson correction was constructed using the MEGA program with the E. coli CRP (GI:16131236) being the outgroup. To construct the tree in Figure S1 (see Additional file 1), we used SyCRP1 as the query sequence to search the RefSeq database using BLASTP with an E-value cutoff 10-7. If there were multiple hits from a species, the hit with the smallest E-value was identified as the CRP in that species. The resulting sequences were used to construct an un-rooted tree in the similar way as described above. To construct the species tree, the DNA sequences of the 16S rRNA genes of the sequenced cyanobacteria and that of E. coli K12 were aligned using ClustalW with manual refinement. After the indels were discarded, the final alignments contain 1311 positions. A Neighbor-Joining tree of the 16 rRNA gene sequences was constructed with the E. coli K12 sequence being the outgroup using Kimura 2-parameter model. Statistical significance at each node in the trees was evaluated using 1000 bootstrap resamplings.
Additional File 1. Figure S1. A protein tree containing 202 SyCRP1 orthologues from a wide range of bacterial species. The red-labeled branch is CRPs from cyanobacteria.
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5. Phylogenetic footprinting and construction of the profile of CRP binding sites in cyanobacteria
A previous screening of the target genes of SyCRP1 in PCC6803 using microarray showed that 18 genes in 13 putative TUs were down-regulated in a sycrp1 disruptant compared with the wild type strain [25]. Our preliminary manual scanning of the upstream promoter regions of these TUs revealed that four of them contain a putative CRP binding site similar to those in E. coli (the first four TUs in Table 1). The rest nine TUs that do not contain putative CRP binding sites are likely to be regulated indirectly by CRP through different regulators. In addition, since the crp gene is auto-regulated in E. coli as well as in many other species [36], we also manually scanned the upstream inter-TU region of sycrp1 (sll1371) and identified a putative CRP binding site in it (Table 1). We thus used the orthologues (if they exist and were identified by the BDBH method) of these five TUs for the phylogenetic footprinting of CRP binding sites in cyanobacterial genomes.
Table 1. Transcriptional units in PCC6803 used for the initial phylogenetic footprinting analysis.
For this purpose, we pooled the entire upstream inter-TU regions of these five TUs in PCC6803, as well as those of TUs containing their orthologous genes in other cyanobacterial genomes which encode at least one crp gene. Motif finding programs including CUBIC [37] and BioProspector [38], were then applied to predict CRP binding sites in these sequences. CUBIC is a graph theoretic based algorithm that identifies highly similar k-mers in a set of pooled sequences; while BioProspector uses a Gibbs sampling strategy to find overrepresented k-mers in a set of pooled sequences. Putative CRP binding sites with high scores were manually picked up from the motif finding results, and were used to build a preliminary profile of the CRP binding sites in cyanobacterial genomes.
Since the number of binding sites used to construct the preliminary profile of CRP binding sites was relatively small (for the reason, see the Results section), in order to minimize possible bias of binding site sampling, we conducted a one-round iteration to obtain a more representative profile of the CRP binding sites. To this end, we scanned cyanobacterial genomes with the preliminary profile using the techniques described below, and picked the high scoring sites from each genome to construct the more representative profile of the CRP binding sites. We then used this final profile for genome-scale predictions of CRP binding sites in the cyanobacterial genomes.
6. Genome-wide prediction of CRP binding sites
The whole genome screening of all possible CRP binding sites were performed using an algorithm that we have developed previously [32]. The design of this algorithm is to enhance the prediction specificity by integrating the information of co-occurrence of multiple binding sites in the upstream region of a gene and that in the upstream regions of its orthologues in related genomes. Briefly, the final profile of the CRP binding sites obtained above was first used to scan all the inter-TU regions of the cyanobacterial genomes. The best motif was returned for each inter-TU region. Then the 19 to 31 bp downstream region of each putative binding site was further scanned for an E. coli -10 σ-70 like box (TAN3T) using a corresponding profile that we have constructed previously from cyanobacterial genomes [32], to which a σ-factor of RNA polymerase is likely to bind to transcribe the downstream TU. Then, the upstream inter-TU regions of the orthologues of the genes in that particular TU in other cyanobacterial genomes were scanned for similar CRP binding sites and -10 like boxes. A score that combines these three pieces of information was computed to rank the putative CRP binding sites for each possible CRP-regulated TU.
Specifically, let M be the final profile of the CRP binding sites obtained above. For each predicted transcription unit U(g1, ..., gn) containing genes g1, ..., gn in a genome G, we extract the entire upstream intergenic region (if that region is larger than 800 bp, then only the 800 bp upstream the translation starting site were extracted) and denoted it as . We also extracted a random DNA sequence with the same length as from the coding region, denoted as . We say that and are associated with U(g1, ..., gn) and with each genes g1, ..., gn as well. All the extracted from one genome are denoted as set IU, and all the in the same genome are denoted as set CU. For each t ∈ IU or t ∈ CU, we scan for possible CRP bindings sites using the profile M. The score of a putative binding site found in t by scanning with profile M is defined as,
where l is the length of the binding sites of M, h any substring of t with length l, h(i) the base at the i-th position of h, p(i, b) the relative frequency of base b occurring at position i in M, q(b) the background frequency of base b, and n the number of sequences used to construct M. When computing p(i, b), a pseudo count 1 is added to the frequency of each base at each position, and a is for normalization to keep Ii within the range of [0,1].
When multiple profiles are considered for scanning t, we sum up the individual SM(t) s as defined by
For this study, we use M1 for the CRP binding sites and M2 for the -10 like box (TAN3T).
For the inter-TU sequence t associated with U(g1, ..., gn) in genome G, if gi has orthologues in mi closely related genomes , we denote ok(gi) as the inter-TU sequence upstream the TU containing gi 's orthologue in genome Gk. When the presence of similar motifs in ok(gi) is also considered, the score of co-occurrence of the multiple binding sites in t is redefined as
where di, j, k is the Hamming distance between the sequence found by using profile Mj in t and ok(gi), lj the length of binding site motif with profile Mj.
7. Statistical evaluation of the predicted binding sites
We evaluated the statistical significance of our predictions by comparing the probability of finding a high scoring binding site in inter-TU sequences with that of finding the same high scoring binding site in randomly extracted coding sequences [32,33]. Let p(St>s) be the probability that an extracted sequence t (t ∈ IU or t ∈ CU) contains a putative binding site with a score (St) larger than s. To avoid possible biased sampling, 300 Cus for each TU in each genome were randomly generated, and SCu was computed for each Cu. Then a log-odd ratio (LOR) function defined as following was used to estimate the confidence of the prediction:
Since is the probability of type-I error for testing the null hypothesis that Iu does not contain a binding site when is greater than a cutoff s, we used it to estimate the false positive rate of the prediction results. In this way, could be also considered as an empirical p-value, and a cutoff of p < 0.01 was used for the CRP binding site prediction in each genome.
1. Conservation of the DBDs of the CRP proteins in cyanobacterial genomes
Using the BDBH algorithm and the criterion described in Methods, we identified orthologues of SyCRP1 (sll1371) of PCC6803 in 12 of the 29 sequenced cyanobacterial genomes. All these 12 genomes encode one SyCRP1 homologue, with the exception that the PCC6803 and PCC7120 genomes contain two: sll1371 and sll1924 (SyCRP2) in PCC6803, and alr0295 (AnCRPA) and alr2325 (AnCRPB) in PCC7120, which are identified by a unidirectional BLASTP search using SyCRP1 of PCC6803 as the query and an E-value cutoff 10-20. The phylogenetic tree of these 12 CRP orthologues shows that they fall into two distinct groups (Figure 1a). There are clear subtle differences between the DBDs of the two groups. Nonetheless, the residues of the DBD that are in direct interaction with the DNA counterpart as revealed by the crystal structure of E. coli CRP/DNA complex [39], are highly conserved in all these cyanobacterial CRP sequences, suggesting that they might bind to similar DNA sequences (Figure 1b).
Figure 1. (a) Phylogenetic relationships of CRPs from 12 cyanobacterial genomes. The tree is rooted with the CRP of E. coli K12, and bootstrap values are shown on the nodes. (b) Multiple sequence alignments of the DNA binding domains of the 12 cyanobacterial CRPs. The DNA binding domains contains a helix-turn-helix motif in which residues 1–9 form the first helix, residues 10–12 form the turn and residues 13–25 form the second helix. Particularly, residues 13–21 form the DNA recognition helix with residues 13, 14, 18 (indicated by solid dots) being in direct contact with DNA through forming hydrogen bonds with it [39]. (c). Logo representation of the profile of the 112 putative CRP binding sites predicted by the phylogenetic footprinting technique. Logo was generated by the Weblogo server [49].
2. Profiles of the CRP binding sites predicted by phylogenetic footprinting
When the 18 genes located in the five TUs (Table 1) in PCC6803 that are likely to be regulated by SyCRP1 in this species, are searched against the 12 cyanobacterial genomes that encode crp genes, we found a total of 30 orthologues in five genomes. These 30 orthologous genes are located in a total of 10 TUs in these five genomes, indicating that these TUs are not well conserved in these 12 genomes. By using the phylogenetic footprinting techniques (see Methods), we predicted a total of eight putative CRP binding sites from the 10 upstream inter-TU regions, suggesting that the CRP binding sites are largely shared by these orthologous genes. In order to increase the representation of the profile of the CRP binding sites and to minimize the possible bias of our original choice of the five TUs, we performed a one-round iteration of putative CRP binding site scanning using this preliminary profile constructed from these eight putative CRP binding sites (see Methods). From this preliminary whole genome scanning results, we selected a total of 112 putative CRP binding sites (Table S1 in Additional file 2) with high scores to construct the final profile of CRP binding sites. These sites display a strong pseudo-palindromic structure with consensus TGTGAN6TCACA (Figure 1c), which is similar to the canonical CRP binding sites in E. coli, suggesting that the pattern of CRP binding sites is well conserved between cyanobacterial and E. coli. This result is also in agreement with the observation that the binding sites of members of the CRP/FNR superfamily maintain a high level of conservation across difference lineages [40].
