Synthetic Biology: A Bridge between Artificial and Natural Cells.

Artificial cells are simple cell-like entities that possess certain properties of natural cells. In general, artificial cells are constructed using three parts: (1) biological membranes that serve as protective barriers, while allowing communication between the cells and the environment; (2) transcription and translation machinery that synthesize proteins based on genetic sequences; and (3) genetic modules that control the dynamics of the whole cell. Artificial cells are minimal and well-defined systems that can be more easily engineered and controlled when compared to natural cells. Artificial cells can be used as biomimetic systems to study and understand natural dynamics of cells with minimal interference from cellular complexity. However, there remain significant gaps between artificial and natural cells. How much information can we encode into artificial cells? What is the minimal number of factors that are necessary to achieve robust functioning of artificial cells? Can artificial cells communicate with their environments efficiently? Can artificial cells replicate, divide or even evolve? Here, we review synthetic biological methods that could shrink the gaps between artificial and natural cells. The closure of these gaps will lead to advancement in synthetic biology, cellular biology and biomedical applications

Hemin Acquisition in Bartonella quintana

Bartonella quintana, a Gram-negative bacterial pathogen, causes Trench fever, bacillary angiomatosis and endocarditis. Transmitted by the human body louse (Pediculus humanus corporis), the agent has a tropism for erythrocytes in humans. In vitro growth requires an extraordinary concentration of hemin, and genomic analyses indicate several potential uptake systems and iron-responsive regulators. Transcription of the hbp genes (hemin binding protein genes) is responsive to alterations in available hemin and an HbpA homolog in B. henselae reportedly functions as a hemin receptor in E. coli hemA strain EB53. B. quintana hbpA was not able to complement EB53, indicating that it is not a hemin receptor. A functional hemin receptor and coordinate uptake system is encoded by the hemin utilization (hut) locus. B. quintana hutA was able to complement a hemA mutation in E. coli EB53 and was shown to be TonB-dependent using an isogenic E. coli hemA tonB strain. Fur (ferric uptake regulator) has been described as a global iron-responsive regulator in &gamma-proteobacteria. If expression is forced, B. quintana fur is able to complement an E. coli fur mutant, but an endogenous promoter for the gene could not be located and native expression in B. quintana was not detected. Overexpression of the iron response regulator (Irr), a Fur family member, in B. quintana repressed hut locus transcription. Previous studies showed that Irr interacted with a consensus motif, the H-box, in the promoter of the hbp genes. A region with homology to the H-box consensus is present in the divergent promoter between hutA and tonB and in the promoter region of hemS. The fate of hemin in the bacterial cytoplasm is not well understood. HemS is a potential hemin storage/degradation enzyme. Initial characterization indicates that HemS is able to bind hemin in a 1:1 fashion with an estimated dissociation constant (Kd) of 5.9 + 1.7 &muM. Complementation analyses using Corynebacterium ulcerans CU712hmuO&delta strain have not been successful but future experiments plan to use an E. coli chuS strain. These studies have characterized the principal hemin uptake system of B. quintana, identified its transcriptional regulator, and initiated investigation of a potential heme oxygenase

The broad-spectrum antimicrobial potential of [Mn(CO)4(S2CNMe(CH2CO2H))], a water-soluble CO-releasing molecule (CORM-401): intracellular accumulation, transcriptomic and statistical analyses, and membrane Polarization

Aims: Carbon monoxide (CO)-releasing molecules (CORMs) are candidates for animal and antimicrobial therapeutics. We aimed to probe the antimicrobial potential of a novel manganese CORM. Results: [Mn(CO)4S2CNMe(CH2CO2H)], CORM-401, inhibits growth of Escherichia coli and several antibiotic-resistant clinical pathogens. CORM-401 releases CO that binds oxidases in vivo, but is an ineffective respiratory inhibitor. Extensive CORM accumulation (assayed as intracellular manganese) accompanies antimicrobial activity. CORM-401 stimulates respiration, polarizes the cytoplasmic membrane in an uncoupler-like manner, and elicits loss of intracellular potassium and zinc. Transcriptomics and mathematical modeling of transcription factor activities reveal a multifaceted response characterized by elevated expression of genes encoding potassium uptake, efflux pumps, and envelope stress responses. Regulators implicated in stress responses (CpxR), respiration (Arc, Fnr), methionine biosynthesis (MetJ), and iron homeostasis (Fur) are significantly disturbed. Although CORM-401 reduces bacterial growth in combination with cefotaxime and trimethoprim, fractional inhibition studies reveal no interaction. Innovation: We present the most detailed microbiological analysis yet of a CORM that is not a ruthenium carbonyl. We demonstrate CO-independent striking effects on the bacterial membrane and global transcriptomic responses. Conclusions: CORM-401, contrary to our expectations of a CO delivery vehicle, does not inhibit respiration. It accumulates in the cytoplasm, acts like an uncoupler in disrupting cytoplasmic ion balance, and triggers multiple effects, including osmotic stress and futile respiration

