Progress Towards Engineering Microbial Surfaces to Degrade Biomass

Lignocellulosic biomass is a promising feedstock to sustainably produce useful biocommodities. However, its recalcitrance to hydrolysis limits its commercial utility. One attractive strategy to overcome this problem is to use consolidated bioprocessing (CBP) microbes to directly convert biomass into chemicals and biofuels. Several industrially useful microbes possess desirable consolidated bioprocessing characteristics, yet they lack the ability to degrade biomass. Engineering these microbes’ surfaces to display cellulases and cellulosome‐like structures could endow them with potent cellulolytic activity, enabling them to be used in CBP. In this chapter, we discuss recent progress in engineering the surfaces of Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and lactic acid bacteria. We discuss the techniques used to display cellulases on their surfaces, their recombinantly achieved cellulolytic activities, and current obstacles that limit their utility

The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin

Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdHN2N3, A326–D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix

Case study: Treatment of oral and locomotory stereotypic behaviors in a mature sow

A 32-month-old female 225-kg nonpregnant cross-bred Newsham sow presented a 6-week history of stereotypic behaviors when housed in a laboratory research facility. A behavioral examination over 12 daylight hours revealed 3 main stereotypic motor patterns, namely (1) oral-nasal gate manipulation defined as placement of the snout between the bars of the pen gate with repetitive, forceful up and down movement; (2) head weaving defined as repetitive lateral head and snout movement toward the pen gates while rocking back and forth on her forequarters with hooves remaining on ground at all times; and (3) body weaving defined as repetitive shifting of body weight from one side to the other with front hooves lifting alternately off the ground. The sow performed the oral-nasal gate manipulation and head and body weaving 4.0%, 12.4%, and 6.8% of her total baseline time budget, respectively. The presumptive diagnosis was oral-nasal and locomotory stereotypies. Three treatments were used to mitigate the duration and frequency of these stereotypic behaviors. Treatment 1—Social treatment (change social stimuli by providing visual and nose-to-nose contact with different neighboring sows); Treatment 2—Forage treatment (change foraging substrates by providing peat moss as a rooting substrate); and Treatment 3—Space treatment (change pen configuration by increasing space). The sow performed the oral-nasal gate manipulation and head and body weaving 0%, 0.4%, and 0.1% of her total time budget, respectively; social treatment: the sow performed the oral-nasal gate manipulation and head and body weaving 0.9%, 15.3%, and 11.3% of her total time budget, respectively; and forage treatment: the sow performed the oral-nasal gate manipulation and head and body weaving 0.5%, 28.0%, and 15.5% of her total time budget, respectively. This study is one of the first reports to evaluate the treatment of established stereotypies in a mature sow. Results suggest the promise of environmental enrichment as an effective treatment strategy. Further research is needed to evaluate the persistence of these behavioral changes and relative importance of different environmental manipulations provided

PTF11eon/SN2011dh: Discovery of a Type IIb Supernova From a Compact Progenitor in the Nearby Galaxy M51

On May 31, 2011 UT a supernova (SN) exploded in the nearby galaxy M51 (the Whirlpool Galaxy). We discovered this event using small telescopes equipped with CCD cameras, as well as by the Palomar Transient Factory (PTF) survey, and rapidly confirmed it to be a Type II supernova. Our early light curve and spectroscopy indicates that PTF11eon resulted from the explosion of a relatively compact progenitor star as evidenced by the rapid shock-breakout cooling seen in the light curve, the relatively low temperature in early-time spectra and the prompt appearance of low-ionization spectral features. The spectra of PTF11eon are dominated by H lines out to day 10 after explosion, but initial signs of He appear to be present. Assuming that He lines continue to develop in the near future, this SN is likely a member of the cIIb (compact IIb; Chevalier and Soderberg 2010) class, with progenitor radius larger than that of SN 2008ax and smaller than the eIIb (extended IIb) SN 1993J progenitor. Our data imply that the object identified in pre-explosion Hubble Space Telescope images at the SN location is possibly a companion to the progenitor or a blended source, and not the progenitor star itself, as its radius (~10^13 cm) would be highly inconsistent with constraints from our post-explosion photometric and spectroscopic data

A Comparative Genome Analysis Identifies Distinct Sorting Pathways in Gram-Positive Bacteria

Surface proteins in gram-positive bacteria are frequently required for virulence, and many are attached to the cell wall by sortase enzymes. Bacteria frequently encode more than one sortase enzyme and an even larger number of potential sortase substrates that possess an LPXTG-type cell wall sorting signal. In order to elucidate the sorting pathways present in gram-positive bacteria, we performed a comparative analysis of 72 sequenced microbial genomes. We show that sortase enzymes can be partitioned into five distinct subfamilies based upon their primary sequences and that most of their substrates can be predicted by making a few conservative assumptions. Most bacteria encode sortases from two or more subfamilies, which are predicted to function nonredundantly in sorting proteins to the cell surface. Only ∼20% of sortase-related proteins are most closely related to the well-characterized Staphylococcus aureus SrtA protein, but nonetheless, these proteins are responsible for anchoring the majority of surface proteins in gram-positive bacteria. In contrast, most sortase-like proteins are predicted to play a more specialized role, with each anchoring far fewer proteins that contain unusual sequence motifs. The functional sortase-substrate linkage predictions are available online (http://www.doe-mbi.ucla.edu/Services/Sortase/) in a searchable database

Native top-down mass spectrometry for the structural characterization of human hemoglobin.

Native mass spectrometry (MS) has become an invaluable tool for the characterization of proteins and noncovalent protein complexes under near physiological solution conditions. Here we report the structural characterization of human hemoglobin (Hb), a 64 kDa oxygen-transporting protein complex, by high resolution native top-down MS using electrospray ionization and a 15-Tesla Fourier transform ion cyclotron resonance mass spectrometer. Native MS preserves the noncovalent interactions between the globin subunits, and electron capture dissociation (ECD) produces fragments directly from the intact Hb complex without dissociating the subunits. Using activated ion ECD, we observe the gradual unfolding process of the Hb complex in the gas phase. Without protein ion activation, the native Hb shows very limited ECD fragmentation from the N-termini, suggesting a tightly packed structure of the native complex and therefore a low fragmentation efficiency. Precursor ion activation allows a steady increase in N-terminal fragment ions, while the C-terminal fragments remain limited (38 c ions and four z ions on the α chain; 36 c ions and two z ions on the β chain). This ECD fragmentation pattern suggests that upon activation, the Hb complex starts to unfold from the N-termini of both subunits, whereas the C-terminal regions and therefore the potential regions involved in the subunit binding interactions remain intact. ECD-MS of the Hb dimer shows similar fragmentation patterns as the Hb tetramer, providing further evidence for the hypothesized unfolding process of the Hb complex in the gas phase. Native top-down ECD-MS allows efficient probing of the Hb complex structure and the subunit binding interactions in the gas phase. It may provide a fast and effective means to probe the structure of novel protein complexes that are intractable to traditional structural characterization tools

A constant-time three-dimensional triple-resonance pulse scheme to correlate intraresidue 1HN, 15N, and 13C' chemical shifts in 15N---13C-labelled proteins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30326/1/0000728.pd