Role of the sRNA GcvB in regulation of cycA in Escherichia coli

Abstract

In Escherichia coli, the gcvB gene encodes a small non-translated RNA that regulates several genes involved in transport of amino acids and peptides (including sstT, oppA and dppA). Microarray analysis identified cycA as an additional regulatory target of GcvB. The cycA gene encodes a permease for the transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in Luria–Bertani broth. In addition, deletion of the gcvB gene resulted in increased sensitivity to d-cycloserine, consistent with increased expression of cycA. A cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA expression in Luria–Bertani broth and that Hfq is required for the GcvB effect. GcvB had no effect on cycA : : lacZ expression in glucose minimal medium supplemented with glycine. However, Hfq still negatively regulated the fusion in the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified a region complementary to this region of cycA mRNA. However, mutations predicted to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of cycA : : lacZ. A model for GcvB function in cell physiology is discussed.

Characterization of Escherichia coli d -Cycloserine Transport and Resistant Mutants

“…In addition, this vector construct lacks the cycA 5= UTR, which is the region regulated by GcvB. Expression constructs lacking the cycA 5= UTR result in increased cycA expression when grown in LB (18). The presence of pcycA in the different cycA mutant backgrounds did not confer increased sensitivity to DCS compared to those strains with the pACYC177 vector when grown in LB or human urine (Table 2).…”

“…coli K-12 and CFT073 gcvB mutants do not display an increase in sensitivity to DCS. Pulvermacher et al recently determined in E. coli K-12 that GcvB, in conjunction with Hfq, negatively regulates cycA expression when grown in LB broth (18). By optical density measurements, they showed that a K-12 gcvB strain (GS1144) lyses earlier and more completely than the parent strain (GS162) when grown in LB containing DCS.…”

“…The results of Pulvermacher et al suggest that when the E. coli K-12 strain GS162 is grown in LB, the small RNA (sRNA) encoded by gcvB, in conjunction with Hfq, interacts with the cycA 5= UTR, which results in the negative regulation of cycA (18). The GS162 gcvB mutant strain lysed at an earlier time point than the parent when growth in LB broth containing DCS was measured by changes in optical density (18). Our results indicate that the loss of gcvB did not affect DCS sensitivity when an E. coli mutant and its respective parent were assayed by disk diffusion on LB agar medium (Table 3).…”

“…GcvB, in conjunction with the RNAbinding protein, Hfq, negatively regulates the expression of genes involved in the transport of amino acids, dipeptides, and oligopeptides (13)(14)(15)(16)(17). Recently, Pulvermacher et al determined in E. coli K-12 that GcvB negatively regulates cycA in an Hfq-dependent manner when grown in LB (18). When grown in minimal glucose medium containing glycine, Hfq, in a GcvB-independent manner, negatively regulates cycA expression.…”

Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

“…Several investigators have used lacZ translational fusions to analyze ncRNA/target interactions (Baker et al, 2007;De Lay and Gottesman, 2009;Mandin and Gottesman, 2009;Pulvermacher et al, 2009). We did a pilot experiment where we inserted test sequence carrying translational signals upstream of the LacZ fusion gene.…”