Screen for Localized Proteins in Caulobacter crescentus

Precise localization of individual proteins is required for processes such as motility, chemotaxis, cell-cycle progression, and cell division in bacteria, but the number of proteins that are localized in bacterial species is not known. A screen based on transposon mutagenesis and fluorescence activated cell sorting was devised to identify large numbers of localized proteins, and employed in Caulobacter crescentus. From a sample of the clones isolated in the screen, eleven proteins with no previously characterized localization in C. crescentus were identified, including six hypothetical proteins. The localized hypothetical proteins included one protein that was localized in a helix-like structure, and two proteins for which the localization changed as a function of the cell cycle, suggesting that complex three-dimensional patterns and cell cycle-dependent localization are likely to be common in bacteria. Other mutants produced localized fusion proteins even though the transposon has inserted near the 5′ end of a gene, demonstrating that short peptides can contain sufficient information to localize bacterial proteins. The screen described here could be used in most bacterial species

CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus.

Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and are ineffective in labeling non-repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method that allows for a nonrepetitive genomic locus to be labeled using one guide RNA. We construct Casilio dual-color probes to visualize the dynamic interactions of DNA elements in single live cells in the presence or absence of the cohesin subunit RAD21. Using a three-color palette, we track the dynamic 3D locations of multiple reference points along a chromatin loop. Casilio imaging reveals intercellular heterogeneity and interallelic asynchrony in chromatin interaction dynamics, underscoring the importance of studying genome structures in 4D

RNaseE and the other constituents of the RNA degradosome are components of the bacterial cytoskeleton

RNaseE is the main component of the RNA degradosome of Escherichia coli, which plays an essential role in RNA processing and decay. Localization studies showed that RNaseE and the other known degradosome components (RNA helicase B, polynucleotide phosphorylase, and enolase) are organized as helical filamentous structures that coil around the length of the cell. These resemble the helical structures formed by the MreB and MinD cytoskeletal proteins. Formation of the RNaseE cytoskeletal-like structure requires an internal domain of the protein that does not include the domains required for any of its known interactions or the minimal domain required for endonuclease activity. We conclude that the constituents of the RNA degradosome are components of the E. coli cytoskeleton, either assembled as a primary cytoskeletal structure or secondarily associated with another underlying cytoskeletal element. This suggests a previously unrecognized role for the bacterial cytoskeleton, providing a mechanism to compartmentalize proteins that act on cytoplasmic components, as exemplified by the RNA processing and degradative activities of the degradosome, to regulate their access to important cellular substrates

Comprehensive methylome characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at single-base resolution

In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N(6)-methyladenine (6 mA) and N(4)-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5'-CTAT-3'), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5'-GAN(7)TAY-3'/3'-CTN(7)ATR-5'). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.This work was supported in part by National Institutes of Health grants 1RC2HG005618-01 (NHGRI) and 1RC2GM092602-01 (NIGMS). The LS group was supported by the European Research Council (ERC) advanced grant, the Fundación Marcelino Botin, and the Spanish Ministry of Research and Innovation to the ICREA researcher LS. VL-R was funded by the Fundacio´n La Caix