Low copy expression vectors for use in Yersinia sp. and related organisms

In Yersinia, the most commonly used expression vectors for genetic studies such as gene complementation do not effectively allow for both induction and repression of gene expression. Additionally, there is no expression system available that can be induced in bacteria growing in vitro as well as in vivo, e.g. in eukaryotic cell lines or in living animal models. Here, we present a series of novel inducible low copy expression vectors that are well suited for use in the Yersinia species. Their tet operator/promoter/repressor system makes them distinct from other vectors, and gene transcription in bacteria can easily be induced by addition of anhydrotetracyline (ATc) either to the growth medium, to tissue culture medium during bacterial infections of cell lines or by injection into animals infected with bacteria. Researchers can choose between two different antibiotic resistances (kanamycin or spectinomycin), between two copy numbers (5 or 12-22) as well as between two different versions for expression from either the native RBS and ATG or RBS and ATG encoded in the plasmid. The whole vector series contains the same multi-cloning site from pBluescript II KS+ that allows for easy subcloning. Moreover, these vectors are built in a modular fashion that makes it simple to adapt them for other purposes. Finally, in addition to their use in Yersinia they are suitable for use in many other Enterobacteriaceae

CcpA-Independent Glucose Regulation of Lactate Dehydrogenase 1 in Staphylococcus aureus

Lactate Dehydrogenase 1 (Ldh1) is a key enzyme involved in Staphylococcus aureus NO·-resistance. Full ldh1-induction requires the presence of glucose, and mutants lacking the Carbon-Catabolite Protein (CcpA) exhibit decreased ldh1 transcription and diminished Ldh1 activity. The redox-regulator Rex represses ldh1 directly by binding to Rex-sites within the ldh1 promoter (Pldh1). In the absence of Rex, neither glucose nor CcpA affect ldh1 expression implying that glucose/CcpA-mediated activation requires Rex activity. Rex-mediated repression of ldh1 depends on cellular redox status and is maximal when NADH levels are low. However, compared to WT cells, the ΔccpA mutant exhibited impaired redox balance with relatively high NADH levels, yet ldh1 was still poorly expressed. Furthermore, CcpA did not drastically alter Rex transcript levels, nor did glucose or CcpA affect the expression of other Rex-regulated genes indicating that the glucose/CcpA effect is specific for Pldh1. A putative catabolite response element (CRE) is located ∼30 bp upstream of the promoter-distal Rex-binding site in Pldh1. However, CcpA had no affinity for Pldh1 in vitro and a genomic mutation of CRE upstream of Pldh1 in S. aureus had no affect on Ldh1 expression in vivo. In contrast to WT, ΔccpA S. aureus preferentially consumes non-glycolytic carbon sources. However when grown in defined medium with glucose as the primary carbon source, ΔccpA mutants express high levels of Ldh1 compared to growth in media devoid of glucose. Thus, the actual consumption of glucose stimulates Ldh1 expression rather than direct CcpA interaction at Pldh1

Identification of YsrT and Evidence that YsrRST Constitute a Unique Phosphorelay System in Yersinia enterocolitica▿

Two-component systems (TCS) and phosphorelay systems are mechanisms used by bacteria and fungi to quickly adapt to environmental changes to produce proteins necessary for survival in new environments. Bacterial pathogens use TCS and phosphorelay systems to regulate genes necessary to establish infection within their hosts, including type III secretion systems (T3SS). The Yersinia enterocolitica ysa T3SS is activated in response to NaCl by YsrS and YsrR, a putative hybrid sensor kinase and a response regulator, respectively. Hybrid TCS consist of a sensor kinase that typically has three well-conserved sites of phosphorylation: autophosphorylation site H1, D1 within a receiver domain, and H2 in the histidine phosphotransferase (HPt) domain. From H2, the phosphoryl group is transferred to D2 on the response regulator. A curious feature of YsrS is that it lacks the terminal HPt domain. We report here the identification of the HPt-containing protein (YsrT) that provides this activity for the Ysr system. YsrT is an 82-residue protein predicted to be cytosolic and α-helical in nature and is encoded by a gene adjacent to ysrS. To demonstrate predicted functions of YsrRST as a phosphorelay system, we introduced alanine substitutions at H1, D1, H2, and D2 and tested the mutant proteins for the ability to activate a ysaE-lacZ reporter. As expected, substitutions at H1, H2, and D2 resulted in a loss of activation of ysaE expression. This indicates an interruption of normal protein function, most likely from loss of phosphorylation. A similar result was expected for D1; however, an intriguing “constitutive on” phenotype was observed. In addition, the unusual feature of a separate HPt domain led us to compare the sequences surrounding the ysrS-ysrT junction in several Yersinia strains. In every strain examined, ysrT is a separate gene, leading to speculation that there is a functional advantage to YsrT being an independent protein

