Proteome partitioning constraints in long-term laboratory evolution.

Adaptive laboratory evolution experiments provide a controlled context in which the dynamics of selection and adaptation can be followed in real-time at the single-nucleotide level. And yet this precision introduces hundreds of degrees-of-freedom as genetic changes accrue in parallel lineages over generations. On short timescales, physiological constraints have been leveraged to provide a coarse-grained view of bacterial gene expression characterized by a small set of phenomenological parameters. Here, we ask whether this same framework, operating at a level between genotype and fitness, informs physiological changes that occur on evolutionary timescales. Using a strain adapted to growth in glucose minimal medium, we find that the proteome is substantially remodeled over 40 000 generations. The most striking change is an apparent increase in enzyme efficiency, particularly in the enzymes of lower-glycolysis. We propose that deletion of metabolic flux-sensing regulation early in the adaptation results in increased enzyme saturation and can account for the observed proteome remodeling.

Inherited chitinases enable sustained growth and rapid dispersal of bacteria from chitin particles.

Many biogeochemical functions involve bacteria utilizing solid substrates. However, little is known about the coordination of bacterial growth with the kinetics of attachment to and detachment from such substrates. In this quantitative study of Vibrio sp. 1A01 growing on chitin particles, we reveal the heterogeneous nature of the exponentially growing culture comprising two co-existing subpopulations: a minority replicating on chitin particles and a non-replicating majority which was planktonic. This partition resulted from a high rate of cell detachment from particles. Despite high detachment, sustained exponential growth of cells on particles was enabled by the enrichment of extracellular chitinases excreted and left behind by detached cells. The 'inheritance' of these chitinases sustains the colonizing subpopulation despite its reduced density. This simple mechanism helps to circumvent a trade-off between growth and dispersal, allowing particle-associated marine heterotrophs to explore new habitats without compromising their fitness on the habitat they have already colonized.

The Histone H1-Like Protein AlgP Facilitates Even Spacing of Polyphosphate Granules in Pseudomonas aeruginosa.

Synthesis of polyphosphate (polyP) is an ancient and universal stress and starvation response in bacteria. In many bacteria, polyP chains come together to form granular superstructures within cells. Some species appear to regulate polyP granule subcellular organization. Despite the critical role of polyP in starvation fitness, the composition of these structures, mechanism(s) underpinning their organization, and functional significance of such organization are poorly understood. We previously determined that granules become transiently evenly spaced on the cell's long axis during nitrogen starvation in the opportunistic human pathogen Pseudomonas aeruginosa. Here, we developed a granule-enrichment protocol to screen for polyP granule-localizing proteins. We identified AlgP as a protein that associates with polyP granules. We further discovered that AlgP is required for the even spacing of polyP granules. AlgP is a DNA-binding protein with a 154 amino acid C-terminal domain enriched in "KPAA" repeats and variants of this repeat, with an overall sequence composition similar to the C-terminal tail of eukaryotic histone H1. Granule size, number, and spacing are significantly perturbed in the absence of AlgP, or when AlgP is truncated to remove the C-terminus. The ΔalgP and algPΔCTD mutants have fewer, larger granules. We speculate that AlgP may contribute to spacing by tethering polyP granules to the chromosome, thereby inhibiting fusion with neighboring granules. Our discovery that AlgP facilitates granule spacing allows us for the first time to directly uncouple granule biogenesis from even spacing, and will inform future efforts to explore the functional significance of granule organization on fitness during starvation. IMPORTANCE The mechanisms underpinning polyP's pleiotropic effects on bacterial starvation physiology remain elusive. This simple polyanion's lack of protein binding specificity has impeded validation of bona fide polyP-binding proteins. However, polyP forms granule superstructures with spatial specificity. Our granule enrichment protocol identified a polyP granule-associated protein in Pseudomonas aeruginosa, AlgP. AlgP was originally reported as a regulator of alginate, an extracellular polysaccharide important in biofilm formation, including in cystic fibrosis (CF) chronic infections. AlgP's putative role in alginate biosynthesis has recently been called into question. We establish a distinct, previously unknown function for AlgP in modulating the subcellular organization of polyP, another polymer important for pathogenesis. In CF clinical isolates, the C-terminal repeat domain of AlgP is a hot spot for genetic rearrangements. Our finding that the C-terminus of AlgP is required for granule organization lays the groundwork for exploring the functional significance of these mutations in the evolutionary trajectory of chronic infections.

Global coordination of metabolic pathways in Escherichia coli by active and passive regulation.

Microorganisms adjust metabolic activity to cope with diverse environments. While many studies have provided insights into how individual pathways are regulated, the mechanisms that give rise to coordinated metabolic responses are poorly understood. Here, we identify the regulatory mechanisms that coordinate catabolism and anabolism in Escherichia coli. Integrating protein, metabolite, and flux changes in genetically implemented catabolic or anabolic limitations, we show that combined global and local mechanisms coordinate the response to metabolic limitations. To allocate proteomic resources between catabolism and anabolism, E. coli uses a simple global gene regulatory program. Surprisingly, this program is largely implemented by a single transcription factor, Crp, which directly activates the expression of catabolic enzymes and indirectly reduces the expression of anabolic enzymes by passively sequestering cellular resources needed for their synthesis. However, metabolic fluxes are not controlled by this regulatory program alone; instead, fluxes are adjusted mostly through passive changes in the local metabolite concentrations. These mechanisms constitute a simple but effective global regulatory program that coarsely partitions resources between different parts of metabolism while ensuring robust coordination of individual metabolic reactions.

An alternative resource allocation strategy in the chemolithoautotrophic archaeon Methanococcus maripaludis.

Most microorganisms in nature spend the majority of time in a state of slow or zero growth and slow metabolism under limited energy or nutrient flux rather than growing at maximum rates. Yet, most of our knowledge has been derived from studies on fast-growing bacteria. Here, we systematically characterized the physiology of the methanogenic archaeon Methanococcus maripaludis during slow growth. M. maripaludis was grown in continuous culture under energy (formate)-limiting conditions at different dilution rates ranging from 0.09 to 0.002 h-1, the latter corresponding to 1% of its maximum growth rate under laboratory conditions (0.23 h-1). While the specific rate of methanogenesis correlated with growth rate as expected, the fraction of cellular energy used for maintenance increased and the maintenance energy per biomass decreased at slower growth. Notably, proteome allocation between catabolic and anabolic pathways was invariant with growth rate. Unexpectedly, cells maintained their maximum methanogenesis capacity over a wide range of growth rates, except for the lowest rates tested. Cell size, cellular DNA, RNA, and protein content as well as ribosome numbers also were largely invariant with growth rate. A reduced protein synthesis rate during slow growth was achieved by a reduction in ribosome activity rather than via the number of cellular ribosomes. Our data revealed a resource allocation strategy of a methanogenic archaeon during energy limitation that is fundamentally different from commonly studied versatile chemoheterotrophic bacteria such as E. coli.

A global resource allocation strategy governs growth transition kinetics of Escherichia coli.

A grand challenge of systems biology is to predict the kinetic responses of living systems to perturbations starting from the underlying molecular interactions. Changes in the nutrient environment have long been used to study regulation and adaptation phenomena in microorganisms and they remain a topic of active investigation. Although much is known about the molecular interactions that govern the regulation of key metabolic processes in response to applied perturbations, they are insufficiently quantified for predictive bottom-up modelling. Here we develop a top-down approach, expanding the recently established coarse-grained proteome allocation models from steady-state growth into the kinetic regime. Using only qualitative knowledge of the underlying regulatory processes and imposing the condition of flux balance, we derive a quantitative model of bacterial growth transitions that is independent of inaccessible kinetic parameters. The resulting flux-controlled regulation model accurately predicts the time course of gene expression and biomass accumulation in response to carbon upshifts and downshifts (for example, diauxic shifts) without adjustable parameters. As predicted by the model and validated by quantitative proteomics, cells exhibit suboptimal recovery kinetics in response to nutrient shifts owing to a rigid strategy of protein synthesis allocation, which is not directed towards alleviating specific metabolic bottlenecks. Our approach does not rely on kinetic parameters, and therefore points to a theoretical framework for describing a broad range of such kinetic processes without detailed knowledge of the underlying biochemical reactions.