Additional File 2. Table S1. Putative CRP binding sites used to construct the profile of CRP binding sites. Table S2. Most conserved putative CRP-regulated genes/TUs in 12 cyanobacterial genomes. Table S3–14. Predicted CRP binding sites in 12 cyanobacterial genome at p-value < 0.01.
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3. Genome-wide prediction of CRP binding sites in the 12 cyanobacterial genomes
We then apply our motif scanning algorithm to predict putative CRP binding sites in the 12 cyanobacterial genomes that encode a crp gene using the profile of the 112 putative CRP binding sites as well as that of the previously prepared -10 like box from cyanobacteria [32]. The log-odds ratio (LOR) function of the predictions in each genome is all high when the score s is high (Figures 2), suggesting that these genomes are likely to contain functional CRP binding sites. The predictions in each of the 12 genomes at p-value < 0.01 are listed in Tables S3-S14 in Additional file 2, and all the prediction results are available upon request. In general, all these predicted CRP promoters contain a pseudo-palindromic CRP binding site and most of them also contain a downstream TA-rich σ-factor binding site, therefore they are likely to be true CRP promoters. As shown in Table S12 (see Additional file 2), the five CRP binding sites associated with the five TUs (shown in bold) in PCC6803, which we selected as the starting point of the our genome-wide prediction, were not necessarily ranked very high (they were ranked 4th, 12th, 14th, 17th, and 43rd) among the 59 predicted CRP binding sites in that genome, suggesting that our initial choice of the five TUs did not bias our prediction to them. Table 2 summarizes the predictions in the 12 cyanobacterial genomes, and the most prevalent genes of predicted CRP regulons are listed in Table S2 in Additional file 2.
Table 2. Summary of the genome-wide CRP binding site predictions.
Figure 2. Evaluation of the genome-wide prediction of CRP regulons in 12 cyanobacterial genomes. The green curves represent the probability and the blue ones represent . The red curves are the log-odd ratio (LOR), defined as (see Methods).
Although it has been estimated that CRP controls the expression of more than 200 TUs in E. coli [2], and the RegulonDB (release 5.8) contains 288 experimentally verified CRP binding sites, the number of predicted CRP binding sites at p-value < 0.01 in each cyanobacterial genomes is relatively small, ranging from 29 in BP-1 (containing 1075 putative TUs) to 249 in ATCC29413 (containing 3300 putative TUs), if one considers that the E. coli K12 genome encodes a total of 2070 putative TUs (predicted by the algorithm described in Methods), and that some of these cyanobacterial genomes contain much more TUs/genes (Table 2) than the E. coli K12 genome does. This result suggests that cyanobacterial CRPs might regulate fewer genes than the E. coli CRP does. We then ask whether the target genes of CRPs are conserved between E. coli and cyanobacteria, as well as among the 12 cyanobacterial genomes, given that the DBDs of CRPs as well as their binding sites are highly conserved.
Although these 12 cyanobacterial genomes share 13.88~32.19% of their genes with E. coli (Table 2), they almost have no common genes in their CRP regulons with that of E. coli K12 (Tables 3, and S3-S14 in Additional file 2), suggesting that the CRPs in cyanobacteria have adapted to regulate very different sets of genes than those in E. coli during the course of evolution. On the other hand, the majority of the CRP targets in these cyanobacterial genomes are not conserved either, rather, only a small portion of the CRP targets are shared by more than 2 of the 12 cyanobacterial genomes (Figure 3), even though our motif scanning algorithm might be biased toward the genes that have orthologues in reference genomes (see Methods). In the extreme cases, many CRP targets are species or lineage specific, suggesting that the targets of CRP have changed very rapidly since the speciation of these cyanobacterial genomes. For example, only 32 out of the 149 putative CRP target genes in the PCC6803 genome are conserved in at least 2 of the 12 cyanobacterial genomes. The only exception is the PCC7120 genome in which the number is 218 out of 442. However, this is largely because the reference genomes include a closely related species ATCC29413. Moreover, the most conserved putative CRP-regulated genes are only shared by five genomes (Figure 3). In addition, 17 of the 29 sequenced cyanobacterial genome do not encode the crp gene, suggesting that CRP is not required for the life of these 17 organisms; alternatively, the function of CRP in these organisms have been replaced by another TF.
Table 3. The CRP regulons are not conserved between PCC6803, PCC7120 and E. coli K12.
Figure 3. Distribution of shared putative CRP-regulated genes in sequenced cyanobacterial genomes. Only two representative examples from PCC6803 and PCC7120 are shown for clarity. The horizontal axis is the number of genomes that share putative CRP-regulated genes with PCC6803 or PCC7120, and the vertical axis is the number of shared genes. Among the 149 predicted CRP-regulated genes (at p < 0.01) in PCC6803, 117 (75%) are species specific, and the rest 32 (25%) are shared with other genomes; of the 442 predicted CRP-regulated genes (at p < 0.01) in PCC7120, 224 (50.7%) are species specific, and the rest 318 (49.3%) are shared with other genomes, most of which (191) are shared with the closely related ATCC29413 genome.
4. Functional classification of CRP regulons in cyanobacteria
Based on the functional annotations of the CRP target genes that we have predicted in this study, CRP seems to be involved in a rather diverse spectrum of functions in cyanobacteria (Table 4) as summarized below.
Table 4. Putative CRP-regulated genes involved in different biological processes.
4.1. Photosynthesis and carbon fixation
Various numbers of genes involved in photosynthesis and carbon fixation were predicted to bear a CRP-regulated promoter in the 12 cyanobacterial genomes that encode crp genes. Specifically, a total of 14 (at p < 0.01) photosystem I and II reaction center genes were predicted to be regulated by CRP, including AM1_0526, asr0847, Ava_4451, Ava_0640, asr0847, CYA_0295, CYB_2824, PMT1665, P9303_22121, P9303_22711, Syncc9605_1640, sll0634, slr1739, and Tery_4669. Several carbon dioxide concentrating mechanism protein ccmK genes, including alr0317-0318 and slr1838-slr1839, were also predicted to bear a CRP binding site. Moreover, putative CRP-regulated promoters were found for the phycobiliprotein family light-harvesting genes, including alr0523-0525, sll1577-1580, ssl3093, slr1459 and tsr0033. In consistent with these results, it has been previously shown that cellular cAMP levels change significantly in response to environmental stimuli such as light-dark cycle [41]. Thus cyanobacterial CRPs are likely to participate in photosynthesis pathways.
4.2. Nitrogen assimilation
Three nitrogenase related proteins AM1_2462, Ava_4669 and alr0874 in MBIC11017, ATCC29413, and PCC7120, respectively, were predicted to be CRP-regulated. Furthermore, an operon encoding a nitrate transporter (all3332-3335) in PCC7120 was predicted to bear a CRP binding site. It has been previously reported that nitrogen starvation resulted in a 3–4-fold increase in intracellular cAMP level in Anabaena variabilis [42,43]. Based on our prediction results, a possible scenario for a role of CRP in the signaling pathway of nitrogen assimilation could be as follows. Nitrogen starvation somehow increases the adenylyl cyclase activity, leading to an increase in the intracellular cAMP level. The activation of CRP by cAMP then lowers the transcription level of genes such as nitrate transporter (all3332-3335), while it enhances the expression of genes like alr0874 (nitrogenase reductase) and nitrogenase (AM1_2462, Ava_4669), as the cell switches to the more energy intensive nitrogen fixation of nitrogen gas. Nonetheless, an in-depth study is needed to elucidate the details of the role that CRP may play in nitrogen fixation in these cyanobacteria. Since not all cyanobacterial species are capable of nitrogen fixation, this role of CRP is unique to the cyanobacterial species capable of nitrogen fixation, such as MBIC11017, ATCC29413, and PCC7120.
4.3. Transporters and porins
A few genes coding for transporters and porins were predicted to be CRP-regulated, including several ion transporter in PCC7120 (alr2210-2213 and alr2118-2120) and PCC6803 (slr1392 and slr1950). Besides, several antiporters and ABC transporters were also predicted to be CRP-regulated in various genomes, e.g. CYA_2315, Ava_0687, sll0240, slr1452-1457, tll0559.
4.4. Kinases and two-component signal transduction systems
Dozens of genes coding for kinases and two-component signal transduction systems were predicted to be CRP-regulated, suggesting that CRP might play an important role in response to environmental changes in cyanobacteria. Interestingly, it has been reported that the CRPs in PCC6803 are involved in phototaxis as both sycrp1 and adenylyl cyclase mutants showed impaired phototaxis [9,29]. However, the genes that are involved in signal transduction for pilus assembly and phototaxis, as listed in [29], were not predicted to be CRP-regulated by our algorithm, therefore, they might be regulated by CRP indirectly.
4.5. Other functions unique to a species/lineage
Among the top hits of our prediction results, a large portion of putative CRP-regulated genes are species or lineage specific. The functions of these genes vary from genome to genome, such as the type IV pilus synthesis in PCC6803 (slr1667-1668 and slr2015-2018) and MBIC11017 (AM1_3323-3324); various transposase in PCC7120 (all3624), BP-1 (tll2385) and IMS101 (Tery_0925); methyltransferase in MBIC11017 (AM1_5474); aldo/keto reductase in A/B-Prime (CYA_0976 and CYB_2928); nblA in BP-1 (tsr0033); TPR repeat containing protein in ATCC29413 (Ava_3483); peptidase in MIT9313 (PMT1940); nuclease in CC9311 (sync_1258), etc. However, the functions of many other species or lineage specific putative CRP-regulated genes are largely unknown, most of them are annotated as hypothetical proteins, such as slr0442, sll1268, ssr2848 and sll1924 in PCC6803, asr4669 in PCC7120, AM1_3950-3951, AM1_4103, AM1_4957 and AM1_2209-2210 in MBIC11017, CYA_0127/CYB_2776 in A/B-Prime, Tery_2530 and Tery_1044 in IMS101, Ava_3757 in ATCC29413, PMT1492, and PMT1223 in MIT9313, P9303_06191, P9303_04111 and P9303_12031 in MIT9303, sync_093, and sync_1261 in CC9311, and Syncc9605_0955 and Syncc9605_0452 in CC9605. It would be interesting to experimentally characterize the functions of these genes as well as the roles that CRP plays in their transcriptional regulation.
5. crp genes were lost in some cyanobacterial genomes during the course of evolution
The presence of crp genes in some, but not all cyanobacterial genomes, raised the question about their evolutionary origin: are crp genes in cyanobacteria acquired through horizontal gene transfer (HGT) from other species, or are they vertically inherited from their last common ancestor, but lost in some cyanobacterial species/strains? To address this question, we constructed a species tree of the 29 sequenced cyanobacterial genomes based on their 16S rRNA gene sequences (Figure 4). Although some nodes in the tree are not well supported by the currently available sequence data, the relationships of the species in the tree are in excellent agreement with a previously inferred cyanobacterial species tree [44]. As shown in Figure 4, the genomes encoding a crp gene do not form a particular monophyletic group, rather, they are sporadically scattered across the tree. On the other hand, all the known cyanobacterial CRPs form a monophyletic group (Figure S1 in Additional file 1), suggesting that they are likely derived from a common ancestor. Therefore, the current sporadic distribution of the crp genes within the sequenced cyanobacteria likely resulted from differential gene losses during the course of evolution. Independent acquisition of the crp gene by individual cyanobacterial lineage/species, though theoretically possible, is less parsimonious since it would entail multiple HGT events from the same or closely related donors. Therefore, we conclude that the 17 cyanobacterial genomes that do not encode a crp gene actually lost their original ones during the course of evolution to adapt to their respective environments. It is interesting to note that most of the species lacking a crp gene belong to marine cyanobacteria; these species often have a reduced genome and inhabit a relative stable oligotrophic environment. On the other hand, the species that contain a crp gene are distributed in both fresh water and terrestrial environment. This again suggests that the crp gene might be beneficial to the species that live in a variable environment, and those that have adapted to a more stable environment such as oligotrophic ocean lost their crp genes.
Figure 4. Relationships between the 29 sequenced cyanobacterial species/strains inferred from their 16S rRNA genes. The tree is rooted with the 16S rRNA gene of E. coli K12. Bootstrap values are shown on the nodes. The cyanobacterial species/strains encoding a crp gene are indicated by bold font.
6. Degradation of CRP binding sites in cyanobacterial genomes after the crp genes were lost
We have previously shown that when a genome lost a TF in the course of evolution, then it would rapidly lose its cognate biding sites in inter-TU regions [32,33]. To extend this conclusion, we applied our CRP binding site prediction algorithm to the 17 cyanobacterial genomes that do not encode a CRP orthologue. Indeed, in four genomes, namely, CC9902, RCC307, WH8102 and WH7803 (Figures S2 in Additional file 3), the LOR function oscillates around zero as the score s increases, indicating that there is no significant difference between the signal of CRP binding sites in the inter-TU regions and that in the randomly selected coding regions (see Methods). These results strongly suggest that these four genomes are unlikely to contain functional CRP binding sites, which is in agreement with our previous observation [32,33]. However, there is a clear CRP binding site signal in the rest of 13 genomes as indicated by their relatively high LOR when the score s is high, suggesting that there exist CRP-like binding sites in the inter-TU regions in these genomes. The reason for this unexpected observation is unknown, but one explanation would be that these sites are bound by a different regulator in these genomes. To identify possible TFs in these genomes that are likely bind these CRP-like binding sites, we analyze the distribution of the CRP/FNR superfamily in these genomes, and found that they all encode at least one member of the superfamily, which are likely to recognize these CRP-like binding sites, as it has been shown that members of the CRP/FRN superfamily recognize similar consensus sequence, and the binding specificity is achieved through competitive binding among the members.
Additional File 3. Figure S2. Genome-wide scanning for CRP-like binding sites in the 17 genomes that do not encode a CRP protein.
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1. CRP is a lineage-specific global regulator
Studies have shown that CRP in E. coli functions as an important global regulator controlling the expression of genes involved in many pathways such as the carbon and energy metabolism pathways. It was also reported that CRP regulates a variety of genes in other bacterial species. For instance, one recent study showed that CRP-like protein regulates genes involved in quorum sensing, motility and intestinal colonization in Vibrio cholerae [7]. In this study, we suggest that CRP in cyanobacteria seems to have distinct functions. First, our results show that the members of the CRP regulons in cyanobacteria have little in common with those in E. coli (Table 3), which is consistent with the observation that genes whose expression is mostly affected by sycrp1 disruption in PCC6803 are involved in the type IV pilus synthesis [8,25,29]. In contrast, genes that are involved in carbon and energy metabolisms as seen in E. coli are not significantly affected by sycrp1 disruption [8,25,29]. Second, cyanobacterial CRPs seem to regulate distinct sets of genes specific to each lineage or strain as we can not clearly identify a particular set of genes common to most of the 12 cyanobacterial genomes that are under CRP control (Figure 3, Table S2 in Additional file 2). Third, more than half (17) of the 29 completely sequenced cyanobacterial genomes do not encode a CRP orthologue, suggesting that CRP is dispensable in these strains/species. For a closely related group of species, it is unlikely that an essential global regulator can be replaced or lost in some genomes while present in the others. Therefore, we conclude that CRP is not a global regulator with conserved functions; instead, it is likely a lineage-specific global regulator.
In fact, it has been shown that global regulators are not necessarily conserved in moderately related species. For instance, among the 7 and 6 global regulators in E. coli and B. subtilis, respectively, none of them is in common [11]. Furthermore, it is not surprising that CRP in cyanobacteria does not function as a conserved global regulator as seen in E. coli, given that CRP is mainly involved in the transcriptional regulation of genes related to carbon metabolism in E. coli, while organic carbon assimilation is no longer a constraint for the growth of autotrophic cyanobacteria. On the other hand, since nitrogen assimilation is a constraint for cyanobacteria, the nitrogen assimilation regulator, NtcA, a member of the CRP/FNR family, which is unanimously encode in all the 29 sequenced genomes, has been characterized as one of the conserved global regulators in cyanobacteria. Thus, it seems that global regulators are often lineage-specific and that the environment plays a vital role in determining which TF functions as a global regulator, and which genes are regulated by the TF.
2. The functions of CRP in cyanobacteria are highly diverse
It has been reported that SyCRP1 regulates the cellular motility in PCC6803, as sycrp1 disruptants were devoid of mobility and showed reduced type IV pilus biogenesis [8]. In another study, it was shown that the operons slr1667-slr1668 and slr2015-slr2018 in PCC6803, which are involved in type IV pilus biosynthesis [29,45], were down-regulated in sycrp1 disruptants using microarray gene expression profiling [25]. In consistent with these findings, we have predicted putative CRP binding sites for these genes. However, the slr1667-slr1668 genes were unique to PCC6803, and the orthologues of slr2015-slr2018 could only be found in MBIC11017 (Table S2 in Additional file 2) among the 29 cyanobacterial genomes. Thus, this function of CRP is likely to be restricted to these two species/strains only. In addition, based on our predictions of CRP regulons in the 12 cyanobacterial genomes, we argue that CRP might be also involved in other functions in different cyanobacterial lineage/strains, including carbon fixation, photosynthesis, nitrogen fixation, ion channels/transporters, and two-component signal transduction, etc. (Table 4, Table S3 – S14 in Additional file 2). Furthermore, as a large portion of our predicted CRP binding sites are associated with hypothetical proteins, CRP might be involved in the regulation of the other novel functions yet to be discovered. In this regard, we have provided a set of candidates for further experimental characterization. However, due to the lack of sufficient information about the functions of the CRP target genes, it is currently rather difficult to derive a general pathway model involving CRP regulons in cyanobacteria. Lastly, AnCrpA was shown to regulate the expression of genes related to nitrogen fixation in PCC7120 [28], including all1517 (nifB), all1439, all1432 (hesA), alr2515 (coax-II), and alr2834 (hepC). However, our algorithm failed to find high scoring CRP binding sites for all these genes, the possible reasons for this are addressed below.
3. Binding sites of AnCRPA in Anabaena sp. PCC7120
Our failure to identify high scoring CRP binding sites in the upstream regions of these nitrogen fixation genes in PCC7120 is actually in agreement with the finding by Suzuki and coworkers who suggested that AnCrpA might have a different binding site pattern from the conventional pseudo-palindromic motif [28]. However, an in vitro binding affinity test using EMSA showed that AnCrpA could bind to the conventional palindromic motif [27]. Thus, a possible explanation of this inconsistency would be that AnCrpA in PCC7120 could form a monomer or a heterodimer in addition to a homodimer, given that two CRP homologues are encoded in this genome, and one of them does not have a DBD. Such a monomer or heterodimer might favor a non-canonical CRP binding site, while the homodimer remains its ability to bind to the conventional palindromic motif. Clearly, a more in-depth study on this topic is needed to verify this hypothesis. This explanation is in agreement with the results that our algorithm predicts many CRP binding sites for other genes in PCC7120 with statistical significance (Table S7 in Additional file 2).
4. Origin of crp genes in cyanobacteria and implications for the evolution of CRP regulatory networks in bacteria
Because the crp gene is widely distributed in many distantly related bacterial groups, including actinobacteria, aquificales, bacteroidetes, chlamydiae, chloroflexi, cyanobacteria, deinococci, firmicutes, fibrobacteres, planctomycetes, proteobacteria, spirochaetes, etc. (Figure S1 in Additional file 1), it was likely present in the common ancestor of the extant eubacterial lineages. Under this scenario, the crp gene in this ancestor was flexible enough to regulate different sets of genes. Alternatively, it is also possible that the crp gene originally evolved in a specific bacterial lineage and was subsequently spread to other groups via HGT. Such HGT events may benefit the recipient organism given the flexibility of CRP in regulating different biochemical activities, which are often coupled to environment stimuli leading to the generation of intracellular signaling molecule cAMP. Therefore, the crp gene in the ancestor of modern cyanobacteria was acquired by either vertical inheritance or an ancient HGT event from other lineages. Some cyanobacteria lost their crp genes since harboring the gene might not necessarily increase their fitness in their new environments. On the other hand, in other cyanobacteria, the crp genes were adapted to better meet their unique physiology and environmental requirements. In other groups of bacteria, crp evolved to regulate other lineage/species specific functions. For instance, it regulates the carbon and energy metabolisms in E. coli, cell-cell communication in Stenotrophomonas maltophilia [46], and quorum sensing in Vibrio cholerae [7], etc
5. Rapid degradation of CRP binding sites
Evolution of cis-regulatory binding sites is an interesting, but not well-studied problem. The 17 cyanobacterial genomes that do not encode a CRP orthologue provided us an excellent opportunity to examine the degradation of the CRP binding site in these genomes. It was expected that high scoring CRP binding sites do not present in those genomes, as previous studies have indicated that binding sites rapidly fade out when the corresponding TF was lost during the course of evolution [32,47]. We do see such fading in the CC9902, RCC307, WH7803, and WH8102 genomes (Figure S2 in Additional file 3). These observations were in consistent with the well-accepted rule that "if no such a TF in a genome, then no corresponding binding sites in the genome". However, surprisingly, in the rest 13 genomes, high scoring CRP-like binding sites seem to appear in the inter-TU regions with a higher probability than in the randomly selected coding regions (Figure S2 in Additional file 3), suggesting that there exist sequence patterns similar to CRP binding sites in these cyanobacterial genomes. A possible explanation of this unexpected observation would be that there exist in these genome TFs that recognize binding sites similar to CRP binding sites. Indeed, at least one member of the CRP/FNR superfamily is encoded in these 13 genomes, and it has been shown that the binding sites of the members of this TF superfamily are well conserved across many species with a wide range of evolutionary distance [40]. Thus, the CRP-like binding sites found in these genomes are likely to be recognized by these non-CRP TFs of the superfamily. The specificity of these similar binding sites is likely to be achieved through the competition among homologous TFs for the same binding site, which is governed by their thermodynamic equilibrium [48].
In this paper, we have predicted CRP binding sites in 12 cyanobacteria genomes that encode a CRP orthologue using a highly accurate motif scanning algorithm. Based on the analysis of these predictions as well as experimental data available to us, we conclude that 1) CRP has rather different functions in cyanobacteria than in E. coli; 2) cyanobacterial CRP also has a very diverse spectrum of functions in different lineages or species/stains, and is even dispensable in some species/strain; 3) CRPs in modern cyanobacteria are likely to be vertically inherited from their last common ancestor, and some cyanobacteria lost their crp genes during the course of evolution to adapt to their new environments; and 4) once the crp gene is lost, its binding sites degrade rapidly. Although many of our predictions still await experimental verification, we should have provided a high quality candidate set for further experimental characterization of the CRP binding sites and regulons in this important group of bacteria.
ORF: Open reading frame; TU: Transcription unit; BDBH: bidirectional best hit; CRP: cAMP receptor protein; DBD: DNA binding domain; HTH: helix-turn-helix; EMSA: electrophoresis mobility shift assay; TF: transcription factor; HGT: horizontal gene transfer; LOR: log-odd ratio; MBIC11017: Acaryochloris marina MBIC11017; ATCC29413: Anabaena variabilis ATCC 29413; A-prime: Synechococcus sp. JA-3-3Ab; B-prime: Synechococcus sp. JA-2-3B'a(2-13); PCC7421: Gloeobacter violaceus PCC 7421; PCC7120: Nostoc sp. PCC 7120; CCMP1375: Prochlorococcus marinus CCMP1375; MED4: Prochlorococcus marinus MED4; AS9601: Prochlorococcus marinus AS9601; MIT9211: Prochlorococcus marinus MIT 9211; MIT9215: Prochlorococcus marinus MIT 9215; MIT9301: Prochlorococcus marinus MIT 9301; MIT9312: Prochlorococcus marinus MIT 9312; MIT9303: Prochlorococcus marinus MIT9303; MIT9313: Prochlorococcus marinus MIT9313; MIT9515: Prochlorococcus marinus MIT 9515; NATL1A: Prochlorococcus marinus NATL1A; NATL2A: Prochlorococcus marinus NATL2A; PCC7942: Synechococcus elongatus PCC 7942; PCC6301: Synechococcus elongatus PCC 6301; RCC307: Synechococcus sp. RCC307; WH7803: Synechococcus sp. WH 7803; WH8102: Synechococcus sp. WH8102; CC9605: Synechococcus sp. CC9605; CC9902: Synechococcus sp. CC9902; CC9311: Synechococcus sp. CC9311; PCC6803: Synechocystis sp. PCC 6803; BF-1: Thermosynechococcus elongates BP-1; and IMS101: Trichodesmium erythraeum IMS101.
Authors' contributions
M.X. designed and conducted the experiments, and wrote the manuscript. Z.S. conceived the project and wrote the manuscript. All authors read and approved the final manuscript.
This research was supported by a start-up fund from the University of North Carolina at Charlotte to Z.S. We would like to thank Drs. Anthony Fodor, Devaki Bhaya, and Jingling Huang for their critical reading of this manuscript and suggestions. We would also like to thank the two anonymous reviewers whose comments have greatly improved this paper.
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Listermann Brewing Co. in Cincinnati will celebrate its five-year anniversary with a customer appreciation and pig roast party at 5 p.m. Saturday (June 8) at the brewery. Dan Listermann launched his small brewery -- which now includes the Triple Digit brand -- in 2008 inside his homebrew shop.
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Here is a sneak peek at a few of the delicious new tastes for Spice Road Table. This all new Disney Dining Table Service Restaurant will be opening its doors in early 2014 at the Morocco Pavilion in Epcot World Showcase.
Spice Road Table will be a wonderful spot for a bite along the waterfront. Inspired by the outdoor cafés along the Mediterranean, the whitewashed façade of Spice Road Table is accented in icy shades of blue from the famous "Blue City" of Chefchaouen in Morocco's Rif Mountains.
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Breakfast and a Cigarette: A Novella in Four Directions
by Bill McLaughlin,
208 pages
(page 14 of 25)
"I'm sorry, Dolores."
"Hey Ray, look, I'm sorry too, I just get..."
"It's all right."
"Have a good sleep. Suarte amigo. Good luck in Miami tomorrow."
"Thanks. I won't forget this—any of it. I promise."
He walked slowly toward the black outline of jumbled shacks a few hundred yards away. He found a grassy patch, smooth and level. The moonlight was sufficient to assemble the tent. He crawled inside and was glad there were no big holes. The roof of the tent was made of netting and when he laid down he watched the stars and tried to find Orion's belt and the Milky Way. The night air was cool; he covered himself with his poncho and wrapped his sneakers in his shirt for a pillow. He watched as the moonlight dappled the sides of his tent.
And that night two men laid down to sleep. One man, in clean sheets and familiar surroundings, lay in the dark thinking again about missing comrades, thunder and sunbursts, mangled instruments and players. He silently cursed his inability to talk about it. Even after all these years, he could not bring himself to admit that what he had seen was real. And he could not forgive himself for getting out alive.
The other man closed his eyes and listened to the sound of crickets and cicadas, felt the cool earth beneath him. He had no time for the past. He was haunted by the present, by a thin troubled voice that tried too hard to sound gay; by a little song that was made for happiness but reaped only sadness. And before the two men slept they thought briefly of each other, sadly realizing that they would never meet again. And what they dreamed was never better or never worse than what they lived.
Ray awoke at first light. He couldn't remember the last time he had slept all night outside. His back was stiff and he felt dirty. He wanted a hot shower and a cup of coffee. He unzipped the tent and crawled out into the day, already warm, and into the middle of an endless row of tomato seedlings, plastic row covers—and alarmingly close—an enormous, spindly irrigation machine on wheels, wide as a football field and spitting out a fine mist of water and chemicals as it slowly rolled toward him.
In the distance he could see lines of workers, stooped over plants. They were dressed in bright colors; some wrapped their heads in cloth, wound round like turbans. Others wore straw hats, neckerchiefs, and bandannas. Yet others were bareheaded and their black skin gleamed in the bright morning air. The sun had already pried itself from the edge of the world and was bearing down upon these women and men and children.
Breakfast and a Cigarette, Part III continues...
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BEN TURNED UP THIS AFTERNOON. I was celebrating the doctor giving me another sicknote and arguing in a nauseous no breakfast Carlsberg haze with Jewish Dave about whether Gordon Brown drank alcohol or not. Ben turned up, unexpected, unannounced,...
by Zack Wilson
by Zack Wilson


text:
Recent answers to Firefox crashes when I try to start it. (BEX CRASH and APPCRASH)https://support.mozilla.org/en-US/questions/9572382013-08-19T22:03:54-07:00Hello, I also have the same problem where Mozilla Firefox won't open due to APPCRASH. I did what you2013-08-19T22:03:54-07:00tiffanyhnghttps://support.mozilla.org/en-US/questions/957238#answer-470188
Hello,

I also have the same problem where Mozilla Firefox won't open due to APPCRASH. I did what you said and got this: bp-80cea7e0-1aa0-4278-8498-ca22c2120120. I scan my computer for viruses and such every time I turn it on and do a disk cleanup. I also uninstalled Mozilla and tried installing it again but it doesn't solve the problem. What can I do?
I was getting the appcrash error and dpl error notes, when closing firefox. I tried a lot of posted 2013-08-17T10:40:59-07:00bill60https://support.mozilla.org/en-US/questions/957238#answer-469023
I was getting the appcrash error and dpl error notes, when closing firefox. I tried a lot of posted remedies. None worked. I deleted microsoft security essentials, and the problems went away. I will now add AVG free antivirus. If it works, I'll use it, otherwise, I'll do without antivirus for a while, until the bug is fixed properly.... In short, it was the antivirus program causing the errors, for me.
Still getting the 2 errors 2013-04-19T10:31:10-07:00Fudqshttps://support.mozilla.org/en-US/questions/957238#answer-429333
Still getting the 2 errors
Certain Firefox problems can be solved by performing a Clean reinstall. This means you remove Firef2013-04-19T10:26:20-07:00Tylerdownerhttps://support.mozilla.org/en-US/questions/957238#answer-429331
Certain Firefox problems can be solved by performing a Clean reinstall. This means you remove Firefox program files and then reinstall Firefox. Please follow these steps:

