The type IV pilin PilA couples surface attachment and cell-cycle initiation in Caulobacter crescentus.

Understanding how bacteria colonize surfaces and regulate cell-cycle progression in response to cellular adhesion is of fundamental importance. Here, we use transposon sequencing in conjunction with fluorescence resonance energy transfer (FRET) microscopy to uncover the molecular mechanism for how surface sensing drives cell-cycle initiation in Caulobacter crescentus We identify the type IV pilin protein PilA as the primary signaling input that couples surface contact to cell-cycle initiation via the second messenger cyclic di-GMP (c-di-GMP). Upon retraction of pili filaments, the monomeric pilin reservoir in the inner membrane is sensed by the 17-amino acid transmembrane helix of PilA to activate the PleC-PleD two-component signaling system, increase cellular c-di-GMP levels, and signal the onset of the cell cycle. We termed the PilA signaling sequence CIP for "cell-cycle initiating pilin" peptide. Addition of the chemically synthesized CIP peptide initiates cell-cycle progression and simultaneously inhibits surface attachment. The broad conservation of the type IV pili and their importance in pathogens for host colonization suggests that CIP peptide mimetics offer strategies to inhibit surface sensing, prevent biofilm formation and control persistent infections.

Chemical synthesis rewriting of a bacterial genome to achieve design flexibility and biological functionality.

Understanding how to program biological functions into artificial DNA sequences remains a key challenge in synthetic genomics. Here, we report the chemical synthesis and testing of Caulobacter ethensis-2.0 (C. eth-2.0), a rewritten bacterial genome composed of the most fundamental functions of a bacterial cell. We rebuilt the essential genome of Caulobacter crescentus through the process of chemical synthesis rewriting and studied the genetic information content at the level of its essential genes. Within the 785,701-bp genome, we used sequence rewriting to reduce the number of encoded genetic features from 6,290 to 799. Overall, we introduced 133,313 base substitutions, resulting in the rewriting of 123,562 codons. We tested the biological functionality of the genome design in C. crescentus by transposon mutagenesis. Our analysis revealed that 432 essential genes of C. eth-2.0, corresponding to 81.5% of the design, are equal in functionality to natural genes. These findings suggest that neither changing mRNA structure nor changing the codon context have significant influence on biological functionality of synthetic genomes. Discovery of 98 genes that lost their function identified essential genes with incorrect annotation, including a limited set of 27 genes where we uncovered noncoding control features embedded within protein-coding sequences. In sum, our results highlight the promise of chemical synthesis rewriting to decode fundamental genome functions and its utility toward the design of improved organisms for industrial purposes and health benefits.

Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

Quantitative Selection Analysis of Bacteriophage φCbK Susceptibility in Caulobacter crescentus.

Classical molecular genetics uses stringent selective conditions to identify mutants with distinct phenotypic responses. Mutations giving rise to less pronounced phenotypes are often missed. However, to gain systems-level insights into complex genetic interaction networks requires genome-wide assignment of quantitative phenotypic traits. In this paper, we present a quantitative selection approach coupled with transposon sequencing (QS-TnSeq) to globally identify the cellular components that orchestrate susceptibility of the cell cycle model bacterium Caulobacter crescentus toward bacteriophage φCbK infection. We found that 135 genes representing 3.30% of the Caulobacter genome exhibit significant accumulation of transposon insertions upon φCbK selection. More than 85% thereof consist of new factors not previously associated with phage φCbK susceptibility. Using hierarchical clustering of dose-dependent TnSeq datasets, we grouped these genes into functional modules that correlate with different stages of the φCbK infection process. We assign φCbK susceptibility to eight new genes that represent novel components of the pilus secretion machinery. Further, we demonstrate that, from 86 motility genes, only seven genes encoding structural and regulatory components of the flagellar hook increase phage resistance when disrupted by transposons, suggesting a link between flagellar hook assembly and pili biogenesis. In addition, we observe high recovery of Tn5 insertions within regulatory sequences of the genes encoding the essential NADH:ubiquinone oxidoreductase complex indicating that intact proton motive force is crucial for effective phage propagation. In sum, QS-TnSeq is broadly applicable to perform quantitative and genome-wide systems-genetics analysis of complex phenotypic traits.