Switching Control of Expression ofptsGfrom the Mlc Regulon to the NagC Regulon

Abstract

The Mlc and NagC transcriptional repressors bind to similar 23-bp operators. The sequences are weakly palindromic, with just four positions totally conserved. There is no cross regulation observed between the repressors in vivo, but there are no obvious bases which could be responsible for operator site discrimination. To investigate the basis for operator recognition and to try to understand what differentiates NagC sites from Mlc sites, we have undertaken mutagenesis experiments to convert ptsG from a gene regulated by Mlc into a gene regulated by NagC. There are two Mlc operators upstream of ptsG, and to switch ptsG to the NagC regulon, it was necessary to change two different characteristics of both operators. Firstly, we replaced the AT base pair at position ؉/؊11 from the center of symmetry of the operators with a GC base pair. Secondly, we changed the sequence of the CG base pairs in the central region of the operator (positions ؊4 to ؉4 around the center of symmetry). Our results show that changes at either of these locations are sufficient to lose regulation by Mlc but that both types of changes in both operators are necessary to convert ptsG to a gene regulated by NagC. In addition, these experiments confirmed that two operators are necessary for regulation by NagC. We also show that regulation of ptsG by Mlc involves some cooperative binding of Mlc to the two operators.Mlc and NagC are homologous proteins, and both act as transcriptional repressors in Escherichia coli. Mlc represses genes involved in the uptake of glucose, while NagC controls the use of N-acetylglucosamine (GlcNAc). Both glucose and GlcNAc are transported into E. coli via the phosphotransferase system (PTS). Mlc represses ptsG, the gene for the major glucose transporter; the ptsHI-crr genes, which encode the soluble components of the PTS; the manXYZ genes, which encode an alternative transporter for glucose, as well as other hexoses; and also malT, the positive transcriptional regulator of the mal regulon (4,11,12,24,26,27). NagC represses the divergent nagE-nagBACD operons for the uptake and degradation of GlcNAc and the chb operon, which contains genes for the transport and degradation of chitobiose (a dimer of GlcNAc) (28, 31). In addition, NagC activates the expression of the glmUS operon, which contains genes of the biosynthetic pathway for UDP-GlcNAc (23), and also the expression of the fimB recombinase necessary for the off-to-on switching of the fim operon for type I fimbriae (39, 40).The Mlc and NagC proteins are 40% identical, with 70% similarity, and are members of the repressor subgroup of the ROK (repressors, open reading frames, kinases) family (44). Although Mlc and NagC have clearly defined and different functions in E. coli, it is surprising that the sequence of the helix-turn-helix (H-T-H) motifs in the N-terminal DNA binding domain are unexpectedly similar (Fig. 1B). The C-terminal domains of Mlc and NagC are also homologous, but the inducing signals which displace Mlc and NagC from their DNA binding sites are very ...

N-Acetyl-glucosamine influences the biofilm formation of Escherichia coli

“…NagC is a repressor of nag operon involved in catabolism of NAG in E. coli [ 34 , 35 ]. The uptake of this sugar leads to the production of intracellular NAG-6-P that will inactivate the regulator NagC [ 36 ]. Our study shows that the inactivation of NagC, whether it is caused by the catabolism of NAG or by a mutation of nagC , is responsible for the decreased biofilm formation.…”

“…Thus, NagC is a positive regulator of biofilm formation in LF82. Interestingly, NagC is also involved in the expression of the locus of enterocytes effacement virulence genes of EHEC as well as type 1 fimbriae of E. coli K-12 [ 13 , 16 , 36 ].…”

The NAG Sensor NagC Regulates LEE Gene Expression and Contributes to Gut Colonization by Escherichia coli O157:H7

“…Yet it is not clear how NagC activates the LEE1 promoter. As proposed by authors working on fimB and galP , NagC could contact RNA polymerase directly (or another regulatory protein bound closer to the LEE1 promoter region) to enhance transcription activation, or that the nucleoprotein complex that includes NagC and other regulators alters the DNA structure nearer the promoter in such a way as to facilitate transcription initiation (Sohanpal et al, 2004 , 2007 ; El Qaidi and Plumbridge, 2008 ; El Qaidi et al, 2009 ).…”

Operator recognition by the ROK transcription factor family members, NagC and Mlc

“…Nevertheless, the HTH is important for operator binding per se . Mutations in the absolutely conserved TT–AA at positions ±5,6 around the center of symmetry of the operator (shown in yellow in Figure 1B and D ) lead to complete loss of repression by Mlc and NagC in vivo ( 10 , 11 ). Contacts between these positions and the HTH are likely to be identical or very similar in Mlc and NagC, due to the quasi-identity of the amino acids in their recognition helices (Figure 1A ) and are presumed to occur in the major groove as observed in other HTH protein–DNA structures (e.g.…”

“…NagC and Mlc are paralogous members of the ROK (Repressors, Open reading frames (ORFs) and kinases) family of proteins ( 7 ), and are responsible for controlling use of amino sugars and uptake of glucose, respectively, in Escherichia coli ( 8 ). Despite very similar DNA operator sites (Figure 1B ) and also very similar amino acid sequences of the recognition helix of the HTH motif (Figure 1A ), there is no cross regulation between the NagC- and Mlc-controlled regulons in vivo , at least not with physiological levels of the proteins ( 9 , 10 ). Overexpressing the proteins from a plasmid does allow heterologous repression, confirming that the two proteins and their targets have a common origin but also demonstrating that the affinity of one protein for the other's target is lower ( 8 ).…”