Reduction of translating ribosomes enables Escherichia coli to maintain elongation rates during slow growth.

Bacteria growing under different conditions experience a broad range of demand on the rate of protein synthesis, which profoundly affects cellular resource allocation. During fast growth, protein synthesis has long been known to be modulated by adjusting the ribosome content, with the vast majority of ribosomes engaged at a near-maximal rate of elongation. Here, we systematically characterize protein synthesis by Escherichia coli, focusing on slow-growth conditions. We establish that the translational elongation rate decreases as growth slows, exhibiting a Michaelis-Menten dependence on the abundance of the cellular translational apparatus. However, an appreciable elongation rate is maintained even towards zero growth, including the stationary phase. This maintenance, critical for timely protein synthesis in harsh environments, is accompanied by a drastic reduction in the fraction of active ribosomes. Interestingly, well-known antibiotics such as chloramphenicol also cause a substantial reduction in the pool of active ribosomes, instead of slowing down translational elongation as commonly thought.

Rational modification of protein stability by targeting surface sites leads to complicated results.

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions.

Islet amyloid: from fundamental biophysics to mechanisms of cytotoxicity.

Pancreatic islet amyloid is a characteristic feature of type 2 diabetes. The major protein component of islet amyloid is the polypeptide hormone known as islet amyloid polypeptide (IAPP, or amylin). IAPP is stored with insulin in the ß-cell secretory granules and is released in response to the stimuli that lead to insulin secretion. IAPP is normally soluble and is natively unfolded in its monomeric state, but forms islet amyloid in type 2 diabetes. Islet amyloid is not the cause of type 2 diabetes, but it leads to ß-cell dysfunction and cell death, and contributes to the failure of islet cell transplantation. The mechanism of IAPP amyloid formation is not understood and the mechanisms of cytotoxicity are not fully defined.

Ionic strength effects on amyloid formation by amylin are a complicated interplay among Debye screening, ion selectivity, and Hofmeister effects.

Amyloid formation plays a role in a wide range of human diseases. The rate and extent of amyloid formation depend on solution conditions, including pH and ionic strength. Amyloid fibrils often adopt structures with parallel, in-register ß-sheets, which generate quasi-infinite arrays of aligned side chains. These arrangements can lead to significant electrostatic interactions between adjacent polypeptide chains. The effect of ionic strength and ion composition on the kinetics of amyloid formation by islet amyloid polypeptide (IAPP) is examined. IAPP is a basic 37-residue polypeptide responsible for islet amyloid formation in type 2 diabetes. Poisson-Boltzmann calculations revealed significant electrostatic repulsion in a model of the IAPP fibrillar state. The kinetics of IAPP amyloid formation are strongly dependent on ionic strength, varying by a factor of >10 over the range of 20-600 mM NaCl at pH 8.0, but the effect is not entirely due to Debye screening. At low ionic strengths, the rate depends strongly on the identity of the anion, varying by a factor of nearly 4, and scales with the electroselectivity series, implicating anion binding. At high ionic strengths, the rate varies by only 8% and scales with the Hofmeister series. At intermediate ionic strengths, no clear trend is detected, likely because of the convolution of different effects. The effects of salts on the growth phase and lag phase of IAPP amyloid formation are strongly correlated. At pH 5.5, where the net charge on IAPP is higher, the effect of different anions scales with the electroselectivity series at all salt concentrations.

Biophysical and functional analyses suggest that adenovirus E4-ORF3 protein requires higher-order multimerization to function against promyelocytic leukemia protein nuclear bodies.

