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

Unexpected diversity in socially synchronized rhythms of shorebirds

The behavioural rhythms of organisms are thought to be under strong selection, influenced by the rhythmicity of the environment. Such behavioural rhythms are well studied in isolated individuals under laboratory conditions, but free-living individuals have to temporally synchronize their activities with those of others, including potential mates, competitors, prey and predators. Individuals can temporally segregate their daily activities (for example, prey avoiding predators, subordinates avoiding dominants) or synchronize their activities (for example, group foraging, communal defence, pairs reproducing or caring for offspring). The behavioural rhythms that emerge from such social synchronization and the underlying evolutionary and ecological drivers that shape them remain poorly understood. Here we investigate these rhythms in the context of biparental care, a particularly sensitive phase of social synchronization where pair members potentially compromise their individual rhythms. Using data from 729 nests of 91 populations of 32 biparentally incubating shorebird species, where parents synchronize to achieve continuous coverage of developing eggs, we report remarkable within-and between-species diversity in incubation rhythms. Between species, the median length of one parent's incubation bout varied from 1-19 h, whereas period length-the time in which a parent's probability to incubate cycles once between its highest and lowest value-varied from 6-43 h. The length of incubation bouts was unrelated to variables reflecting energetic demands, but species relying on crypsis (the ability to avoid detection by other animals) had longer incubation bouts than those that are readily visible or who actively protect their nest against predators. Rhythms entrainable to the 24-h light-dark cycle were less prevalent at high latitudes and absent in 18 species. Our results indicate that even under similar environmental conditions and despite 24-h environmental cues, social synchronization can generate far more diverse behavioural rhythms than expected from studies of individuals in captivity. The risk of predation, not the risk of starvation, may be a key factor underlying the diversity in these rhythms.</p

Unexpected diversity in socially synchronized rhythms of shorebirds

The behavioural rhythms of organisms are thought to be under strong selection, influenced by the rhythmicity of the environment1, 2, 3, 4. Such behavioural rhythms are well studied in isolated individuals under laboratory conditions1, 5, but free-living individuals have to temporally synchronize their activities with those of others, including potential mates, competitors, prey and predators6, 7, 8, 9, 10. Individuals can temporally segregate their daily activities (for example, prey avoiding predators, subordinates avoiding dominants) or synchronize their activities (for example, group foraging, communal defence, pairs reproducing or caring for offspring)6, 7, 8, 9, 11. The behavioural rhythms that emerge from such social synchronization and the underlying evolutionary and ecological drivers that shape them remain poorly understood5, 6, 7, 9. Here we investigate these rhythms in the context of biparental care, a particularly sensitive phase of social synchronization12 where pair members potentially compromise their individual rhythms. Using data from 729 nests of 91 populations of 32 biparentally incubating shorebird species, where parents synchronize to achieve continuous coverage of developing eggs, we report remarkable within- and between-species diversity in incubation rhythms. Between species, the median length of one parent’s incubation bout varied from 1–19 h, whereas period length—the time in which a parent’s probability to incubate cycles once between its highest and lowest value—varied from 6–43 h. The length of incubation bouts was unrelated to variables reflecting energetic demands, but species relying on crypsis (the ability to avoid detection by other animals) had longer incubation bouts than those that are readily visible or who actively protect their nest against predators. Rhythms entrainable to the 24-h light–dark cycle were less prevalent at high latitudes and absent in 18 species. Our results indicate that even under similar environmental conditions and despite 24-h environmental cues, social synchronization can generate far more diverse behavioural rhythms than expected from studies of individuals in captivity5, 6, 7, 9. The risk of predation, not the risk of starvation, may be a key factor underlying the diversity in these rhythms

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

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

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