An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis.

MreB proteins play a major role during morphogenesis of rod-shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane-associated MreB polymers have been shown to be associated to elongation-specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso-diaminopimelate (m-DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane-associated cell wall synthesizing machineries.

Rewiring capsule production by CRISPRi-based genetic oscillators demonstrates a functional role of phenotypic variation in pneumococcal-host interactions.

Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence1-8, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes9-14, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation15-20. In this study, we used synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference together with live cell microscopy and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.

Synthetic genetic oscillators demonstrate the functional importance of phenotypic variation in pneumococcal-host interactions.

Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation. In this study, we use synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference (CRISPRi) together with live cell imaging and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.

Mechanistic basis of choline import involved in teichoic acids and lipopolysaccharide modification.

Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have fundamental roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. Streptococcus pneumoniae is a prominent pathogen where phosphocholine decoration plays a fundamental role in virulence. Here, we present cryo-electron microscopy and crystal structures of S. pneumoniae LicB, revealing distinct conformational states and describing architectural and mechanistic elements essential to choline import. Together with in vitro and in vivo functional characterization, we found that LicB displays proton-coupled import activity and promiscuous selectivity involved in adaptation to choline deprivation conditions, and describe LicB inhibition by synthetic nanobodies (sybodies). Our results provide previously unknown insights into the molecular mechanism of a key transporter involved in bacterial pathogenesis and establish a basis for inhibition of the phosphocholine modification pathway across bacterial phyla.

MiniBacillus PG10 as a Convenient and Effective Production Host for Lantibiotics.

Efficient bacterial cell factories are important for the screening and characterization of potent antimicrobial peptides such as lantibiotics. Although lantibiotic production systems have been established in Lactococcus lactis and Escherichia coli, the industrial workhorse Bacillus subtilis has been left relatively unexplored as a lantibiotic production host. Therefore, we tested different B. subtilis strains for their ability to produce lantibiotic peptides by using the subtilin modification and transport enzymes derived from the natural subtilin producer B. subtilis ATCC 6633. Our study shows that although B. subtilis ATCC 6633 and 168 are able to produce various processed lantibiotic peptides, an evident advantage of using either the 8-fold protease-deficient strain WB800 or the genome-minimized B. subtilis 168 strain PG10 is the lack of extracellular serine protease activity. Consequently, leader processing of lantibiotic precursor peptides is circumvented and thus potential toxicity toward the production host is prevented. Furthermore, PG10 provides a clean secondary metabolic background and therefore appears to be the most promising B. subtilis lantibiotic production host. We demonstrate the production of various lantibiotic precursor peptides by PG10 and show different options for their in vitro activation. Our study thus provides a convenient B. subtilis-based lantibiotic production system, which facilitates the search for novel antimicrobial peptides.

Three New Integration Vectors and Fluorescent Proteins for Use in the Opportunistic Human Pathogen Streptococcus pneumoniae.

Here, we describe the creation of three integration vectors, pPEPX, pPEPY and pPEPZ, for use with the opportunistic human pathogen Streptococcus pneumoniae. The constructed vectors, named PEP for Pneumococcal Engineering Platform (PEP), employ an IPTG-inducible promoter and BglBrick and BglFusion compatible multiple cloning sites allowing for fast and interchangeable cloning. PEP plasmids replicate in Escherichia coli and harbor integration sites that have homology in a large set of pneumococcal strains, including recent clinical isolates. In addition, several options of antibiotic resistance markers are available, even allowing for selection in multidrug resistant clinical isolates. The transformation efficiency of these PEP vectors as well as their ability to be expressed simultaneously was tested. Two of the three PEP vectors share homology of the integration regions with over half of the S. pneumoniae genomes examined. Transformation efficiency varied among PEP vectors based on the length of the homology regions, but all were highly transformable and can be integrated simultaneously in strain D39V. Vectors used for pneumococcal cloning are an important tool for researchers for a wide range of uses. The PEP vectors described are of particular use because they have been designed to allow for easy transfer of genes between vectors as well as integrating into transcriptionally silent areas of the chromosome. In addition, we demonstrate the successful production of several new spectrally distinct fluorescent proteins (mTurquoise2, mNeonGreen and mScarlet-I) from the PEP vectors. The PEP vectors and newly described fluorescent proteins will expand the genetic toolbox for pneumococcal researchers and aid future discoveries.

Binding-site identification and genotypic profiling of hepatitis C virus polymerase inhibitors.

The search for hepatitis C virus polymerase inhibitors has resulted in the identification of several nonnucleoside binding pockets. The shape and nature of these binding sites differ across and even within diverse hepatitis C virus genotypes. These differences confront antiviral drug discovery with the challenge of finding compounds that are capable of inhibition in variable binding pockets. To address this, we have established a hepatitis C virus mutant and genotypic recombinant polymerase panel as a means of guiding medicinal chemistry through the elucidation of the site of action of novel inhibitors and profiling against genotypes. Using a genotype 1b backbone, we demonstrate that the recombinant P495L, M423T, M414T, and S282T mutant enzymes can be used to identify the binding site of an acyl pyrrolidine analog. We assess the inhibitory activity of this analog and other nonnucleoside inhibitors with our panel of enzyme isolates generated from clinical sera representing genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a.