Identification of d -arabinan-degrading enzymes in mycobacteria

Bacterial cell growth and division require the coordinated action of enzymes that synthesize and degrade cell wall polymers. Here, we identify enzymes that cleave the D-arabinan core of arabinogalactan, an unusual component of the cell wall of Mycobacterium tuberculosis and other mycobacteria. We screened 14 human gut-derived Bacteroidetes for arabinogalactan-degrading activities and identified four families of glycoside hydrolases with activity against the D-arabinan or D-galactan components of arabinogalactan. Using one of these isolates with exo-D-galactofuranosidase activity, we generated enriched D-arabinan and used it to identify a strain of Dysgonomonas gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan, including members of the DUF2961 family (GH172) and a family of glycoside hydrolases (DUF4185/GH183) that display endo-D-arabinofuranase activity and are conserved in mycobacteria and other microbes. Mycobacterial genomes encode two conserved endo-D-arabinanases with different preferences for the D-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall

Southeastern Association of Law Libraries Annual Meeting

The 2009 SEAALL Annual Meeting was held in Athens Georgia, April 16-18, 2009

Book review: Women and Human Development: the Capabilities Approach

Review of: Women and Human Development : The Capabilities Approach by Martha C. Nussbaum. New York: Cambridge University Press, 2000. ISBN: 0521660866

Effect of <i>osm-9</i> and <i>osm-12</i> mutations and genetic ablation of ASH osmosensory neurons on survival during hypertonic stress.

<p>(A) Survival of wild type worms and <i>osm-9(ky10)</i> loss-of-function mutants exposed to different concentrations of NaCl for 24 h. Values are means ± S.E. of four independent and blinded experiments. *P<0.05 and **P<0.001 compared to wild type animals. (B) Survival of wild type worms and <i>osm-9(ok1677)</i> and <i>osm-12(n1606)</i> loss-of-function mutants exposed to different concentrations of NaCl for 24 h. Values are means ± S.E. of four independent experiments. *P<0.05 for both <i>osm-9(ok1677)</i> and <i>osm-12(n1606)</i> mutants compared to wild type animals. (C) Survival of wild type and ASH ablated (i.e., <i>Is[sra-6p</i>::<i>mCasp1]</i> transgenic strain) worms exposed to 500 or 600 mM NaCl for 24 h. Values are means ± S.E. of 6–12 independent experiments. *P<0.002 and **P<0.02 compared to wild type worms. (D) Survival of wild type, <i>gpa-4(pk381)</i> mutants and ASI ablated (i.e., <i>oyIs84</i>[<i>gpa-4p</i>::<i>TU#813 + gcy-27p</i>::<i>TU#814 + gcy-27p</i>::<i>GFP + unc-122p</i>::<i>dsRed</i>] transgenic strain) worms exposed to 500 or 600 mM NaCl for 24 h. Values are means ± S.E. of 8–9 experiments. *</p

Effect of loss of ASH osmosensory neuron function on hypertonic stress induced whole animal water loss and volume recovery.

<p>(A) Whole worm volume changes. Wild type worms and <i>osm-9(ok1677)</i> and <i>osm-12(n1606)</i> loss-of-function mutants were transferred to agar plates containing 200 mM NaCl at time 0. Values are means ± S.E. (n = 8). <i>gpdh-1</i> mRNA expression (B) and whole animal glycerol levels (C) in wild type and <i>osm-9(ok1677)</i> and <i>osm-12(n1606)</i> worms. Values are means ± S.E. (n = 3). *P<0.002 and **P<0.007 compared to wild type worms. (D) Time course changes in <i>gpdh-1</i> expression. Wild type worms and <i>osm-9(ok1677)</i> and <i>osm-12(n1606)</i> loss-of-function mutants were transferred to agar plates containing 200 mM NaCl at time 0. Values are means ± S.E. (n = 3–5). *P<0.01 compared to <i>osm-9(ok1677)</i> and <i>osm-12(n1606)</i> mutants.</p

Osmotic avoidance behavior in wild type and ASH ablation worms and <i>osm</i> mutants.

<p>Osmotic avoidance behavior in wild type and ASH ablation worms and <i>osm</i> mutants.</p

Abnormal Osmotic Avoidance Behavior in <i>C</i>. <i>elegans</i> Is Associated with Increased Hypertonic Stress Resistance and Improved Proteostasis - Fig 5

<p><b>Lifespan and (A) and resistance to heat shock (B), cadmium (C) and oxidative stress (D) in <i>osm-9(ok1677)</i> worms.</b> Median lifespan (A) was 21 days for wild type and <i>osm-9(ok1677)</i> worms (n = 100 worms for both groups). Values in B, C and D are means ± S.E. (n = 5). *P<0.007 and **P<0.04 compared to wild type worms.</p

Abnormal Osmotic Avoidance Behavior in <i>C</i>. <i>elegans</i> Is Associated with Increased Hypertonic Stress Resistance and Improved Proteostasis

<div><p>Protein function is controlled by the cellular proteostasis network. Proteostasis is energetically costly and those costs must be balanced with the energy needs of other physiological functions. Hypertonic stress causes widespread protein damage in <i>C</i>. <i>elegans</i>. Suppression and management of protein damage is essential for optimal survival under hypertonic conditions. ASH chemosensory neurons allow <i>C</i>. <i>elegans</i> to detect and avoid strongly hypertonic environments. We demonstrate that mutations in <i>osm-9</i> and <i>osm-1</i>2 that disrupt ASH mediated hypertonic avoidance behavior or genetic ablation of ASH neurons are associated with enhanced survival during hypertonic stress. Improved survival is not due to altered systemic volume homeostasis or organic osmolyte accumulation. Instead, we find that <i>osm-9(ok1677)</i> mutant and <i>osm-9(RNAi)</i> worms exhibit reductions in hypertonicity induced protein damage in non-neuronal cells suggesting that enhanced proteostasis capacity may account for improved hypertonic stress resistance in worms with defects in osmotic avoidance behavior. RNA-seq analysis revealed that genes that play roles in managing protein damage are upregulated in <i>osm-9(ok1677)</i> worms. Our findings are consistent with a growing body of work demonstrating that intercellular communication between neuronal and non-neuronal cells plays a critical role in integrating cellular stress resistance with other organismal physiological demands and associated energy costs.</p></div

Putative proteostasis genes differentially upregulated in <i>osm-9(ok1677)</i> worms following a 6 h exposure to 200 mM NaCl.

<p>Putative proteostasis genes differentially upregulated in <i>osm-9(ok1677)</i> worms following a 6 h exposure to 200 mM NaCl.</p

Effect of RNAi induced loss of <i>osm-9</i> function on <i>let-60(ga89)</i> induced egg hatching defects and larval arrest.

<p><i>let-60(ga89)</i> encodes a ts mutant of ras GTPase. The mutant phenotype can be induced at permissive temperatures (16°C) by exposing worms to 300 mM NaCl [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154156#pone.0154156.ref014" target="_blank">14</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154156#pone.0154156.ref016" target="_blank">16</a>]). <i>let-60(ga89)</i> mutants were crossed with <i>uIs60</i>[<i>Punc-119</i>::<i>yfp</i> + <i>Punc-119</i>::<i>sid-1</i>] worms to increase the sensitivity of neurons to RNAi (46). <i>let-60(ga89);uIs60</i>[<i>Punc-119</i>::<i>yfp</i> + <i>Punc-119</i>::<i>sid-1</i>] worms were fed bacteria expressing scrambled (control) or <i>osm-9</i> dsRNA and maintained at 16°C on agar plates containing either 51 or 300 mM NaCl. Values are means ± S.E. (n = 3–5 experiments with 100–300 eggs). *P<0.006 compared to control worms exposed to 51 mM NaCl. **P<0.005 compared to control worms exposed to 300 mM NaCl.</p