Regulation and release of VSH-1, a prophage and gene transfer agent of Brachyspira hyodysenteriae

Brachyspira hyodysenteriae is an anaerobic spirochete and an important pathogen of swine. B. hyodysenteriae cells harbor VSH-1, a mitomycin C-inducible prophage that mediates generalized transduction between B. hyodysenteriae strains. VSH-1 virions package random fragments of B. hyodysenteriae chromosomal DNA rather than a viral genome, which has complicated genetic investigations. N-terminal amino acid sequences generated for proteins from purified VSH-1 whole virions and tailless heads facilitated the identification of VSH-1 structural genes and allowed their assignment as head or tail associated. It remained uncertain however, as to whether or not additional genes important to virion production existed within the prophage sequence. While mitomycin C-induction of the VSH-1 prophage resulted in cell lysis pointed to bacteriophage genes for lytic growth, the identity of these genes was unknown. The ability of VSH-1 virions to mediate horizontal gene transfer in B. hyodysenteriae populations coupled with the recent observation that VSH-1 was common among Brachyspira strains suggested that VSH-1 might play an important role in Brachyspira ecology.;This dissertation is a continuation of research on VSH-1 to understand the biology of this bacteriophage. In the following studies, additional VSH-1 genes were identified including an endolysin involved in VSH-1 escape from B. hyodysenteriae cells. The organization of genes for head and tail structures and lytic functions was found to be similar to other bacteriophage operons. Characteristics of VSH-1 transcription were investigated, leading to the identification of an alternative to mitomycin C as VSH-1 inducing agent

The Broad-Spectrum Antimicrobial Potential of [Mn(CO)(4)(S2CNMe(CH2CO2H))], a Water-Soluble CO-Releasing Molecule (CORM-401): Intracellular Accumulation, Transcriptomic and Statistical Analyses, and Membrane Polarization

Aims: Carbon monoxide (CO)-releasing molecules (CORMs) are candidates for animal and antimicrobial therapeutics. We aimed to probe the antimicrobial potential of a novel manganese CORM. Results: [Mn(CO)(4)S2CNMe(CH2CO2H)], CORM-401, inhibits growth of Escherichia coli and several antibiotic-resistant clinical pathogens. CORM-401 releases CO that binds oxidases in vivo, but is an ineffective respiratory inhibitor. Extensive CORM accumulation (assayed as intracellular manganese) accompanies antimicrobial activity. CORM-401 stimulates respiration, polarizes the cytoplasmic membrane in an uncoupler-like manner, and elicits loss of intracellular potassium and zinc. Transcriptomics and mathematical modeling of transcription factor activities reveal a multifaceted response characterized by elevated expression of genes encoding potassium uptake, efflux pumps, and envelope stress responses. Regulators implicated in stress responses (CpxR), respiration (Arc, Fnr), methionine biosynthesis (MetJ), and iron homeostasis (Fur) are significantly disturbed. Although CORM-401 reduces bacterial growth in combination with cefotaxime and trimethoprim, fractional inhibition studies reveal no interaction. Innovation: We present the most detailed microbiological analysis yet of a CORM that is not a ruthenium carbonyl. We demonstrate CO-independent striking effects on the bacterial membrane and global transcriptomic responses. Conclusions: CORM-401, contrary to our expectations of a CO delivery vehicle, does not inhibit respiration. It accumulates in the cytoplasm, acts like an uncoupler in disrupting cytoplasmic ion balance, and triggers multiple effects, including osmotic stress and futile respiration

The Role of Burkholderia thailandensis-Encoded MarR Homologs in Response to Host-Derived Signals

