One out of Four: HspL but No Other Small Heat Shock Protein of <em>Agrobacterium tumefaciens</em> Acts as Efficient Virulence-Promoting VirB8 Chaperone

<div><p>Alpha-crystallin-type small heat shock proteins (sHsps) are ubiquitously distributed in most eukaryotes and prokaryotes. Four sHsp genes named <em>hspL</em>, <em>hspC</em>, <em>hspAT1</em>, and <em>hspAT2</em> were identified in <em>Agrobacterium tumefaciens</em>, a plant pathogenic bacterium capable of unique interkingdom DNA transfer via type IV secretion system (T4SS). HspL is highly expressed in virulence-induced growth condition and functions as a VirB8 chaperone to promote T4SS-mediated DNA transfer. Here, we used genetic and biochemical approaches to investigate the involvement of the other three sHsps in T4SS and discovered the molecular basis underlying the dominant function of HspL in promoting T4SS function. While single deletion of <em>hspL</em> but no other sHsp gene reduced T4SS-mediated DNA transfer and tumorigenesis efficiency, additional deletion of other sHsp genes in the <em>hspL</em> deletion background caused synergistic effects in the virulence phenotypes. This is correlated with the high induction of <em>hspL</em> and only modest increase of <em>hspC</em>, <em>hspAT1</em>, and <em>hspAT2</em> at their mRNA and protein abundance in virulence-induced growth condition. Interestingly, overexpression of any single sHsp gene alone in the quadruple mutant caused increased T4SS-mediated DNA transfer and tumorigenesis. Thermal aggregation protecting assays <em>in vitro</em> indicated that all four sHsps exhibit chaperone activity for the model substrate citrate synthase but only HspL functions as efficient chaperone for VirB8. The higher VirB8 chaperone activity of HspL was also demonstrated <em>in vivo,</em> in which lower amounts of HspL than other sHsps were sufficient in maintaining VirB8 homeostasis in <em>A. tumefaciens.</em> Domain swapping between HspL and HspAT2 indicated that N-terminal, central alpha-crystallin, and C-terminal domains of HspL all contribute to HspL function as an efficient VirB8 chaperone. Taken together, we suggest that the dominant role of HspL in promoting T4SS function is based on its higher expression in virulence-induced condition and its more efficient VirB8 chaperone activity as compared to other sHsps.</p> </div

Chaperone activity assay of sHsps.

<p>(A) Thermal aggregation protection assays using model substrate CS (600 nM) was carried out at 43°C in the absence (□) or presence of HspL-His₆ (♦), HspC-His₆ (•), HspAT1-His₆ (▴) or HspAT2-His₆ (▾) at concentration of 1.2 µM respectively. (B) 1 µM GST-VirB8 was used as substrate and was carried out at 50°C in the absence (□) or presence of HspL-His₆ (♦), HspC-His₆ (•), HspAT1-His₆ (▾) or HspAT2-His₆ (▴) at concentration of 2 µM respectively. (C) 1 µM GST-VirB8 was used as substrate and was carried out at 50°C in the absence (□) or presence of HspAT2-His₆ with concentration at 1 µM(•), 2 µM(▾), 4 µM(▴), 8 µM(▪) and 16 µM (<>\raster="rg1"<>), or of 2 µM HspL-His₆ (♦). Aggregation was monitored in absorbance at 360 nm and presented as a function of time.</p

AS-induced mRNA and protein expression of four sHsp genes in <i>A. tumefaciens</i>.

<p>(A) Quantitative RT-PCR analysis of sHsp mRNA levels in <i>A. tumefaciens</i> wild type (WT) strain NT1RE(pJK270) and the <i>ΔhspL</i> mutant. Relative expression level is normalized by 16S rRNA as an internal control and the mean values of fold-change relative to the DMSO control of WT. Data are mean with standard deviation (SD) of 2 biological replicates, each of which contains 3 technical replicates. (B) Each of the sHsp genes expressing proteins tagged with HA and driven by its upstream promoter region on plasmid was expressed in wild type (WT) strain NT1RE(pJK270) and the <i>ΔhspL</i> mutant. The agrobacterial cells were grown in ABMES medium (pH5.5) at 25°C in the absence (−, DMSO control) or the presence of AS for 16 hrs. The total cell lysates were detected by western blot analysis with specific antibody against HA, VirB2, or RpoA. Numbers on the left are molecular masses of reference proteins in kDa.</p

Effects of IPTG-induced His-tagged HspL, HspC, HspAT1 or HspAT2 on VirB8 accumulation.

<p>(A) <i>ΔhspL</i>(pTrc-HspL-His), <i>ΔhspL</i>(pTrc-HspC-His), <i>ΔhspL</i>(pTrc-HspAT1-His), and <i>ΔhspL</i>(pTrc-HspAT2-His) were grown in AB-MES (pH 5.5) containing 200 µM AS at 28°C with or without 0.4 mM IPTG for 24 h. The total cell lysates were detected by western blot analysis with specific antiserum. (B) <i>ΔhspL</i>(pTrc-HspL-His) and<i>ΔhspL</i>(pTrc-HspAT2-His) were grown in AB-MES (pH 5.5) containing 200 µM AS at 28°C with different concentrations of IPTG (0∼80 µM) for 24 h. The signal of VirB8 or sHsp-His was determined by western blot using anti-VirB8 or anti-His antiserium and the intensity of signal was quantified. The signal of VirB8 or sHsp-His was normalized by the signal of RpoA (loading control). The regression curve was created using the increase of signal intensity of VirB8 as Y-axis and sHsp-His as X-axis. The lineal range of signals was selected to calculate regression curve, the increase of signal intensity of VirB8 as Y-axis and sHsp-His as X-axis.</p

Mobilization efficiency of RSF1010 derivative PML122<i>Δkm</i>::Tc^R between <i>Agrobacterium</i> strains.