Note: You might want to print these steps or view them in another browser.

Download the latest Desktop version of Firefox from http://www.mozilla.org and save the setup file to your computer.

After the download finishes, close all Firefox windows (click Exit from the Firefox or File menu).

Delete the Firefox installation folder, which is located in one of these locations, by default:

Windows:

C:\Program Files\Mozilla Firefox

C:\Program Files (x86)\Mozilla Firefox

Mac: Delete Firefox from the Applications folder.

Linux: If you installed Firefox with the distro-based package manager, you should use the same way to uninstall it - see Install Firefox on Linux. If you downloaded and installed the binary package from the Firefox download page, simply remove the folder firefox in your home directory.

Now, go ahead and reinstall Firefox:

Double-click the downloaded installation file and go through the steps of the installation wizard.

Once the wizard is finished, choose to directly open Firefox after clicking the Finish button.

Please report back to see if this helped you!
I did all the scans but i didn't work when i tried to open mozilla i had the same problem 2013-04-19T10:22:28-07:00Fudqshttps://support.mozilla.org/en-US/questions/957238#answer-429328
I did all the scans but i didn't work when i tried to open mozilla i had the same problem
Ty very much for the quick feedback, I will start doing every scan and report the results back to yo2013-04-19T08:06:26-07:00Fudqshttps://support.mozilla.org/en-US/questions/957238#answer-429292
Ty very much for the quick feedback, I will start doing every scan and report the results back to you!
BEX = Buffer Overflow Exception or Data Execution Prevention (DEP) exception. DEP is a security feat2013-04-19T07:49:19-07:00jscher2000https://support.mozilla.org/en-US/questions/957238#answer-429285
BEX = Buffer Overflow Exception or Data Execution Prevention (DEP) exception. DEP is a security feature that seeks to prevent exploits that inject program code into Windows memory. Firefox doesn't trigger this error on its own, but add-ons or external programs that tweak Firefox might. Please see cor-el's recommended tools.
When i open Crash Reports I only have the folder pending.. 2013-04-19T07:43:58-07:00Fudqshttps://support.mozilla.org/en-US/questions/957238#answer-429284
When i open Crash Reports I only have the folder pending..
Do a malware check with some malware scanning programs on the Windows computer. Please scan with all2013-04-19T07:41:02-07:00cor-elhttps://support.mozilla.org/en-US/questions/957238#answer-429283
Do a malware check with some malware scanning programs on the Windows computer.
 Please scan with all programs because each program detects different malware.

Make sure that you update each program to get the latest version of their databases before doing a scan.

Malwarebytes' Anti-Malware:
http://www.malwarebytes.org/mbam.php

SuperAntispyware:
http://www.superantispyware.com/

Microsoft Safety Scanner:
http://www.microsoft.com/security/scanner/en-us/default.aspx

Windows Defender: Home Page:
http://www.microsoft.com/windows/products/winfamily/defender/default.mspx

Spybot Search & Destroy:
http://www.safer-networking.org/en/index.html

Kasperky Free Security Scan:
http://www.kaspersky.com/security-scan

You can also do a check for a rootkit infection with TDSSKiller.

http://support.kaspersky.com/viruses/solutions?qid=208280684

See also:

"Spyware on Windows": http://kb.mozillazine.org/Popups_not_blocked

We're sorry to hear that your Firefox seems to be crashing when you open it. Please perform the fol2013-04-19T07:36:42-07:00Tylerdownerhttps://support.mozilla.org/en-US/questions/957238#answer-429282
We're sorry to hear that your Firefox seems to be crashing when you open it. Please perform the following steps to give us a crash report ID that helps us find out more about the cause of the crash.

Press the following shortcut to get a Run window: [Windows] + [R]. This should bring up a window that contains a text field.

In that text field, enter %APPDATA% and press Enter. An explorer window should open.

From that explorer window, double-click on the Mozilla folder, then double-click on Firefox and then on Crash reports. Double-click on submitted.

Now, you should see a list of files that contain reports. Go to View > Arrange Icons by > Modified to get the most recent files at the top of the window.

Open the most recent 5 files with a text editor and copy the IDs.

Paste each ID with bp- into the reply window on the forums.

Thanks in advance!

You can find more information and troubleshooting steps in the Firefox crashes - Troubleshoot, prevent and get help fixing crashes article.


text:
Sep 29, 2012 at 07:41AM EDT
Now that you changed your name and picture that cake thing makes very less sense, shame on you :D
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The host of the hit Travel Channel series 'Man v. Food Nation,' Adam Richman and his protégé go toe to toe with The Comfy Cow's 15-scoop behemoth.
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Is this Lea Michele the Actor?
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26th March 2015 @ 06:43pm
It has been confirmed that Kate Upton will join Lea Michele in the new movie The Layover
13th January 2015 @ 04:44am
Lea Michele performs the song "Let It Go" in Glee's most recent season premiere, which will happen tonight.
4th July 2014 @ 11:02am
It has been claimed that Lea Michele is very happy in her relationship with her new boyfriend Matthew Paetz


