High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

Synthetic Genomes.

DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.

Characterizing the mechanosensitive response of Paraburkholderia graminis membranes.

Bacterial mechanosensitive channels gate in response to membrane tension, driven by shifts in environmental osmolarity. The mechanosensitive channels of small conductance (MscS) and large conductance (MscL) from Escherichia coli (Ec) gate in response to mechanical force applied to the membrane. Ec-MscS is the foundational member of the MscS superfamily of ion channels, a diverse family with at least fifteen subfamilies identified by homology to the pore lining helix of Ec-MscS, as well as significant diversity on the N- and C-termini. The MscL family of channels are homologous to Ec-MscL. In a rhizosphere associated bacterium, Paraburkholderia graminis C4D1M, mechanosensitive channels are essential for cell survival during changing osmotic environments such as a rainstorm. Utilizing bioinformatics, we predicted six MscS superfamily members and a single MscL homologue. The MscS superfamily members fall into at least three subfamilies: bacterial cyclic nucleotide gated, multi-TM, and extended N-terminus. Osmotic downshock experiments show that wildtype P. graminis cells contain a survival mechanism that prevents cell lysis in response to hypoosmotic shock. To determine if this rescue is due to mechanosensitive channels, we developed a method to create giant spheroplasts of P. graminis to explore the single channel response to applied mechanical tension. Patch clamp electrophysiology on these spheroplasts shows two unique conductances: MscL-like and MscS-like. These conductances are due to likely three unique proteins. This indicates that channels that gate in response to mechanical tension are present in the membrane. Here, we report the first single channel evidence of mechanosensitive ion channels from P. graminis membranes.

An experiment-informed signal transduction model for the role of the Staphylococcus aureus MecR1 protein in ß-lactam resistance.

The treatment of hospital- and community-associated infections by methicillin-resistant Staphylococcus aureus (MRSA) is a perpetual challenge. This Gram-positive bacterium is resistant specifically to ß-lactam antibiotics, and generally to many other antibacterial agents. Its resistance mechanisms to ß-lactam antibiotics are activated only when the bacterium encounters a ß-lactam. This activation is regulated by the transmembrane sensor/signal transducer proteins BlaR1 and MecR1. Neither the transmembrane/metalloprotease domain, nor the complete MecR1 and BlaR1 proteins, are isolatable for mechanistic study. Here we propose a model for full-length MecR1 based on homology modeling, residue coevolution data, a new extensive experimental mapping of transmembrane topology, partial structures, molecular simulations, and available NMR data. Our model defines the metalloprotease domain as a hydrophilic transmembrane chamber effectively sealed by the apo-sensor domain. It proposes that the amphipathic helices inserted into the gluzincin domain constitute the route for transmission of the ß-lactam-binding event in the extracellular sensor domain, to the intracellular and membrane-embedded zinc-containing active site. From here, we discuss possible routes for subsequent activation of proteolytic action. This study provides the first coherent model of the structure of MecR1, opening routes for future functional investigations on how ß-lactam binding culminates in the proteolytic degradation of MecI.

Comparison of gene expression levels of appA, ppsR, and EL368 in Erythrobacter litoralis spheroplasts under aerobic and anaerobic conditions, and under blue light, red light, and dark conditions.

We compared the gene expression levels of the blue-light-responsive genes, appA (encoding photosynthesis promoting protein AppA), ppsR (encoding photosynthesis suppressing protein PpsR), and EL368 (encoding a blue-light-activated histidine kinase with a light, oxygen, or voltage domain) between aerobic and anaerobic conditions in spheroplasts of the aerobic photosynthetic bacterium Erythrobacter litoralis. The spheroplasts conducted photosynthesis under red light but not under blue light. All three blue-light-responsive genes showed higher expression under aerobic conditions than under anaerobic conditions under blue light. In contrast, under red light, although the expression level of appA was higher in the presence of oxygen than in the absence of oxygen, the expression levels of ppsR and EL368 were similar in the presence and absence of oxygen. Our findings demonstrate that the expression of blue-light-responsive genes is strongly affected by oxygen in E. litoralis spheroplasts.

In Saccharomyces cerevisiae gene targeting fidelity depends on a transformation method and proportion of the overall length of the transforming and targeted DNA.