Multiple antibiotic resistance regulator (MarR) family is the most ubiquitous category of transcriptional regulators that exists among different bacteria and archaea. MarR family transcriptional regulators have been studied for their involvement in various biological processes, such as environmental chemical response, pathogenicity, and environmental stress responses. This work elucidates the role of MarR homologs MftR (major facilitator transcriptional regulator) and BifR (biofilm regulator) in the soil bacterium Burkholderia thailandensis. Burkholderia thailandensis-encoded MarR homolog MftR is divergently oriented to a gene that encodes the efflux pump MftP (major facilitator transport protein). MftR binds two cognate sites (each site consist of 9 bp imperfect inverted repeats) in the mftR-mftP intergenic region with equivalent affinity. For each site, urate attenuates DNA binding by MftR with equivalent sensitivity. MftR shows two-step unfolding transition (dimerization and DNA binding region) and urate binding to MftR and variants (mutagenesis of four conserved residues previously reported to be involved in urate binding to Deinococcus radiodurans HucR and Rhizobium radiobacter (now known as Agrobacterium fabrum) PecS) results in one step thermal unfolding transition. Further, data suggest the binding of urate in the cleft between the dimer interface and the DNA-binding lobes. DNA binding by MftR is attenuated by urate. MftR binds DNA with lower affinity at 37 °C. Collectively, this study suggests that MftR upregulates the genes under its control by responding to urate and by thermal upshift. Secondary metabolites are often produced during host invasion by a pathogen and function as virulence factors to survive in host. In normal condition biosynthetic gene cluster that produces drug or drug like molecules remains inactive for unknown reason. The signal required to activate these biosynthetic gene clusters is hard to identify. Global gene expression data of mftR strains suggests that MftR is a master regulator, which represses the various biosynthetic gene clusters required for the production of antimicrobial bactobolins, the iron siderophore malleobactin, and the virulence factor malleilactone among others. Along with that this study also identifies urate as a physiologically relevant inducer of biosynthetic gene clusters responsible for producing virulence factors. Burkholderia thailandensis also encodes a redox-sensitive MarR homolog, BifR that represses biofilm formation. Binding of BifR at two sites between the intergenic region of ecsC and emrB-bifR represses the expression of ecsC (putative LasA protease) and emrB-bifR. Oxidized BifR also binds to the intergenic region with nM affinity. However, oxidizing conditions further represses the expression. Oxidized BifR forms dimer-of-dimers. BifR also represses an operon that is required for the enzymatic synthesis of phenazine antibiotic. Phenazine acts as an alternative respiratory electron acceptor. Biofilm formation generates oxygen-limiting environment. This study suggests that BifR regulates LasA protease and expression of genes, which are involved in biofilm formation. Overall, my study identifies novel properties of MarR homologs in B. thailandensis, which suggest the role of MarR homologs in awakening of cryptic gene clusters that facilitate identification of novel pharmaceuticals and regulation of synthesis of alternate electron acceptor to survive in oxygen-limiting environment in biofilm

Urate responsive MarR homologs

Differential gene expression in response to internal and external stimuli is studied in detail to understand the intricate mechanisms underlying response to various environmental stressors in microorganisms. MarR family transcriptional regulators have been studied for their involvement in such mechanisms. This work elucidates the mechanism of urate-induced attenuation of DNA binding of HucR, a MarR homolog, and extends this mechanism to describe a novel subfamily of MarR homologs responsive to urate, proposing a physiological relevance of utilizing urate as a signaling molecule. HucR (hypothetical urate regulator) binds to the shared promoter region between uricase and hucR genes. It has high specificity for urate in attenuation of DNA binding. The ligand-binding site in HucR was identified using molecular-dynamics guided mutational analysis, leading to a proposed mechanism for the attenuation of DNA binding upon interaction of urate. According to this model, urate is anchored in the binding pocket by W20 and R80 while a charge-repulsion displaces D73, which propagates the conformational change to the DNA recognition helix. A possible extension of this mechanism to other MarR homologs was examined through homology search where a number of MarR homologs were identified as conserving the residues involved in urate binding. Further, they show high sequence identity in helix-3, which includes the conserved aspartic acid residue and in the DNA recognition helix, a sequence conservation that correlates to the conservation of bases in their proposed 18 bp consensus dyadic-binding site. To further investigate this phenomenon, Agrobacterium tumefaciens-encoded PecS, which conserves these residues, was studied in detail. PecS binds to the shared promoter region between the genes pecS and pecM while urate attenuates DNA binding in vitro and elevates the transcript levels in vivo. This study thus identifies a novel subfamily of MarR family transcription factors that bind urate and proposes a novel signalling function of urate, wherein invading bacteria utilize urate produced by the host to promote successful host colonization

Nitrous oxide metabolism in nitrate-reducing bacteria: Physiology and regulatory mechanisms

Nitrous oxide (N2O) is an important greenhouse gas (GHG) with substantial global warming potential and also contributes to ozone depletion through photochemical nit- ric oxide (NO) production in the stratosphere. The negative effects of N2O on climate and stratospheric ozone make N2O mitigation an international challenge. More than 60% of global N2O emissions are emitted from agricultural soils mainly due to the appli- cation of synthetic nitrogen-containing fertilizers. Thus, mitigation strategies must be developed which increase (or at least do not negatively impact) on agricultural effi- ciency whilst decrease the levels of N2O released. This aim is particularly important in the context of the ever expanding population and subsequent increased burden on the food chain. More than two-thirds of N2O emissions from soils can be attributed to bacterial and fungal denitrification and nitrification processes. In ammonia-oxidizing bacteria, N2O is formed through the oxidation of hydroxylamine to nitrite. In denitrifiers, nitrate is reduced to N2 via nitrite, NO and N2O production. In addition to denitrification, respiratory nitrate ammonification (also termed dissimilatory nitrate reduction to ammonium) is another important nitrate-reducing mechanism in soil, responsible for the loss of nitrate and production of N2O from reduction of NO that is formed as a by-product of the reduction process. This review will synthesize our current understand- ing of the environmental, regulatory and biochemical control of N2O emissions by nitrate-reducing bacteria and point to new solutions for agricultural GHG mitigation