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Stories from around the World
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By now, we have completed thousands of film portraits from all over the world, covering more than 100 countries and hundreds of different jobs. This year, we sent our storyhunters on a roadtrip...
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"Tokyo, here we are!" Yes, people on the other side of the world have really interesting stories to tell as well. Stay tuned and find out what locals and expats have to tell you about...
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Step up, be active! Watch as our storytellers inspire you to discover new perspectives and expand your horizon. Leave your comfort zone and be the master of your own destiny! Get your...
Employers on whatchado
naseba offers business facilitation platforms in emerging and high growth markets. We conduct exhaustive research, match buyers and sellers and then produce high-profile events, all with a...
AIESEC is present in 113 countries and has over 85.000 members. Every year, 24.000 young people develop themselves by taking the opportunity to lead a team & to strive to bring a positive...
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New Testament to present Christmas plays
New Testament Christian School in Alexandria invites family and friends to the K-6 Christmas musical Checkin' It Twice, which includes Wise Men played by (left to right) Alex Greseth, Michael Jackson and Andrew Lenz, on Thursday, December 12 at 6:30 p.m. at the school. The musical will be followed by the 7th-12th grade students' performance of the one-act play Give the Gift Back.
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Trident Creates Multimedia for IBM's Top of the Line ThinkPad 770x.
MOUNTAIN VIEW, Calif., Nov. 5 /PRNewswire/ -- Trident Microsystems, Inc. (Nasdaq: TRID) today announced that IBM's notebook PC, the ThinkPad 770X uses the Cyber9397 DVD multimedia accelerator.
The notebook is one of the first designs to use Intel's Pentium II 300MHz processor and be equipped with a DVD ROM drive, advanced audio capability and a 56Kbps modem. Delivered standard with an 8.1 GB hard drive, 64 or 128 MB of SDRAM, and 4MB or 8MB of video SGRAM, the IBM ThinkPad 770X will outperform most available desktop units.
The Cyber9397 DVD incorporates a number of industry firsts including:
* Windows 98 WDM DVD (Windows Driver Model with DirectShow). A driver model based on the Windows NT model designed to provide a common architecture of I/O services for both Windows NT and Windows operating systems for specific classes of drivers. These include USB and IEEE 1394 buses, audio, still-image capture, video capture, and HID-compliant devices.
* Windows98 MHS (Multiple Head Standard), a Microsoft standard covering the chip's ability to handle multiple outputs to various display devices such as TVs, LCD or a monitor at the same time with different rates, and resolutions.
* Windows 98 WDM Video Capture, a WDM-based kernel-mode extension of Microsoft DirectShow, providing kernel connection and streaming services as used by the WDM Stream class driver.
* AGP (Accelerated Graphics Port), an Intel port for graphics display.
* PC98, the 1998-99 requirements for PC system and peripheral design that serve as the basis for the "Designed for Windows" logo, as defined in the PC 98 System Design Guide.
* DX6, the new API (Application Programming Interface) from Microsoft that covers 3D graphics advancement.
* ACPI (Advanced Configuration and Power Interface) a specification that defines a new interface to the system board that enables the operating system to implement operating system directed power management and system configuration and allows instant on PCs.
* APM (Advanced Power Management), A software interface, defined by Microsoft and Intel, between hardware-specific power management software and an operating system power management driver. It has recently been replaced on Windows 98 compliant systems by the ACPI.
"Because it meets the latest and most stringent specifications, the Cyber9397 DVD is probably the best videographics solution for the advanced notebook manufacturer today. As part of our Cyber line of notebook products which includes both embedded memory devices for the entry-level PC and sophisticated discrete ICs, it is the result of Trident's eleven years of experience in producing multimedia chips," said Frank Lin, president and CEO of Trident. "We are extremely pleased that Trident devices once again are chosen for the multimedia graphics solution of the IBM ThinkPad notebook product line. We, at Trident, are committed to making IBM notebooks successful in the marketplace."
The single-cycle 3D graphics engine can perform premium 3D functions at more than 500k triangles per second. The 3D engine off-loads the CPU of 50% of its 3D tasks, enabling the ThinkPad's CPU to concentrate on other processing. The 3D engine, integrated with a triangle setup engine, provides Gouraud shading, texture mapping, alpha blending, Z-buffering, fogging and anti-aliasing, resulting in arcade-quality graphics on notebook PCs.
The Cyber9397 DVD allows the display shown on the LCD and the CRT or TV to run at different refresh rates, different resolutions and different color depths. For example, while the slides of a presentation are being projected onto a screen, the presenter can read notes off his/her notebook.
The Cyber9397 DVD provides flexible and extensive mobile power management capabilities. The chip operates at 3.3V with independent power planes for different functions in the chip. This ensures that each function can be individually powered down when not in use. With an appropriate configuration, a suitable balance can be achieved between graphics power consumption and performance by adjusting the frame buffer size, and speed.
Trident Microsystems, Inc., with headquarters in Mountain View, California, designs, develops and markets graphics controllers and multimedia integrated circuits for PCs. Trident's products are sold through a network of OEMs, original design manufacturers and system integrators worldwide. For further information about Trident and its products please consult our web site: http://www.tridentmicro.com .
Note: Trident is a registered trademark of Trident Microsystems, Inc. ClearTV and Cyber9397 are trademarks of Trident Microsystems, Inc. All other trademarks used herein are the property of their respective owners.
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text: POPSUGAR Food Peppers Stuffed With Feta Peppers Stuffed With Feta by partysugar 8/13/10 0 Shares Like us on Facebook Previous Photo 1 of 10 View on One Page ADVERTISEMENT ( more slides) Next Share This Link Copy 1 Peppers Stuffed With Feta Previous Photo 1 of 10 View on One Page ADVERTISEMENT ( more slides) Next Read More Peppers Stuffed With Feta Get inspired with our daily newsletter Enjoy gorgeous decor ideas, fast and easy recipes, and more in your inbox. DIY Home Food Tech Love & Sex Vegetarian Sign up with Link Love How to make the perfect pie crust — Fork + Plate McDonald's will test all-day breakfast — HuffPost Taste Noodle technology: a pasta that doesn't require you to pre-boil — Grub Street A history of the Frappuccino — Yahoo! Food Easy make-ahead Easter brunch — Rachael Ray Watch the reactions of these adults eating lunchables — Buzzfeed Video The best vegan sandwich you've ever tasted — POPSUGAR Fitness Latest Food


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Dans le centre d'Ampasimena, le village vit au rythme des enfants. Le matin vers 7h30 ils quittent le village pour l'école qui se situe dans le village voisin. Ampasimena retrouve alors son calme jusqu'à leur retour vers 16h30.
In the center of Ampasimena, the town lives to the rhythm of the children. In the morning towards 7h30 they leave the town for the school that is situated in the town neighbor. Ampasimena rediscovers then his calm until their return towards 16h30.
Welcome to Africa, AKA the motherland! Check out African Internet Radio while you're scoping the panoramas.The earliest fossil of the homo sapiens family (human beings) was found in Ethiopia, dating back more than 200,000 years. Compared to this length of time, even the "ancient Sumerians" from 6000 B.C. are drooling toddlers.Let's mention a few African heroes you may have heard of, for inspiration in the face of the continued economic inequality and violence which plague Africa today: Nelson Mandela, first democratically elected President of South Africa, who fought against apartheid and served 27 years in prison while advocating freedom and peace. Haile Salassie, Emperor of Ethiopia, who resisted Mussolini and the fascist Italian invasion of WWII, and who is worshipped as an incarnation of God by the Rastafari movement. Kwame Nkrumah, first Prime Minister of Ghana, advocate of uniting Africa in Pan-Africanism. Fela Kuti, inventor of Afrobeat music, who declared his home to be an independent state, ran for president of Nigeria, and to whose funeral ONE MILLION PEOPLE came to pay their respects.In June 2001 the African Union was formed, consisting of 53 African States organized, like in the EU, around common economic and political development.Text by Steve Smith.