Gene replacement is one of the most essential approaches in construction of the genetically modified yeast strains. However, the fidelity of gene targeting and the effort needed for construction of a particular strain can vary significantly. We investigated the influence of two important factors-the choice of the transformation method and the design of the transforming DNA fragment, which can vary in overall length (including flanking regions and selectable marker) compared to the length of the targeted region in the genome. Gene replacement fidelity was determined in several assays using electroporation and spheroplast transformation, and compared with our previous results obtained by lithium acetate. We have demonstrated clearly that gene targeting fidelity depends on the transformation protocol, being highest for lithium acetate method. In contrast, lower fidelity was observed with electroporation and spheroplast transformation. Additionally, the fidelity also depends on a design of the transformation assay, since a higher overall length ratio of the transforming DNA and targeted region results in higher fidelity. Moreover, the karyotype analysis of the aberrant transformants by qPCR demonstrates that gene targeting can result in diploidisation of haploid strains, most likely via targeted chromosome duplication followed by subsequent duplication of other chromosomes.

The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum.

The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes.

Caenispirillum deserti sp. nov., a spheroplast-forming bacterium isolated from a salt desert.

A Gram-stain-negative, vibrio-shaped, spheroplast-forming, motile, aerobic bacterium was isolated from the soil of a salt desert in Kutch, Gujarat, India. The strain, designated JC232(T), was oxidase- and catalase-positive. 16S rRNA gene sequence analysis indicated that strain JC232(T) was a member of the genus Caenispirillum and was related most closely to Caenispirillum salinarum AK4(T) (98.9% similarity) and Caenispirillum bisanense K92(T) (96.8%). Genome relatedness based on DNA-DNA hybridization of strain JC232(T) with the type strains of closely related species was less than 40%. The DNA G+C content of strain JC232(T) was 70 mol%. Phosphatidylglycerol, phosphatidylethanolamine, phosphotidylcholine, diphosphatidylglycerol, two unidentified amino lipids (AL1 and 2) and four unidentified lipids (UL1-4) were the polar lipids of strain JC232(T). C18 : 1ω7c/C18 : 1ω6c, C16 : 0 and C16 : 1ω7c/C16 : 1ω6c were the major (>15%) fatty acids of strain JC232(T), with minor amounts of C12 : 0, C14 : 0 3-OH/iso-C16 : 0 I, C18 : 1 2-OH, C18 : 0, C16 : 0 3-OH and C19 : 0cycloω8c. Although strain JC232(T) shared the predominant ubiquinone system (Q10) with the type strains of C. salinarum and C. bisanense, it differed from the latter in polar lipid profile, NaCl growth range and other phenotypic/physiological properties. On the basis of morphological, physiological, genotypic, phylogenetic and chemotaxonomic analyses, strain JC232(T) is considered to represent a novel species of the genus Caenispirillum, for which the name Caenispirillum deserti sp. nov. is proposed. The type strain is JC232(T) ( = KCTC 42064(T) = NBRC 110150(T)).

The mechanosensitive channel of small conductance (MscS) functions as a Jack-in-the box.

Phenotypical analysis of the lipid interacting residues in the closed state of the mechanosensitive channel of small conductance (MscS) from Escherichia coli (E. coli) has previously shown that these residues are critical for channel function. In the closed state, mutation of individual hydrophobic lipid lining residues to alanine, thus reducing the hydrophobicity, resulted in phenotypic changes that were observable using in vivo assays. Here, in an analogous set of experiments, we identify eleven residues in the first transmembrane domain of the open state of MscS that interact with the lipid bilayer. Each of these residues was mutated to alanine and leucine to modulate their hydrophobic interaction with the lipid tail-groups in the open state. The effects of these changes on channel function were analyzed using in vivo bacterial assays and patch clamp electrophysiology. Mutant channels were found to be functionally indistinguishable from wildtype MscS. Thus, mutation of open-state lipid interacting residues does not differentially stabilize or destabilize the open, closed, intermediate, or transition states of MscS. Based on these results and other data from the literature, we propose a new gating paradigm for MscS where MscS acts as a "Jack-In-The-Box" with the intrinsic bilayer lateral pressure holding the channel in the closed state. In this model, upon application of extrinsic tension the channel springs into the open state due to relief of the intrinsic lipid bilayer pressure.

From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human cells.

Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed.

Quantitative analysis of chromosomal and plasmid DNA during the growth of spheroplasts of Escherichia coli.

We generated spheroplasts from Escherichia coli carrying a broad-host-range plasmid. In the presence of penicillin, the spheroplasts did not divide but grew and enlarged in marine broth, whereas, in the absence of penicillin, they elongated. We quantified cellular DNA at different time points by using real-time quantitative PCR. Both chromosomal and plasmid DNA had replicated during spheroplast growth not only in the absence but also in the presence of penicillin. Thus, plasmid DNA and chromosomal DNA replication might be regulated synchronously during the growth of spheroplasts.

Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast-embryonic stem cell fusion.

Introducing human genes into mice offers the opportunity to analyze their in vivo function or to obtain therapeutic molecules. For proper gene regulation, or in case of multigene families, megabase (Mb)-sized DNA fragments often have to be used. Yeast artificial chromosome (YAC)-mediated transgenesis is irreplaceable for this purpose, because alternative methods such as the use of bacterial artificial chromosomes (BACs) cannot introduce DNA fragments larger than 500 kb into the mouse germ line. However, YAC libraries often contain only partial gene loci. Time-consuming reconstruction of YACs, genetic instability and the difficulty in obtaining intact YAC DNA above a certain size impede the generation of humanized mice. Here we describe how to reconstruct YACs containing Mb-sized human DNA, such as the T cell receptor-α (TRA) gene locus, thus facilitating the introduction of large DNA fragments into the mouse germ line. Fusion of YAC-containing yeast and embryonic stem (ES) cells avoids the need for YAC DNA purification. These ES cells are then used to stably introduce the functional TRA gene locus into the mouse germ line. The protocol takes ∼1 year to complete, from reconstruction of the entire TRA gene locus from YACs containing partial but overlapping TRA regions to germline transmission of the YAC.

A stable hybrid containing haploid genomes of two obligate diploid Candida species.

Candida albicans and Candida dubliniensis are diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains of C. albicans of the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploid C. albicans generated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n + 2n) of C. albicans and C. dubliniensis by spheroplast fusion. Heterodiploid (n + n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicans Cse4/C. dubliniensis Cse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of either C. albicans or C. dubliniensis. In keeping with the recent discovery of the existence of haploid C. albicans strains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny of C. albicans or C. dubliniensis in future.

The Rcs stress response and accessory envelope proteins are required for de novo generation of cell shape in Escherichia coli.

Interactions with immune responses or exposure to certain antibiotics can remove the peptidoglycan wall of many Gram-negative bacteria. Though the spheroplasts thus created usually lyse, some may survive by resynthesizing their walls and shapes. Normally, bacterial morphology is generated by synthetic complexes directed by FtsZ and MreBCD or their homologues, but whether these classic systems can recreate morphology in the absence of a preexisting template is unknown. To address this question, we treated Escherichia coli with lysozyme to remove the peptidoglycan wall while leaving intact the inner and outer membranes and periplasm. The resulting lysozyme-induced (LI) spheroplasts recovered a rod shape after four to six generations. Recovery proceeded via a series of cell divisions that produced misshapen and branched intermediates before later progeny assumed a normal rod shape. Importantly, mutants defective in mounting the Rcs stress response and those lacking penicillin binding protein 1B (PBP1B) or LpoB could not divide or recover their cell shape but instead enlarged until they lysed. LI spheroplasts from mutants lacking the Lpp lipoprotein or PBP6 produced spherical daughter cells that did not recover a normal rod shape or that did so only after a significant delay. Thus, to regenerate normal morphology de novo, E. coli must supplement the classic FtsZ- and MreBCD-directed cell wall systems with activities that are otherwise dispensable for growth under normal laboratory conditions. The existence of these auxiliary mechanisms implies that they may be required for survival in natural environments, where bacterial walls can be damaged extensively or removed altogether.

Fluorescent system based on bacterial expression of hybrid KcsA channels designed for Kv1.3 ligand screening and study.

Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.

Reassembly of functionally intact environmental DNA-derived biosynthetic gene clusters.

Only a small fraction of the bacterial diversity present in natural microbial communities is regularly cultured in the laboratory. Those bacteria that remain recalcitrant to culturing cannot be examined for the production of bioactive secondary metabolites using standard pure-culture approaches. The screening of genomic DNA libraries containing DNA isolated directly from environmental samples (environmental DNA (eDNA)) provides an alternative approach for studying the biosynthetic capacities of these organisms. One drawback of this approach has been that most eDNA isolation procedures do not permit the cloning of DNA fragments of sufficient length to capture large natural product biosynthetic gene clusters in their entirety. Although the construction of eDNA libraries with inserts big enough to capture biosynthetic gene clusters larger than ∼40kb remains challenging, it is possible to access large gene clusters by reassembling them from sets of smaller overlapping fragments using transformation-associated recombination in Saccharomyces cerevisiae. Here, we outline a method for the reassembly of large biosynthetic gene clusters from captured sets of overlapping soil eDNA cosmid clones. Natural product biosynthetic gene clusters reassembled using this approach can then be used directly for functional heterologous expression studies.

