Prashanth P. Parmar

CREATION OF A BACTERIAL CELL CONTROLLED BY A CHEMICALLY SYNTHESIZED GENOME

Let There Be Life The DNA sequence information from thousands of genomes is stored digitally as ones and zeros in computer memory. Now, Gibson et al. (p. 52, published online 20 May; see the cover; see the Policy Forum by Cho and Relman) have brought together technologies from the past 15 years to start from digital information on the genome of Mycoplasma mycoides to chemically synthesize the genomic DNA as segments that could then be assembled in yeast and transplanted into the cytoplasm of another organism. A number of methods were also incorporated to facilitate testing and error correction of the synthetic genome segments. The transplanted genome became established in the recipient cell, replacing the recipient genome, which was lost from the cell. The reconstituted cells were able to replicate and form colonies, providing a proof-of-principle for future developments in synthetic biology. A synthetic Mycoplasma mycoides genome transplanted into M. capricolum was able to control the host cell. We report the design, synthesis, and assembly of the 1.08–mega–base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including “watermark” sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.

Temperature and growth phase influence the outer-membrane proteome and the expression of a type VI secretion system in Yersinia pestis.

Yersinia pestis cells were grown in vitro at 26 and 37 degrees C, the ambient temperatures of its flea vector and its mammalian hosts, respectively, and subjected to subcellular fractionation. Abundance changes at 26 vs 37 degrees C were observed for many outer-membrane (OM) proteins. The cell adhesion protein Ail (y1324) and three putative small beta-barrel OM proteins (y1795, y2167 and y4083) were strongly increased at 37 degrees C. The Ail/Lom family protein y1682 (OmpX) was strongly increased at 26 degrees C. Several porins and TonB-dependent receptors, which control small molecule transport through the OM, were also altered in abundance in a temperature-dependent manner. These marked differences in the composition of the OM proteome are probably important for the adaptation of Y. pestis to its in vivo life stages. Thirteen proteins that appear to be part of an intact type VI secretion system (T6SS) were identified in membrane fractions of stationary-phase cells grown at 26 degrees C, but not at 37 degrees C. The corresponding genes are clustered in the Y. pestis KIM gene locus y3658-y3677. The proteins y3674 and y3675 were particularly abundant and co-fractionated in a Mr range indicative of participation in a multi-subunit complex. The soluble haemolysin-coregulated protein y3673 was even more abundant. Its release into the extracellular medium was triggered by treatment of Y. pestis cells with trypsin. Proteases and other stress-response-inducing factors may constitute environmental cues resulting in the activation of the T6SS in Y. pestis.

The Shigella dysenteriae serotype 1 proteome, profiled in the host intestinal environment, reveals major metabolic modifications and increased expression of invasive proteins

Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. We present the first comprehensive proteome analysis of this pathogen, profiling proteins from bacteria cultured in vitro and bacterial isolates from the large bowel of infected gnotobiotic piglets (in vivo). Overall, 1061 distinct gene products were identified. Differential display analysis revealed that SD1 cells switched to an anaerobic energy metabolism in vivo. High in vivo abundances of amino acid decarboxylases (GadB and AdiA) which enhance pH homeostasis in the cytoplasm and protein disaggregation chaperones (HdeA, HdeB and ClpB) were indicative of a coordinated bacterial survival response to acid stress. Several type III secretion system effectors were increased in abundance in vivo, including OspF, IpaC and IpaD. These proteins are implicated in invasion of colonocytes and subversion of the host immune response in S. flexneri. These observations likely reflect an adaptive response of SD1 to the hostile host environment. Seven proteins, among them the type III secretion system effectors OspC2 and IpaB, were detected as antigens in Western blots using piglet antisera. The outer membrane protein OmpA, the heat shock protein HtpG and OspC2 represent novel SD1 subunit vaccine candidates and drug targets.

Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Shigella dysenteriae proteome

The in vitro stationary phase proteome of the human pathogen Shigella dysenteriae serotype 1 (SD1) was quantitatively analyzed in Coomassie Blue G250 (CBB)-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method APEX for absolute protein expression measurements. Of the 4502 genome-predicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A squared Pearson correlation analysis revealed a reasonably good correlation (R2= 0.67) for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. Stoichiometric ratios of subunits of protein complexes characterized in E. coli were compared with APEX quantitative ratios of orthologous SD1 protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, demonstrating versatile applications of the APEX method in quantitative proteomics.

