Automated Discovery and Interpretation of ADA-Compliant Door Placards

A familiar difficulty to any new student on campus is making one’s way from classroom A to classroom B. Facilities with different wings, multiple floors, and irregular floorplans can magnify this challenge, while students with vision impairments are impacted even more by the challenge of identifying the destination. This thesis explored different methods of discovering Americans with Disabilities Act (ADA)- compliant room identifying placards (“plaques”) and identifying the text on the sign. The plaque detection was accomplished with both standard image manipulation techniques and a Histogram of Oriented Gradients (HOG) (Dalal & Triggs, 2005) object detector. The text reading utilized both standard image manipulation tools as well as an implementation of the Efficient and Accurate Scene Text detector (EAST) (Zhou et al., 2017) to isolate text, while Tesseract (Smith, 2007) was used to interpret the text. Different methods of dataset generation were utilized to train the detectors, including manual gathering, internet search scraping, and dataset generation. Results of testing these different methods on a dataset of image frames gathered from filming the Computer Science/Information Technology (CSIT) hallway of Kutztown University’s Old Main building proved the combination of HOG and EAST to be an effective method for identifying and transcribing room identification plaques. In the case of consistent visual design of rooms signs, the generated dataset proved to be nearly as effective as training the detector on real annotated images

Effects of flooding and temperature stress on soybean germination

The potential for uniform soybean [Glycine max (L.) Merr.] emergence may be reduced when fields are periodically saturated with water due to heavy rains and poor drainage during the germination phase of soybean growth. A laboratory study was conducted to determine the effects of timing and duration of flooding, germination temperature, and mechanical damage on the germination and emergence of soybean. Seeds were more susceptible to longer durations of flooding when flooded at a later time during the germination process. When flooding seeds 1 day after the start of imbibition, no differences in germination and emergence percentage were observed among the flooding duration treatments. However, when flooding seeds 2 or 3 days after the start of imbibition a significant drop in germination and emergence percentage occurred when seeds were flooded for 48 h. Effects of temperature on seed germination also interacted with the timing and the duration of flooding. Flooding of seeds 1 or 2 days after the start of imbibition at 150 C reduced germination and emergence percentage more than flooding at 250 C. Flooding of seeds 3 days after the start of imbibition also caused a significant decline in germination and emergence percentage, but the differences between germination temperatures were minimized. Initiating flooding on seed 3 days, compared with 1 day, after the start of imbibition was more detrimental at either temperature studied. Seed injury due to flooding for I h was observed at 15' C, but flooding for up to 48 h did not increase seed injury. However, at 25' C there was not a significant decline in germination and emergence percentage until seeds were flooded for 12 hours and there was further decline in germination and emergence percentage when seeds were flooded for 48 h. Impact damaged seeds sustained a greater loss in viability due to impact damage at 250 C compared with 150 C. Overall, seeds at 150 C were more susceptible to flooding stress than seeds at 250 C, but temperature differences were not significant when flooding seeds that had imbibed water for 3 days or when flooding persisted for 48 h. These results suggest flooding soybean seeds 3 days after the start of imbibition for as little as 48 h in the laboratory can cause significant reductions in the ability of seeds to germinate and produce healthy seedlings. Additional studies correlating these results with flooding in field situations are needed

The green fluorescent protein functions as a reporter for protein localization in Escherichia coli

The use of the green fluorescent protein (GFP) of Aequorea victoria as a reporter for protein localization in Escherichia coli has been investigated. This was accomplished by constructing gene fusions of gfp[Subscript uv] (an allele of gfp optimized for expression in E coli) to the 3' end of genes encoding various exported proteins, including malE(maltose binding protein, MBP), phoA(alkaline phosphatase, PhoA) and bla ([Beta]-lactamase, Bla). It was observed that GFP functions as an effective reporter for protein localization, in that GFP is fluorescent when cytoplasmically localized but non-fluorescent when localized to the periplasmic space. Our results also showed that the GFP hybrid fusion proteins are not toxic to the cell. By utilizing export defective sec mutants, we observed that inhibition of protein export restored fluorescence of the fusion protein. Also, signal sequence mutations that rendered the fusion protein export-defective were isolated by screening for fluorescent colonies. The observation that GFP fusion proteins can be exported from the cytoplasm and that GFP is fluorescent only in the cytoplasm demonstrates that GFP is a useful reporter for studies of bacterial protein export and folding

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Membrane Topology Mapping of the O-Antigen Flippase (Wzx), Polymerase (Wzy), and Ligase (WaaL) from Pseudomonas aeruginosa PAO1 Reveals Novel Domain Architectures