CcpA affect at P<i>_ldh1</i> is indirect.

<p><b>A.</b> EMSA with His-tagged CcpA using P<i>_ldh</i>₁ (LEFT) or P<i>_rocD</i>₂ (RIGHT) as probes and an internal <i>hmp</i> fragment as a non-specific probe (N.S. band). Only at highest ratios of CcpA::DNA did non-specific shifted bands become evident using P<i>_ldh</i>₁ as a probe (white arrows). 250 fmol of DNA probes were used in all wells. <b>B.</b> Alignment of CRE from P<i>_ldh</i>₁, P<i>_rocD</i>₂, the <i>B. subtilis</i> consensus sequence and the mutated CRE^*. <b>C.</b> Q RT-PCR analyses of <i>ldh</i>1 transcript levels normalized to those of <i>rpoD</i> in WT, Δ<i>ccpA</i> and CRE^* derivatives of <i>S. aureus</i> strain COL following 15 min. NO· exposure (2 mM DEA-NO).</p

CcpA-Independent Glucose Regulation of Lactate Dehydrogenase 1 in <em>Staphylococcus aureus</em>

<div><p>Lactate Dehydrogenase 1 (Ldh1) is a key enzyme involved in <em>Staphylococcus aureus</em> NO·-resistance. Full <em>ldh</em>1-induction requires the presence of glucose, and mutants lacking the Carbon-Catabolite Protein (CcpA) exhibit decreased <em>ldh</em>1 transcription and diminished Ldh1 activity. The redox-regulator Rex represses <em>ldh</em>1 directly by binding to Rex-sites within the <em>ldh</em>1 promoter (P<em>_ldh</em>₁). In the absence of Rex, neither glucose nor CcpA affect <em>ldh</em>1 expression implying that glucose/CcpA-mediated activation requires Rex activity. Rex-mediated repression of <em>ldh</em>1 depends on cellular redox status and is maximal when NADH levels are low. However, compared to WT cells, the Δ<em>ccpA</em> mutant exhibited impaired redox balance with relatively high NADH levels, yet <em>ldh</em>1 was still poorly expressed. Furthermore, CcpA did not drastically alter Rex transcript levels, nor did glucose or CcpA affect the expression of other Rex-regulated genes indicating that the glucose/CcpA effect is specific for P<em>_ldh</em>₁. A putative catabolite response element (CRE) is located ∼30 bp upstream of the promoter-distal Rex-binding site in P<em>_ldh</em>₁. However, CcpA had no affinity for P<em>_ldh</em>₁<em>in vitro</em> and a genomic mutation of CRE upstream of P<em>_ldh</em>₁ in <em>S. aureus</em> had no affect on Ldh1 expression <em>in vivo.</em> In contrast to WT, Δ<em>ccpA S. aureus</em> preferentially consumes non-glycolytic carbon sources. However when grown in defined medium with glucose as the primary carbon source, Δ<em>ccpA</em> mutants express high levels of Ldh1 compared to growth in media devoid of glucose. Thus, the actual consumption of glucose stimulates Ldh1 expression rather than direct CcpA interaction at P<em>_ldh</em>₁.</p> </div

Ldh1 expression in <i>S. aureus</i> is dependent on glucose and CcpA.