<p>Mobilization efficiency of RSF1010 derivative PML122<i>Δkm</i>::Tc^R between <i>Agrobacterium</i> strains.</p

Tumorigenesis and RSF1010 transfer efficiency assays of single and multiple sHsp deletion mutants.

<p>(A) Tumorigenesis efficiency is presented as number of tumor per disc averaged from more than 60 potato discs, with standard errors. Three independent experiments were carried out with similar results with the representative result shown. (B) The RSF1010 transfer efficiency was evaluated as number of transconjugants per input donor. Average values for relative RSF1010 transfer efficiency from three independent experiments are shown with standard errors, in which the efficiency of wild type strain NT1RE(pJK270) (WT) was set at 100% and that of other strains is shown relative to that of NT1RE(pJK270). Means annotated with the same letter (a-d) are not significantly different; those with different letters are significantly different (<i>P</i><0.05) according to Duncan’s multiple range test. The absolute RSF1010 transfer efficiency and the numbers of transconjugants and input donors obtained from three independent experiments were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049685#pone-0049685-t001" target="_blank">Table 1</a>.</p

Effect of overproduced-small heat shock proteins on mobilization efficiency of RS1010 derivative 122<i>Δkm</i>::Tc^R between <i>Agrobacterium</i> strains.

a<p>expressed as number of transconjugants per input donor.</p

Effect of overproduced sHsp in tumorigenesis and T4SS-mediated DNA transfer.

<p>Wild type NT1RE(pJK270) (WT), the quadruple sHsp deletion mutant <i>Δ4sHsps</i> harboring pRL662 (V) or pRL662::<i>hspL</i> (HspL), pRL662::<i>hspC</i> (HspC), pRL662::<i>hspAT1</i> (HspAT1), pRL662::<i>hspAT2</i> (HspAT2) were assayed for their tumorigenesis efficiency on potato tuber discs and RSF1010 transfer efficiency as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049685#pone-0049685-g001" target="_blank">Figure 1</a>. The absolute RSF1010 transfer efficiencies obtained from each of the three independent experiments were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049685#pone-0049685-t002" target="_blank">Table 2</a>.</p

Chaperone activity assay of HspL/HspAT2 chimeric proteins using GST-VirB8 as the substrate.

<p>(A) Amino acid sequences of HspL and HspAT2 were aligned and the conserved amino acids are highlighted in yellow blocks and the blue and green blocks indicate the identical and similar amino acids, respectively. The boundary for N-terminal arm, α-crystallin domain, and C-terminal arm was indicated by arrows. (B) Diagram of the HspL/HspAT2 chimeric proteins. The HspL sequence is represented by thin lines, whereas HspAT2 is represented by thick lines with the respective position of amino acid residues indicated. The amino acid length of each protein is indicated to the right. (C) Thermal aggregation protection assays using GST-VirB8 (1 µM) was carried out at 50°C in the absence (□), or presence of HspL-His₆ (♦), _NAT2-HspL-His₆ (•), HspL-AT2_C-His₆ (▾), _NCHspL-AT2α-His₆ (▴) and HspAT2–His₆ (▪) at concentration of 2 µM (for GST-VirB8) or 1.2 µM (for CS) respectively. Aggregation was monitored in absorbance at 360 nm and presented as a function of time.</p

An Oxygenase-Independent Cholesterol Catabolic Pathway Operates under Oxic Conditions

<div><p>Cholesterol is one of the most ubiquitous compounds in nature. The 9,10-<i>seco</i>-pathway for the aerobic degradation of cholesterol was established thirty years ago. This pathway is characterized by the extensive use of oxygen and oxygenases for substrate activation and ring fission. The classical pathway was the only catabolic pathway adopted by all studies on cholesterol-degrading bacteria. <i>Sterolibacterium denitrificans</i> can degrade cholesterol regardless of the presence of oxygen. Here, we aerobically grew the model organism with ¹³C-labeled cholesterol, and substrate consumption and intermediate production were monitored over time. Based on the detected ¹³C-labeled intermediates, this study proposes an alternative cholesterol catabolic pathway. This alternative pathway differs from the classical 9,10-<i>seco</i>-pathway in numerous important aspects. First, substrate activation proceeds through anaerobic C-25 hydroxylation and subsequent isomerization to form 26-hydroxycholest-4-en-3-one. Second, after the side chain degradation, the resulting androgen intermediate is activated by adding water to the C-1/C-2 double bond. Third, the cleavage of the core ring structure starts at the A-ring via a hydrolytic mechanism. The ¹⁸O-incorporation experiments confirmed that water is the sole oxygen donor in this catabolic pathway.</p></div