Biosynthesis of B-band lipopolysaccharide (LPS) in Pseudomonas aeruginosa follows the Wzy-dependent pathway, requiring the integral inner membrane proteins Wzx (O-antigen [O-Ag] flippase), Wzy (O-Ag polymerase), and WaaL (O-Ag ligase). For an important first step in deciphering the mechanisms of LPS assembly, we set out to map the membrane topology of these proteins. Random and targeted 3′ wzx, wzy, and waaL truncations were fused to a phoA-lacZα dual reporter capable of displaying both alkaline phosphatase and β-galactosidase activity. The results from truncation fusion expression and the corresponding differential enzyme activity ratios allowed for the assignment of specific regions of the proteins to cytoplasmic, transmembrane (TM), or periplasmic loci. Protein orientation in the inner membrane was confirmed via C-terminal fusion to green fluorescent protein. Our data revealed unique TM domain properties in these proteins, particularly for Wzx, indicating the potential for a charged pore. Novel periplasmic and cytoplasmic loop domains were also uncovered, with the latter in Wzy and WaaL revealing tracts consistent with potential Walker A/B motifs

Cell Wall Antibiotics Provoke Accumulation of Anchored mCherry in the Cross Wall of Staphylococcus aureus

A fluorescence microscopy method to directly follow the localization of defined proteins in Staphylococcus was hampered by the unstable fluorescence of fluorescent proteins. Here, we constructed plasmid (pCX) encoded red fluorescence (RF) mCherry (mCh) hybrids, namely mCh-cyto (no signal peptide and no sorting sequence), mCh-sec (with signal peptide), and mCh-cw (with signal peptide and cell wall sorting sequence). The S. aureus clones targeted mCh-fusion proteins into the cytosol, the supernatant and the cell envelope respectively; in all cases mCherry exhibited bright fluorescence. In staphylococci two types of signal peptides (SP) can be distinguished: the +YSIRK motif SPlip and the −YSIRK motif SPsasF. mCh-hybrids supplied with the +YSIRK motif SPlip were always expressed higher than those with −YSIRK motif SPsasF. To study the location of the anchoring process and also the influence of SP type, mCh-cw was supplied on the one hand with +YSIRK motif (mCh-cw1) and the other hand with -YSIRK motif (mCh-cw2). MCh-cw1 preferentially localized at the cross wall, while mCh-cw2 preferentially localized at the peripheral wall. Interestingly, when treated with sub-lethal concentrations of penicillin or moenomycin, both mCh-cw1 and mCh-cw2 were concentrated at the cross wall. The shift from the peripheral wall to the cross wall required Sortase A (SrtA), as in the srtA mutant this effect was blunted. The effect is most likely due to antibiotic mediated increase of free anchoring sites (Lipid II) at the cross wall, the substrate of SrtA, leading to a preferential incorporation of anchored proteins at the cross wall

Septal and lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment

The distribution of PBP5, the major D,D-carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild-type cells and in mutants lacking one or more D,D-carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane-bound form localized to the developing septum and restored wild-type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment

Laboratory Evolution of Fast-Folding Green Fluorescent Protein Using Secretory Pathway Quality Control

Green fluorescent protein (GFP) has undergone a long history of optimization to become one of the most popular proteins in all of cell biology. It is thermally and chemically robust and produces a pronounced fluorescent phenotype when expressed in cells of all types. Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics. Here we report that unlike other well-folded variants of GFP (e.g., GFPmut2), superfolder GFP was spared from elimination when targeted for secretion via the SecYEG translocase. This prompted us to hypothesize that the folding quality control inherent to this secretory pathway could be used as a platform for engineering similar ‘superfolded’ proteins. To test this, we targeted a combinatorial library of GFPmut2 variants to the SecYEG translocase and isolated several superfolded variants that accumulated in the cytoplasm due to their enhanced folding properties. Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP. Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function. Overall, we demonstrate that laboratory evolution combined with secretory pathway quality control enables sampling of largely unexplored amino-acid sequences for the discovery of artificial, high-performance proteins with properties that are unparalleled in their naturally occurring analogues

Feedback Inhibition in the PhoQ/PhoP Signaling System by a Membrane Peptide

The PhoQ/PhoP signaling system responds to low magnesium and the presence of certain cationic antimicrobial peptides. It regulates genes important for growth under these conditions, as well as additional genes important for virulence in many gram-negative pathogens. PhoQ is a sensor kinase that phosphorylates and activates the transcription factor PhoP. Since feedback inhibition is a common theme in stress-response circuits, we hypothesized that some members of the PhoP regulon may play such a role in the PhoQ/PhoP pathway. We therefore screened for PhoP-regulated genes that mediate feedback in this system. We found that deletion of mgrB (yobG), which encodes a 47 amino acid peptide, results in a potent increase in PhoP-regulated transcription. In addition, over-expression of mgrB decreased transcription at both high and low concentrations of magnesium. Localization and bacterial two-hybrid studies suggest that MgrB resides in the inner-membrane and interacts directly with PhoQ. We further show that MgrB homologs from Salmonella typhimurium and Yersinia pestis also repress PhoP-regulated transcription in these organisms. In cell regulatory circuits, feedback has been associated with modulating the induction kinetics and/or the cell-to-cell variability in response to stimulus. Interestingly, we found that elimination of MgrB-mediated feedback did not have a significant effect on the kinetics of reporter protein production and did not decrease the variability in expression among cells. Our results indicate MgrB is a broadly conserved membrane peptide that is a critical mediator of negative feedback in the PhoQ/PhoP circuit. This new regulator may function as a point of control that integrates additional input signals to modulate the activity of this important signaling system

Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways