#bacterial proteins

Transgenic Plant -2

TALENs are derived from proteins of:

Transcription activator-like effector nucleases originate from plant pathogenic bacteria Xanthomonas genus comprising Xanthomonas oryzae pv. oryzae causing bacterial blight of rice and Xanthomonas citri causing citrus canker. Pathogen delivers effector proteins via type III secretion apparatus into plant cell cytoplasm mimicking eukaryotic transcription factors: N-terminal type III secretion signal, central repeat domain 12-30 tandem repeats of 33-35 amino acids differing at two positions conferring DNA specificity, C-terminal activation domain and nuclear localization signals. Once in nucleus, TALE binds promoter of susceptibility gene such as SWEET sucrose efflux transporters OsSWEET11,14 inducing expression to feed bacteria. Boch and Bonas 2009 cracked code linking RVD to bases. TAL effector scaffold repurposed by replacing activation domain with FokI nuclease catalytic domain creating TALEN able to cut at designer loci. Rice edited to mutate SWEET promoter EBE element preventing Xanthomonas activation while preserving endogenous function achieved bacterial blight resistance demonstrating direct application of pathogen biology to crop improvement before CRISPR adoption.

Ref: Boch Science 2009 326:1509 TALE Xanthomonas; Bogdanove Curr Opin Microbiol; Moscou Bogdanove 2009.

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Microbial Physiology

Which of the following bacterial proteins is a tubulin homolog?

Cytoskeletal homology across domains revealed by structural biology although sequence similarity low. FtsZ protein essential division highly conserved tubulin homolog. Crystal structures show globular N terminal domain with Rossmann fold GTPase active site containing T1 to T6 loops coordinating GTP plus magnesium, H7 helix central, C terminal domain involved filament contacts. Organization identical to alpha beta tubulin dimer architecture. Both polymerize head to tail forming protofilaments, binding GTP promotes assembly, hydrolysis to GDP promotes disassembly causing dynamic instability. Tubulin assembles hollow microtubules 13 protofilaments lateral contacts forming 25 nanometer tube providing tracks for kinesin dynein motors and spindle fibers. FtsZ forms single stranded protofilaments bundling into Z ring at midcell scaffolding peptidoglycan synthases. MreB actin homolog and Crescentin intermediate filament like are other bacterial cytoskeletal types but tubulin homolog uniquely FtsZ. Therefore bacterial protein list identifies FtsZ as tubulin counterpart, essential gene, target for antimicrobial PC190723, chlorinated benzamide derivatives disrupting GTPase and filament assembly blocking cytokinesis causing filamentous nonviable cells.

Ref: Erickson et al., Microbiol Rev 2010, FtsZ tubulin homolog structural and functional homology.

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Microbial Physiology

The ParM-ParR-ParC system in bacteria is responsible for:

Type II partition system ParM ParR ParC extensively studied molecularly via electron microscopy and total internal reflection fluorescence revealing filaments pushing plasmids. parC centromere comprises two clusters five 11 base pair direct repeats separated binding ParR dimers forming helical clamp around DNA. ParR directly interacts with ParM filament ends increasing elongation rate. Filaments grow bipolarly inserting monomers at parC ParR junction producing pushing force separating plasmids by up to cell length. After plasmids reach poles ParM hydrolyzes ATP destabilizing filaments recycling monomers. While some literature loosely describes adaptor interactions with membrane, primary function not membrane anchoring but segregation ensuring each daughter inherits plasmid. System analogous to eukaryotic anaphase B spindle elongation. Mutations in ParM Walker A lysine abolish ATPase eliminate plasmid stability increasing loss rate thousand fold. Understanding ParM ParR ParC mechanism informs synthetic biology construction stable low copy vectors and insight how bacteria solve problem genome distribution without nuclear envelope and mitotic apparatus using cytoskeletal polymers.

Ref: Moller-Jensen et al., EMBO J 2002, ParM-ParR-ParC segregation pushing plasmids - Type II system.

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Microbial Physiology

Which protein plays a role in bacterial plasmid segregation?

Low copy plasmids require faithful segregation to avoid loss during division that would be frequent if distribution random. Actin like protein ParM encoded by many IncE plasmids R1 provides pushing mechanism. ParM ATPase polymerizes into left handed double helical filaments structurally resembling F actin despite low sequence identity, two protofilaments wound. Filaments exhibit dynamic instability both ends grow ATP bound, hydrolysis induces catastrophe. In plasmid context, centromere site parC contains repeat sequences bound by adaptor ParR which forms helical oligomer encircling parC forming ring. ParR ring caps plus ends ParM filaments stabilizing them promoting elongation that pushes two plasmid ParR parC complexes apart towards opposite cell poles prior septation. Thus ParM plays role bacterial plasmid segregation via actin like spindle. FtsZ tubulin homolog cell division, MreB maintains rod shape via elongasome, MamK positions magnetosome chain chain. Discovery ParM demonstrated bacteria possess true cytoskeleton capable force generation similar eukaryotic mitosis but simplified, target for understanding plasmid stability biotechnology maintaining industrial plasmids without antibiotic selection pressure.

Ref: Garner et al., Science 2007, ParM role in bacterial plasmid segregation - actin homolog.

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