Characterization of Escherichia coli nucleoids released by osmotic shock.

Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concentrations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50-5 µg/ml), were shocked by 100-fold dilution of the sucrose buffer. Liberated nucleoids stained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI), the dimeric cyanine dye TOTO-1, or fluorescent DNA-binding protein appeared as cloud-like structures, in the absence of phase contrast. Because UV-irradiation disrupted the DAPI-stained nucleoids within 5-10 s, they were imaged by time-lapse microscopy with exposure times less than 2 s. The volume of nucleoids isolated from ampicillin- or low-lysozyme spheroplasts and minimally exposed to UV (<2 s) was on average ∼42 µm(3). Lysozyme at concentrations above 1 µg/ml in the lysate compacted the nucleoids. Treatment with protease E or K (20-200 µg/ml) and sodium dodecyl sulfate (SDS; 0.001-0.01%) caused a twofold volume increase and showed a granular nucleoid at the earliest UV-exposure; the expansion could be reversed with 50 µM ethidium bromide, but not with chloroquine. While DNase (1 µg/ml) caused a rapid disruption of the nucleoids, RNase (0.1-400 µg/ml) had no effect. DAPI-stained nucleoids treated with protease, SDS or DNase consisted of granular substructures at the earliest exposure similar to UV-disrupted nucleoids obtained after prolonged (>4 s) UV irradiation. We interpret the measured volume in terms of a physical model of the nucleoid viewed as a branched DNA supercoil crosslinked by adhering proteins into a homogeneous network.

Quantitative proteomic analysis of yeast DNA replication proteins.

Chromatin is dynamically regulated, and proteomic analysis of its composition can provide important information about chromatin functional components. Many DNA replication proteins for example bind chromatin at specific times during the cell cycle. Proteomic investigation can also be used to characterize changes in chromatin composition in response to perturbations such as DNA damage, while useful information is obtained by testing the effects on chromatin composition of mutations in chromosome stability pathways. We have successfully used the method of stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomic analysis of normal and pathological changes to yeast chromatin. Here we describe this proteomic method for analyzing changes to Saccharomyces cerevisiae chromatin, illustrating the procedure with an analysis of the changes that occur in chromatin composition as cells progress from a G1 phase block (induced by alpha factor) into S phase (in the presence of DNA replication inhibitor hydroxyurea).

IcmF family protein TssM exhibits ATPase activity and energizes type VI secretion.

The type VI secretion system (T6SS) with diversified functions is widely distributed in pathogenic Proteobacteria. The IcmF (intracellular multiplication protein F) family protein TssM is a conserved T6SS inner membrane protein. Despite the conservation of its Walker A nucleotide-binding motif, the NTPase activity of TssM and its role in T6SS remain obscure. In this study, we characterized TssM in the plant pathogen Agrobacterium tumefaciens and provided the first biochemical evidence for TssM exhibiting ATPase activity to power the secretion of the T6SS hallmark protein, hemolysin-coregulated protein (Hcp). Amino acid substitutions in the Walker A motif of TssM caused reduced ATP binding and hydrolysis activity. Importantly, we discovered the Walker B motif of TssM and demonstrated that it is critical for ATP hydrolysis activity. Protein-protein interaction studies and protease susceptibility assays indicated that TssM undergoes an ATP binding-induced conformational change and that subsequent ATP hydrolysis is crucial for recruiting Hcp to interact with the periplasmic domain of the TssM-interacting protein TssL (an IcmH/DotU family protein) into a ternary complex and mediating Hcp secretion. Our findings strongly argue that TssM functions as a T6SS energizer to recruit Hcp into the TssM-TssL inner membrane complex prior to Hcp secretion across the outer membrane.

Cloning whole bacterial genomes in yeast.

Many bacterial and archaeal genomes are of a similar size to molecules that have been cloned in the yeast Saccharomyces cerevisiae and thus might be clonable as single, circular episomes in this host. Yeast offers a variety of efficient tools for the manipulation and study of cloned DNA. One strategy to clone a genome in yeast is to cotransform yeast spheroplasts with the genome of interest and a linear yeast vector whose termini are homologous to a spot in the genome. Clones are selected on auxotrophic medium and then screened for completeness and size; they may also be sequenced.