Mechthild Pohlschroder

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Graduate Group Affiliations

Contact information
201 Leidy Labs
415 University Avenue
Philadelphia, PA 19104-6018
Office: 215 573-2283
Fax: 215 898-8780
Education:
Vordiplom (Biology)
University of Muenster, Germany, 1989.
PhD (Microbiology)
University of Massachusetts, Amherst, 1994.
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Description of Research Expertise

Research Interests
Biosynthesis and function of prokaryotic cell envelopes and their surface structures

Key words: archaea, protein secretion, prokaryotes, type IV pili, flagella, biofilm, sortase, exosortase, archaeosortase, twin arginine transport.

Description of Research
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Description of Research

Bacteria and archaea have complex cell envelopes that have several important functions, including providing a barrier that protects the cytoplasm from the environment. Along with its associated proteinaceous structures, it also ensures cell stability, facilitates motility and mediates adherence to biotic and abiotic surfaces. Although archaea are ubiquitous, present in all habitats examined thus far, including the human microbiome, compared to the bacteria, little is yet known about this domain of life. Using the genetically and biochemically amenable model archaeon Haloferax volcanii, my lab employs well-established techniques of molecular biology, biochemistry, and microscopy, as well as cutting edge technologies, such as RNAseq, to characterize the unique archaeal, as well as the highly conserved, aspects of archaeal cell surface biology. Our lab’s extensive characterization of the evolutionarily conserved Sec pathway, as well as the Tat pathway, advanced the understanding of protein transport across both domains of prokaryotes. Exciting results from our recent analyses of cell surface anchoring strategies of archaeal proteins have uncovered a seemingly unique archaeal surface anchoring strategy and also revealed a novel membrane anchoring mechanism that is conserved between archaea and bacteria. Furthermore, our analyses of archaeal surface filaments led to the identification of previously unknown roles of type IV pilins in the regulation of early steps in biofilm formation. The lab has used the data gleaned from these in vivo studies to develop novel software programs, as well as to improve existing ones, that predict the substrates that use specific transport and cell surface anchoring pathways. Information gleaned from these studies has invariably been used to further develop our understanding of the molecular mechanisms that have allowed haloarchaea to adapt to high salt conditions.


Cell Surface anchoring mechanisms
Following transport to the cell surface, secreted proteins involved in such critical cell processes as surface adhesion and mating are anchored to the cell surface. A better understanding of the molecular machineries involved in protein anchoring will facilitate the development of effective approaches for interfering with surface anchoring of such specific targets as virulence factors.

We have discovered that the N-terminal region of H. volcanii Tat substrates often contain a lipobox, a motif that is modified by the covalent attachment of a lipid to an invariant cysteine. Although archaea do not have homologues of the bacterial enzymes involved in lipid modification of lipobox-containing proteins, we have identified two candidates that might be involved in this process, as they are found in all archaea that express proteins containing a lipobox but not found in other archaea.

Recently, my lab confirmed that some archaea, including H. volcanii, also employ another type of protein anchoring mechanism that is mediated through a lipid-modified amino acid residue. In this case, an enzyme known as the archaeosortase processes, and attaches a lipid to, the C-terminus of substrates containing a PGF motif. One such substrate, the Surface Layer Glycoprotein (SLG), perhaps the best studied of all archaeal proteins, was long believed to be anchored to the cell membrane by intercalation of a protein segment. We are now characterizing additional archaeosortase substrates, including several proteins that contain a low-complexity domain, a type of domain often found in proteins involved in mediating surface adhesion or cell-cell interactions. In fact, we have already shown that cells lacking one of these substrates have significantly diminished mating efficiencies. The lab has also begun to characterize bacterial archaeosortase homologs, the exosortases, and their substrates in Nitrosomonas europea, the first in vivo study of these enzymes in bacteria.

Type IV pili biosynthesis and their roles in biofilm formation
My lab’s research places special emphasis on type IV pili, cell surface structures that mediate, and, in some cases, regulate, the processes involved in establishing and maintaining biofilms, microbial aggregates that allow cells to survive severe environmental stresses.

While investigating the roles played by pili in surface adhesion, we discovered six conserved H. volcanii adhesion pilins are also involved in regulating flagella-dependent motility. This previously unknown mechanism that plays a role in mediating this transition from the planktonic to sessile state, underscores the importance of the ability to quickly adapt to severe changes in the local environment. Complementation experiments indicate that pilin regulation of motility does not require assembled pili and that pilins do not interact directly with the flagella. We are now attempting to identify an intermediate through which pilin-dependent motility regulation operates.

Surprisingly, while some type IV pilins promoted microcolony formation, as has previously been reported, a distinct subset inhibits this early step in biofilm formation. We are also investigating the effects that N-glycosylation, as well as other post-translation modifications, has on this regulatory mechanism.

Rotation Projects for 2014-2015
1. Characterization of H. volcanii archaeosortase substrates.

2. Genetic screens using transposon mutagenesis to identify components involved in archaeal surface adhesion or inhibition of flagella-dependent motility

3. Biochemical analysis of archaeal type IV pilus biosynthesis machinery, which presumably is less complex than that of well-studied gram negative bacteria, hence providing a great model

Lab personnel:
Farid Abdul, Graduate Student
Rianne Esquivel, Graduate Student
Yitin Zhou, Graduate Student
Vanessa Munoz, Rotation Student
Evan Yang, Undergraduate Student
Melanie Santos, Undergraduate Student
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Last updated: 09/25/2014
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