RESUMO
RT-PCR and DNA microarrays were used to probe for Zn(II)-responsive genes in E. coli cells that were made Zn(II) deficient. Microarray data revealed 114 genes were significantly up-regulated and 146 genes were significantly down-regulated in Zn(II) deficient conditions. The three most up-regulated genes were (1) znuA, which encodes for a periplasmic protein known to be involved with Zn(II) import, (2) yodA, which encodes for a periplasmic protein with unknown function, and (3) ykgM, which encodes for a ribosomal protein that is thought to be a paralog of ribosomal protein L31. YodA was over-expressed and purified as a maltose binding protein (MBP) fusion protein and shown to tightly bind 4 equivalents of Zn(II). Metal analyses showed that MBP-YkgM does not bind Zn(II). On the other hand, MBP-L31 tightly binds 1 equivalent of Zn(II). EXAFS studies on MBP-L31 suggest a ligand field of 1 histidine, 1 cysteine, and 2 additional N/O scatterers. Site-directed mutagenesis studies suggest that Cys16 coordinates Zn(II) in MBP-L31 and that the other three cysteines do not bind metal. These results are discussed in light of Zn(II) starvation model that has been postulated for B. subtilis.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Proteínas Ribossômicas/genética , Zinco/farmacologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Histidina/química , Histidina/genética , Histidina/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Fatores de Tempo , Espectroscopia por Absorção de Raios X/métodos , Zinco/metabolismoRESUMO
ZnuA is the periplasmic Zn(2+)-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn(2+)-bound, and Co(2+)-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn(2+) with Co(2+) results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn(2+) periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn(2+) (estimated K (d) < 20 nM), Co(2+), Ni(2+), Cu(2+), Cu(+), and Cd(2+), but not Mn(2+). Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn(2+) substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Metais/metabolismo , Periplasma/metabolismo , Absorção , Naftalenossulfonato de Anilina/química , Sítios de Ligação , Dicroísmo Circular , Cobalto/metabolismo , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/citologia , Fluorescência , Modelos Moleculares , Estrutura Secundária de Proteína , Espectrofotometria Ultravioleta , Especificidade por Substrato , Zinco/metabolismoRESUMO
Zinc homeostasis is not well understood beyond methods of import and export. In order to better understand zinc homeostasis in Escherichia coli by identifying Zn(II)-responsive proteins, a proteomic approach was taken. Through the use of two-dimensional gel electrophoresis, we were able to show that the levels of OmpF, AspC, YcdO, Eno, and CysE increased after 30 min of Zn(II) stress, while the levels of Tig, TufA, SelA, and LeuC decreased relative to non-stressed controls. After 4 h of Zn(II) stress, the levels of three proteins (DnaK, YeaU, and Mdh) were found to be up-regulated, while the levels of seven amino acid importers (HisJ, ArgT, LivJ, DppA, OppA, RbsB, and GinH) were found to be decreased. None of these proteins had been reported to be up- or down-regulated in any previously published cDNA microarray experiments. This result raises questions about the validity of cDNA arrays when they are used to make assumptions concerning protein levels within bacterial cells. These data also suggest that time is a factor when characterizing how the E. coli proteome responds to Zn(II) stress.
Assuntos
Escherichia coli/genética , Biossíntese de Proteínas/genética , Zinco/fisiologia , Cátions Bivalentes , Regulação para Baixo/genética , Eletroforese em Gel Bidimensional , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteômica , Fatores de TempoRESUMO
DNA microarrays were used to probe the transcriptional response of Escherichia coli to N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Fifty-five transcripts were significantly up-regulated, including all of the genes that are regulated by Zur and many that are regulated by Fur. In the same TPEN-treated cells, 46 transcripts were significantly down-regulated.
Assuntos
Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Etilenodiaminas/farmacologia , Regulação Bacteriana da Expressão Gênica , Quelantes de Ferro/farmacologia , Transcrição Gênica/efeitos dos fármacos , Adaptação Fisiológica , Regulação para Baixo , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para CimaRESUMO
The adaptive response of Escherichia coli cells to differing intracellular and extracellular Zn(II) concentrations was evaluated by two-dimensional gel electrophoresis and peptide identifications. Twenty-one Zn(II)-responsive proteins, which were previously not known to be associated with Zn(II), were identified. Most of the proteins were related to cellular metabolism and include membrane transporters and glycolytic and TCA-associated enzymes. The expression levels of no known Zn(II) transporters were identified with these studies. The results of these studies suggest a role of Zn(II) in the expression levels of several E. coli proteins, and the results are discussed in light of recent genomic profiling studies on the adaptive response of E. coli cells to stress by Zn(II) excess.