Proteomic analysis of iron acquisition, metabolic and regulatory responses of Yersinia pestis to iron starvation

BackgroundThe Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26°C and 37°C).ResultsDifferential protein display was performed for three Y. pestis subcellular fractions. Five characterized Y. pestis iron/siderophore acquisition systems (Ybt, Yfe, Yfu, Yiu and Hmu) and a putative iron/chelate outer membrane receptor (Y0850) were increased in abundance in iron-starved cells. The iron-sulfur (Fe-S) cluster assembly system Suf, adapted to oxidative stress and iron starvation in E. coli, was also more abundant, suggesting functional activity of Suf in Y. pestis under iron-limiting conditions. Metabolic and reactive oxygen-deactivating enzymes dependent on Fe-S clusters or other iron cofactors were decreased in abundance in iron-depleted cells. This data was consistent with lower activities of aconitase and catalase in iron-starved vs. iron-rich cells. In contrast, pyruvate oxidase B which metabolizes pyruvate via electron transfer to ubiquinone-8 for direct utilization in the respiratory chain was strongly increased in abundance and activity in iron-depleted cells.ConclusionsMany protein abundance differences were indicative of the important regulatory role of the ferric uptake regulator Fur. Iron deficiency seems to result in a coordinated shift from iron-utilizing to iron-independent biochemical pathways in the cytoplasm of Y. pestis. With growth temperature as an additional variable in proteomic comparisons of the Y. pestis fractions (26°C and 37°C), there was little evidence for temperature-specific adaptation processes to iron starvation.

Characterizing the dynamic nature of the Yersinia pestis periplasmic proteome in response to nutrient exhaustion and temperature change

The periplasmic proteome of Yersinia pestis strain KIM6+ was characterized using differential 2‐DE display of proteins isolated from several subcellular fractions. Circa 160 proteins were designated as periplasmic, including 62 (putative) solute‐binding proteins of ATP‐binding cassette (ABC) transporters (SBPs) and 46 (putative) metabolic enzymes. More than 30 SBPs were significantly increased in abundance during stationary phase cell growth, compared to the exponential phase. The data suggest that nutrient exhaustion in the stationary phase triggers cellular responses resulting in the induced expression of numerous ABC transporters, which are responsible for the import of solutes/nutrients. Limited availability of inorganic phosphate (Pi) also caused dramatic proteomic changes. Nine proteins were functionally linked to the mobilization and import of three small molecules (Pi, phosphonate and glycerol‐3‐phosphate) and accounted for nearly half of the total protein mass in the periplasm of Pi‐starved cells. When cells were grown at 26°C versus 37°C, corresponding to ambient temperatures in the flea vector and mammalian hosts, respectively, several periplasmic proteins with no known roles in the Y. pestis life cycle were strongly altered in abundance. This included a putative nitrate/sulfonate/bicarbonate‐specific SBP (Y1004), encoded by the virulence‐associated plasmid pMT1 and increased in abundance at 37°C.

Analysis of the Proteome of Intracellular Shigella flexneri Reveals Pathways Important for Intracellular Growth

ABSTRACT Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu.

Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na2CO3, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14). Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries). Thirty-one proteins were experimentally associated with the outer membrane (OM). Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM) proteins with two or more predicted transmembrane domains (TMDs) were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

Proteomic View of Interactions of Shiga Toxin-Producing Escherichia coli with the Intestinal Environment in Gnotobiotic Piglets.

Background Shiga toxin (Stx)-producing Escherichia coli cause severe intestinal infections involving colonization of epithelial Peyer’s patches and formation of attachment/effacement (A/E) lesions. These lesions trigger leukocyte infiltration followed by inflammation and intestinal hemorrhage. Systems biology, which explores the crosstalk of Stx-producing Escherichia coli with the in vivo host environment, may elucidate novel molecular pathogenesis aspects. Methodology/Principal Findings Enterohemorrhagic E. coli strain 86–24 produces Shiga toxin-2 and belongs to the serotype O157:H7. Bacterial cells were scrapped from stationary phase cultures (the in vitro condition) and used to infect gnotobiotic piglets via intestinal lavage. Bacterial cells isolated from the piglets’ guts constituted the in vivo condition. Cell lysates were subjected to quantitative 2D gel and shotgun proteomic analyses, revealing metabolic shifts towards anaerobic energy generation, changes in carbon utilization, phosphate and ammonia starvation, and high activity of a glutamate decarboxylase acid resistance system in vivo. Increased abundance of pyridine nucleotide transhydrogenase (PntA and PntB) suggested in vivo shortage of intracellular NADPH. Abundance changes of proteins implicated in lipopolysaccharide biosynthesis (LpxC, ArnA, the predicted acyltransferase L7029) and outer membrane (OM) assembly (LptD, MlaA, MlaC) suggested bacterial cell surface modulation in response to activated host defenses. Indeed, there was evidence for interactions of innate immunity-associated proteins secreted into the intestines (GP340, REG3-γ, resistin, lithostathine, and trefoil factor 3) with the bacterial cell envelope. Significance Proteomic analysis afforded insights into system-wide adaptations of strain 86–24 to a hostile intestinal milieu, including responses to limited nutrients and cofactor supplies, intracellular acidification, and reactive nitrogen and oxygen species-mediated stress. Protein and lipopolysaccharide compositions of the OM were altered. Enhanced expression of type III secretion system effectors correlated with a metabolic shift back to a more aerobic milieu in vivo. Apparent pathogen pattern recognition molecules from piglet intestinal secretions adhered strongly to the bacterial cell surface.