Abstract
Zinc homeostasis is critical for bacterial survival and is mediated largely at the transcriptional level by the regulation of zinc uptake and efflux genes. Here we use RNA-seq to assess transcriptional changes as a result of zinc limitation in the denitrifying bacterium Paracoccus denitrificans. The results identify the differential expression of 147 genes, most of which were upregulated in zinc-depleted medium. Included in this set of genes are a large number of transition metal transporters, several transcription factors, and hypothetical proteins. Intriguingly, genes encoding nitric oxide reductase (norCB) and nitrite reductase (nirS) were also upregulated. A Zur consensus binding motif was identified in the promoters of the most highly upregulated genes. The zinc uptake regulator (Zur) from this organism was also characterized and shown to bind to the Zur motif in a zinc-dependent manner. This work expands our current understanding of the transcriptional response of gram-negative bacteria to zinc limitation and identifies genes involved in denitrification as part of the Zur regulon.
Highlights
Zinc, an essential trace element, is indispensable for the survival of all organisms and mediates crucial roles as a catalytic center of many enzymes and in organizing protein structure (Coleman, 1992)
If present in excess, zinc is toxic to the cell due to its high affinity for divalent metal binding sites and displacement of elements such as manganese or iron required for protein function (Waldron and Robinson, 2009)
Invading bacteria may encounter severe zinc limitation as a result of “nutritional immunity” (Weinberg, 1975; Hood and Skaar, 2012), where zinc and manganese are sequestered by the host protein calprotectin in response to infection (Clohessy and Golden, 1995; Damo et al, 2013; KehlFie et al, 2013)
Summary
An essential trace element, is indispensable for the survival of all organisms and mediates crucial roles as a catalytic center of many enzymes and in organizing protein structure (Coleman, 1992). Global transcription changes in response to zinc limitation alone have been investigated for only a few WT bacterial species including E. coli (Graham et al, 2009), Neisseria meningitides (Pawlik et al, 2012), Pseudomonas protegens Pf-5 (Lim et al, 2013) and Streptococcus pneumoniae (Shafeeq et al, 2011) These investigations demonstrate differential regulation of no more than ∼100 genes, which invariably include ATP binding cassette (ABC) transporters for zinc. Recent ChiP-on-chip experiments identified Zur binding sites within or near a number of genes involved in oxidative or disulfide stress response in Bacillus subtilis (Prestel et al, 2015) These studies highlight the diversity of functions regulated by Zur in response to zinc limitation among different bacterial species. Comparison of promoter regions of upregulated genes allowed for the identification of a conserved Zur consensus sequence that was subsequently confirmed by electrophoretic mobility shift assay (EMSA) using purified P. denitrificans Zur
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