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Regulation of sialic acid catabolism by the DNA binding protein NanR in Escherichia coli.

ABSTRACT: All Escherichia coli strains so far examined possess a chromosomally encoded nanATEK-yhcH operon for the catabolism of sialic acids. These unique nine-carbon sugars are synthesized primarily by higher eukaryotes and can be used as carbon, nitrogen, and energy sources by a variety of microbial pathogens or commensals. The gene nanR, located immediately upstream of the operon, encodes a protein of the FadR/GntR family that represses nan expression in trans. S1 analysis identified the nan transcriptional start, and DNA footprint analysis showed that NanR binds to a region of approximately 30 bp covering the promoter region. Native (nondenaturing) polyacrylamide gel electrophoresis, mass spectrometry, and chemical cross-linking indicated that NanR forms homodimers in solution. The region protected by NanR contains three tandem repeats of the hexameric sequence GGTATA. Gel shift analysis with purified hexahistidine-tagged or native NanR detected three retarded complexes, suggesting that NanR binds sequentially to the three repeats. Artificial operators carrying different numbers of repeats formed the corresponding number of complexes. Among the sugars tested that were predicted to be products of the nan-encoded system, only the exogenous addition of sialic acid resulted in the dramatic induction of a chromosomal nanA-lacZ fusion or displaced NanR from its operator in vitro. Titration of NanR by the nan promoter region or artificial operators carrying different numbers of the GGTATA repeat on plasmids in this fusion strain supported the binding of the regulator to target DNA in vivo. Together, the results indicate that GGTATA is important for NanR binding, but the precise mechanism remains to be determined.

SUBMITTER: Kalivoda KA

PROVIDER: S-EPMC166481 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Regulation of sialic acid catabolism by the DNA binding protein NanR in Escherichia coli.

Kalivoda Kathryn A KA Steenbergen Susan M SM Vimr Eric R ER Plumbridge Jacqueline J

Journal of bacteriology 20030801 16

All Escherichia coli strains so far examined possess a chromosomally encoded nanATEK-yhcH operon for the catabolism of sialic acids. These unique nine-carbon sugars are synthesized primarily by higher eukaryotes and can be used as carbon, nitrogen, and energy sources by a variety of microbial pathogens or commensals. The gene nanR, located immediately upstream of the operon, encodes a protein of the FadR/GntR family that represses nan expression in trans. S1 analysis identified the nan transcrip ...[more]

PMID: 12897000

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Project description:UnlabelledCorynebacterium glutamicum metabolizes sialic acid (Neu5Ac) to fructose-6-phosphate (fructose-6P) via the consecutive activity of the sialic acid importer SiaEFGI, N-acetylneuraminic acid lyase (NanA), N-acetylmannosamine kinase (NanK), N-acetylmannosamine-6P epimerase (NanE), N-acetylglucosamine-6P deacetylase (NagA), and glucosamine-6P deaminase (NagB). Within the cluster of the three operons nagAB, nanAKE, and siaEFGI for Neu5Ac utilization a fourth operon is present, which comprises cg2936, encoding a GntR-type transcriptional regulator, here named NanR. Microarray studies and reporter gene assays showed that nagAB, nanAKE, siaEFGI, and nanR are repressed in wild-type (WT) C. glutamicum but highly induced in a ΔnanR C. glutamicum mutant. Purified NanR was found to specifically bind to the nucleotide motifs A[AC]G[CT][AC]TGATGTC[AT][TG]ATGT[AC]TA located within the nagA-nanA and nanR-sialA intergenic regions. Binding of NanR to promoter regions was abolished in the presence of the Neu5Ac metabolism intermediates GlcNAc-6P and N-acetylmannosamine-6-phosphate (ManNAc-6P). We observed consecutive utilization of glucose and Neu5Ac as well as fructose and Neu5Ac by WT C. glutamicum, whereas the deletion mutant C. glutamicum ΔnanR simultaneously consumed these sugars. Increased reporter gene activities for nagAB, nanAKE, and nanR were observed in cultivations of WT C. glutamicum with Neu5Ac as the sole substrate compared to cultivations when fructose was present. Taken together, our findings show that Neu5Ac metabolism in C. glutamicum is subject to catabolite repression, which involves control by the repressor NanR.ImportanceNeu5Ac utilization is currently regarded as a common trait of both pathogenic and commensal bacteria. Interestingly, the nonpathogenic soil bacterium C. glutamicum efficiently utilizes Neu5Ac as a substrate for growth. Expression of genes for Neu5Ac utilization in C. glutamicum is here shown to depend on the transcriptional regulator NanR, which is the first GntR-type regulator of Neu5Ac metabolism not to use Neu5Ac as effector but relies instead on the inducers GlcNAc-6P and ManNAc-6P. The identification of conserved NanR-binding sites in intergenic regions within the operons for Neu5Ac utilization in pathogenic Corynebacterium species indicates that the mechanism for the control of Neu5Ac catabolism in C. glutamicum by NanR as described in this work is probably conserved within this genus.

Project description:Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015, https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS5 insertion or IS-mediated deletion in cadC, while population B11 had a point mutation affecting the arginine activator adiY The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR, yhiM, and Gad). Other strains showed downregulation of H2 consumption mediated by hydrogenases (hya and hyb) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes (dmsABC, frdABCD, hybABO, nikABCDE, and nrfAC). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism.IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain.

Dataset Information

Regulation of sialic acid catabolism by the DNA binding protein NanR in Escherichia coli.

Publications

Regulation of sialic acid catabolism by the DNA binding protein NanR in Escherichia coli.

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