text:
How To Paint And Upholster A Chair: Part 2
Well, we can check cheerful desk chairs off of our wish list for the office. They make me beam:
You can read all about how I prepped, primed, & painted them here. And now for the quick upholstery deets. I had some leftover fabric from the dining room curtains on hand so I laid it over the detached seats and centered one of the grellow thingies (that's a technical term) and trimmed the fabric around the cushion, leaving enough that I could easily pull and staple to the backside.
Making sure it was staying centered, I carefully turned it over and shot four staples into it while pulling it nice and tight (I have a basic manual staple gun for around $20 from Home Depot). Those staples went in at noon, three o'clock, six o'clock, and nine o'clock.
That way I could flip it back over and ensure things still looked straight and that the fabric was pulled tight enough (if it wasn't, it would just be four staples to pop out instead of a dozen). After confirming the pattern still looked nice and straight from the front, I flipped it back over and spaced out about six staples per side (while pulling the fabric tight along the way).
When it came to the corners, I just treat those like wrapping a present. It's hard to describe in words that don't make it sound like a super complicated ten step process (it's not) but I basically just fold the fabric into little triangles like the side of a present and staple them until they look good from the front. The front is all that matters – so the back can look all weird like this:
As long as you turn it over and things look great, you're golden.
So I just did the same thing with the next cushion (lining up the grellow thingies in the same way so it would look like Cushion #1's twin). About 30 staples later that cushion was done too.
The entire upholstery project probably took me thirty minutes from start to finish. Maybe 45 because I kept pausing to watch Housewives of Beverly Hills. Oh and the reason I just went over the fabric that was there was because we didn't have any cushion/fabric related issues (other than the fact that we didn't like the fabric). So it wasn't like we had odor problems or a flat lifeless cushion to deal with (which would have involved removing the fabric and the cushion and getting new foam from a place like JoAnn Fabric).
Then of course the hard part was waiting for my spray painted chairs to fully cure so they were nice and hard and ready for every day
use abuse. I gave them two sunny days outside (which also helps speed up off-gassing since spray paint can be stinky until it's fully cured)…
… and a third day in the sunroom (with the windows cracked and the fan going to continue to allow them to completely dry before bringing them inside). Then late last night I danced a giddy little chair-time jig and slipped the newly dressed cushions onto those so-fresh-and-so-green chairs (securing them with the same four screws that I removed from the underside to free them here). The result was nothing short of magic. But the cool kind involving levitation and mind-reading. Nothing weird like a bad card trick or a double sided quarter.
This photo's coloring is probably the most true to life (below). They look so great with the white cabinets and the dark wood counters.
Yeah, we're kind of obsessed. We love how the more intense greeny-yellow in the chairs' paint & fabric relates to the soft pop of color in the stencil. Instead of taking away or competing with the stencil, they just seem to bring out the grellow pop on the wall- so they enhance/add to it (if that makes sense). Although I'm not sure how well it'll come across in pics (colors seem to vary by monitor). In person the pairing is pretty darn amazing.
Since the stencil is so subtle and the chairs are so bold, they sort of layer in together – so it's not chaotic and weird (which would probably happen if the stencil were more high contrast or the chairs were more washed out). The key is to keep things at different levels (ex: bold pillows on a more subtle tone on tone rug or sofa). Since if everything textile, accessory, and furnishing were fighting to be the star it might get kinda… Lady Gaga. Not that there's anything wrong with that if it's what you're going for.
The best view of all is through the dining room. Me-ow. And I love that the table runner is easily switched if I feel like cutting the green… but for now I dig it. Can't wait to add some large scale art above each chair for some much needed height in the office (but nothing too crazy to compete with the chair + stencil combo).
But now, back to the close ups.
Our office just got a whole lot happier. And we did too. Picture me serving up a nice cheesy grin.
These $35 secondhand babies have certainly come a long way. Here they are right after we bought them from a local secondhand store:
And since we already had the fabric on hand, our makeover total was just $18 (the cost of three cans of Ivy Leaf spray paint from Michael's). But if you wanted to add the initial cost of the chairs ($35 a pop) and the fabric ($12.99 for a yard, which was all I needed for both seats) it comes to a nice even $50 (and fifty cents) per chair. Which makes me all sorts of hyper since similar chairs (like these and these) are in the $200-$250 range. And don't look now, but this guy is $694 on ebay. Woah nellie.
Oh and just to keep it random, I snapped another pic of the stencil. Because it makes me happy, and it's so hard to capture. Elusive little guy. Of course the pop of color looks yellow in this pic, but it's a lighter shade of the chair in real life.
Have you painted or upholstered any chairs of late? Do you have extra fabric laying around that you're itching to use? Do you have the same magic hierarchy as I do (with levitation and mind reading at the top and bad card tricks and double sided coin gags at the bottom)?
Psst- Check out the first half of this chair makeover here.
Psssst- We just announced this week's giveaway winners. Click here to see if you're one of them.
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328 Comments
Love the chairs! They're so fresh and fun!
By the way, can I please borrow some of your patience? I rarely wait the 72 hours to let something cure – I'm usually tapping my fingers after 15 minutes!!
Haha–Mrs. Cautious loves Captain Careful.
They look great! Did you ever consider upholstering with Sue the Napkin?
So cute, I love green, great color choice!
wow, those look great guys! i'm loving all the pops of green. :)
They look great! I like that they "go" with the dining room, too, because they can be seen from in there. The lime green is so cheery, too. What a great place to work!
So fresh and so clean clean!
Have you seen this chair at Urban Outfitters? http://www.urbanoutfitters.com/urban/catalog/productdetail.jsp?id=17995671&color=045&itemdescription=true&navAction=jump&search=true&isProduct=true&parentid=A_FURN_FURNITURE
They've really got some fun furniture right now. Just need to win that old lottery that we never enter.
Ya know, I saw this too, and was wondering if $329 is a decent price? No, it's not a YHL deal, but if Target is selling same style chair (probably not as well made) and it's covered in a Designer fabric which is not cheap…get my drift? What do you guys think?
The chairs look amazing, thanks to you I now have a grellow fixation and I am finding it everywhere, literally EVERYWHERE right now.
Oh my goodness I absolutely love how these chairs came out!!! They make me smile and the view from the dining room is amazing:-) I must admit though that the whole time I was admiring the chairs my eyes where also drawn to the new lamps. THEY STOLE THE SHOW (where did you find those beauties???)
I noticed and admired those lamps right off the bat, too! Those are gorgeous and as for the chairs, in the words of my four-year-old, I LUB dem!
Brilliant! It ties both spaces together wonderfully.
Love these chairs! They're so happy! Are they super comfortable? I would love to do something similar in my home office, but I sit in my desk chair for 9 hours a day. I'm afraid I may be doomed to the ugly ergonomic desk chair til the end of time!
Love love love the colors! And I love the even though they are seperate spaces, the two rooms have some unity which makes them flow together so nicely! Great job!
Why yes, as a matter of fact, I have upholstered a chair lately. I did this one for the local newspaper (they photographed and interviewed as I worked for a "how-to" article):
Your grellow chairs look amazing. You can never have too much green!
I haven't commented in a while but this is the BEST!!! OMG!!! The chairs are so wonderful and rich looking. Love them. This room is fast becoming my favorite room. It is B.R.I.L.L.I.A.N.T!!!!
Bravo to you and such a great eye for style, color, and design. You are simply the BEST, Sherry!!
OHH BOY! They look so soo freakin' amazing!! I still can't get over that stencil, I am soo in love with it. And now with the chairs in the room, it just makes the little grellow's pop!!! LOVE, LOVE, LOVE!!!
Seriously, how are you going to get any work done?? I would just be sitting there, staring at my stenciled walls, petting the pretty chairs!!! LOL!
Love love love. You've totallly made me re-think my sewing room colour scheme. Which is good because painting begins this weekend!
Nice job! I'm in the middle of some chair seats myself. I hope they turn out as well as yours did!
I have to say though that (maybe I'm really slow) but it just now clicked with me that "grellow" was green+yellow. When you first mentioned it, for whatever reason, I was thinking grey+yellow — and was really confused when the color looked green on my screen! Doh!
OMG they look amazing!!!! Now, I wish I had chairs like that to do in my house…
Do you ever worry about bedbugs when bringing in used furniture? That is something I'm paranoid about after a friend got some from furniture. Love the chairs! Great job :)
Love this makeover Sherry – especially the stenciling. I just can't seem to get over how cute the walls look!
Oh my goodness! To be honest, I kind of expected you to paint it that color because it made perfect sense and knowing how much you love this color. Fabulous chairs, soo you (and soo johnatan adler;-). I've decided that for my new house once I am done purchaing main items like sofas I will DIY accents myself just because it brings soul into the house! You guys should have a show on HGTV, like both of you, and do on TV what you do at home. It will be a natural progression for you to fill that hole on HGTV, i.e. lack of a show that would relate to young families. I mean who can afford Candice or Color Splash makeover realy? The challenge is to realistically squish months long decision/DIY process into a 30 min show. Oh and I wish you still did the mood boards just for fun and just because ;-)
Interesting choice! Being a bit more of a minimalist, I would have gone for a solid colour seat cover, maybe a lighter, greyish version of the lamp colour. But I enjoy the experimentation you guys have got going.
I think there's a typo – (but nothing too crazy to complete with the chair + stencil combo) – should read '…compete..'?
The chairs look great! I love the color scheme that you have used…it's so bright and cheerful! What a fun place to live. ;)
Oh! I see. They're more "grrrr" than "ello". I love it. If you guys decide to go a shade darker below the chair rail, then the whole thing will just be "meow"!
I actually did just do a chair – with wonderful, bright red script fabric. I have a thing for chairs – and I am like you in that I want them to be "happy". Great job.
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Games for Small Lawns
Publisher: Philadelphia : J. B. Lippincott company, [1933]
Branch Call Number: 796 H45
Characteristics: xiii, 132p.,incl. front., illus. (incl.plans) diagrs.,20 cm
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List - Multcolib My Librarian Matt: Games people play by: multcolib_matt Mar 06, 2014
Balloon tennis anyone?
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