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  • Erwin Santana posted an update 1 week, 6 days ago

    ?putida. Estimations of the number of IHF binding sites EGFR inhibitor in the genome of E.?coli have yielded disparate results ranging from 70 to over 600 locations (Boffini and Prentki, 1991; Freundlich et?al., 1992; Ussery et?al., 2001; Grainger et?al., 2006; Grainger and Busby, 2008). In our case, we resorted to use the functional information regarding the IHF site of the Pu promoter presented above to estimate the upper limit of this figure in the chromosome of P.?putida KT2440. Using the consensus sequence determined before, we found 2653 sites, which matched the motif WWCARnnnnWTR and thus represent the maximum edge of the functional extent of the factor. Evidently, these reflect core target DNA sequences that are necessary for specific recognition, but functional binding may not occur unless there are other less defined characteristics in the adjacent sequences, e.g. an upstream AT-rich track and overall bendability of the region (see above). However, given the increasing number of IHF molecules of P.?putida along growth (approx. from 100 to >?4000; Valls et?al., 2002) it could well happen that many of the sites are in fact occupied in cells grown to stationary phase. Out of them, 330 WWCARnnnnWTR sequences were found in intergenic regions (Table?S1) and are thus likely to have a role in regulating expression of contiguous genes. The set of sites encompasses ORFs for functions belonging to different categories (Table?S2), including amino acid transport/metabolism, cell envelope and outer membrane biogenesis, coenzyme metabolism, energy production and conversion, signal transduction mechanisms, inorganic ion transport and metabolism, transcription and translation, ribosomal structure and biogenesis (summary in Fig.?6). Also, a large share of IHF sites includes hypothetical proteins within or close to mobile elements, suggesting an important role of the factor in the occurrence of horizontal gene transfer (Fass and Groisman, 2009). Surprisingly, we did not find IHF sites in any regulatory position near genes encoding central carbon metabolism (i.e. core pathways for consumption/production of carbohydrates and amino acids), an issue that is discussed below. Finally, we examined the positioning of IHF binding sites in respect to repetitive extragenic palindromic (REP) sequences from P.?putida. Enterobacterial REP sequences are associated to IHF (Boccard and Prentki, 1993; Oppenheim et?al., 1993), but the counterparts found in P.?putida may not follow the E.?coli paradigm (Aranda-Olmedo et?al., 2002; Ramos-Gonzalez et?al., 2006). Inspection of the ??800 REP sites of P.?putida (Aranda-Olmedo et?al., 2002) in respect to predicted IHF sites revealed that there was not one case where the two occurred on top of each other. Moreover, only in 49 instances the distance between IHF and REP sites was <?100?bp (not shown).

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