<p><b>A.</b> WT <i>S. aureus</i> strain Newman and an isogenic Δ<i>ccpA</i> mutant harboring P<i>_ldh</i>₁::GFP promoter fusions were grown in chemically defined medium with 0.5% casamino acids as primary carbon/energy sources. Glucose (0.5%) was added when indicated. Once cultures reached early exponential phase, NO· was administered (1 mM DETA/NO) and fluorescence and optical density were monitored for two hours. <b>B.</b> Quantitative Real-Time Reverse Transcriptase PCR (Q RT-PCR) was used to determine <i>ldh</i>1 transcript levels relative to <i>rpoD</i> in WT <i>S. aureus</i> strain COL and an isogenic isogenic Δ<i>ccpA</i> mutant grown in media as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054293#pone-0054293-g001" target="_blank">Figure 1A</a>. NO· was administerd as 2 mM DEA-NO. <b>C.</b> Ldh1 enzyme activity from cell extracts of WT and isogenic Δ<i>ccpA S. aureus</i> strain COL lacking <i>ldh</i>2. Cells were cultured in BHI and stimulated with NO·(2 mM DEA-NO) 15 minutes prior to obtaining lysates.</p

Strains, Plasmids and Primers.

<p>Strains, Plasmids and Primers.</p

Alteration of Rex levels and/or activity cannot explain the Rex-dependency of ldh1 glucose-induction.

<p><b>A.</b> Q RT-PCR analyses of <i>rex</i> transcript levels normalized to those of <i>rpoD</i> in WT versus Δ<i>ccpA S. aureus</i> strain COL following NO· stimulation. <b>B.</b> Q RT-PCR analyses of other Rex-regulated genes normalized to <i>rpoD</i> upon NO·-stimulation. Only <i>ldh</i>1 exhibits CcpA-dependent activation. <b>C.</b> Redox status depicted as NAD⁺/NADH ratios of WT versus Δ<i>ccpA S. aureus</i> COL prior to and after stimulation with NO·.</p

Both Rex and CcpA are required for glucose-mediated induction of <i>ldh</i>1.

<p><b>A</b>. Q RT-PCR of <i>ldh1</i> transcript from <i>S. aureus</i> strain COL normalized to <i>rpoD</i> in cells exposed/unexposed to NO· administered as 2 mM DEA-NO 15 minutes prior to RNA isolation. <b>B</b>. ElectroMobility Shift Assay (EMSA) of P_<i>ldh1</i> using purified His-Rex at increasing molar ratios of Rex:DNA (250 fmol promoter DNA in all wells). Internal fragment of <i>hmp</i> was used as a non-specific probe (N.S. band). As predicted by the presence of two Rex sites in P_<i>ldh1</i>, two independent shifted bands appear with increasing Rex::DNA molar ratios. <b>C</b>. Schematic representation of P_<i>ldh1</i> and fragments used for GFP:fusions and their relative activity following stimulation with NO· (2 mM DETA-NO). The activity of Fusion 4 was indistinguishable form that of a promoterless control. <b>D</b>. Reverse transcriptase PCR using two different forward primers depicted in Figure 1C to amplify products using both cDNA (cD) and genomic (G) DNA as templates.</p

Low copy expression vectors for use in Yersinia sp. and related organisms

In Yersinia, the most commonly used expression vectors for genetic studies such as gene complementation do not effectively allow for both induction and repression of gene expression. Additionally, there is no expression system available that can be induced in bacteria growing in vitro as well as in vivo, e.g. in eukaryotic cell lines or in living animal models. Here, we present a series of novel inducible low copy expression vectors that are well suited for use in the Yersinia species. Their tet operator/promoter/repressor system makes them distinct from other vectors, and gene transcription in bacteria can easily be induced by addition of anhydrotetracyline (ATc) either to the growth medium, to tissue culture medium during bacterial infections of cell lines or by injection into animals infected with bacteria. Researchers can choose between two different antibiotic resistances (kanamycin or spectinomycin), between two copy numbers (5 or 12-22) as well as between two different versions for expression from either the native RBS and ATG or RBS and ATG encoded in the plasmid. The whole vector series contains the same multi-cloning site from pBluescript II KS+ that allows for easy subcloning. Moreover, these vectors are built in a modular fashion that makes it simple to adapt them for other purposes. Finally, in addition to their use in Yersinia they are suitable for use in many other Enterobacteriaceae