The early region 4 open reading frame 3 protein (E4-ORF3; UniProt ID P04489) is the most highly conserved of all adenovirus-encoded gene products at the amino acid level. A conserved attribute of the E4-ORF3 proteins of different human adenoviruses is the ability to disrupt PML nuclear bodies from their normally punctate appearance into heterogeneous filamentous structures. This E4-ORF3 activity correlates with the inhibition of PML-mediated antiviral activity. The mechanism of E4-ORF3-mediated reorganization of PML nuclear bodies is unknown. Biophysical analysis of the purified WT E4-ORF3 protein revealed an ordered secondary/tertiary structure and the ability to form heterogeneous higher-order multimers in solution. Importantly, a nonfunctional E4-ORF3 mutant protein, L103A, forms a stable dimer with WT secondary structure content. Because the L103A mutant is incapable of PML reorganization, this result suggests that higher-order multimerization of E4-ORF3 may be required for the activity of the protein. In support of this hypothesis, we demonstrate that the E4-ORF3 L103A mutant protein acts as a dominant-negative effector when coexpressed with the WT E4-ORF3 in mammalian cells. It prevents WT E4-ORF3-mediated PML track formation presumably by binding to the WT protein and inhibiting the formation of higher-order multimers. In vitro protein binding studies support this conclusion as demonstrated by copurification of coexpressed WT and L103A proteins in Escherichia coli and coimmunoprecipitation of WT·L103A E4-ORF3 complexes in mammalian cells. These results provide new insight into the properties of the Ad E4-ORF3 protein and suggest that higher-order protein multimerization is essential for E4-ORF3 activity.

Sensitivity of amyloid formation by human islet amyloid polypeptide to mutations at residue 20.

Islet amyloid polypeptide (IAPP, amylin) is responsible for amyloid formation in type 2 diabetes and in islet cell transplants. The only known natural mutation found in mature human IAPP is a Ser20-to-Gly missense mutation, found with small frequency in Chinese and Japanese populations. The mutation appears to be associated with increased risk of early-onset type 2 diabetes. Early measurements in the presence of organic co-solvents showed that S20G-IAPP formed amyloid more quickly than the wild type. We confirm that the mutant accelerates amyloid formation under a range of conditions including in the absence of co-solvents. Ser20 adopts a normal backbone geometry, and the side chain makes no steric clashes in models of IAPP amyloid fibers, suggesting that the increased rate of amyloid formation by the mutant does not result from the relief of steric incompatibility in the fiber state. Transmission electronic microscopy, circular dichroism, and seeding studies were used to probe the structure of the resulting fibers. The S20G-IAPP peptide is toxic to cultured rat INS-1 (transformed rat insulinoma-1) ß-cells. The sensitivity of amyloid formation to the identity of residue 20 was exploited to design a variant that is much slower to aggregate and that inhibits amyloid formation by wild-type IAPP. An S20K mutant forms amyloid with an 18-fold longer lag phase in homogeneous solution. Thioflavin T binding assays, together with experiments using a p-cyanophenylalanine (p-cyanoPhe) variant of human IAPP, show that the designed S20K mutant inhibits amyloid formation by human IAPP. The experiments illustrate how p-cyanoPhe can be exploited to monitor amyloid formation even in the presence of other amyloidogenic proteins.

Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands.

Cyanovirin-N (CVN) is an 11 kDa pseudosymmetric cyanobacterial lectin that has been shown to inhibit infection by the human immunodeficiency virus by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work, we describe rationally designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson-Boltzmann calculations and protein repacking algorithms were used to select stabilizing mutations in the protein core. By substituting the buried polar side chains of Ser11, Ser20, and Thr61 with aliphatic groups, we stabilized CVN by nearly 12 °C against thermal denaturation, and by 1 M GuaHCl against chemical denaturation, relative to a previously characterized stabilized mutant. Glycan microarray binding experiments confirmed that the specificity profile of carbohydrate binding is unperturbed by the mutations and is identical for all variants. In particular, the variants selectively bound glycans containing the Manα(1→2)Man linkage, which is the known minimal binding unit of CVN. We also report the slow denaturation kinetics of CVN and show that they can complicate thermodynamic analysis; in particular, the unfolding of CVN cannot be described as a fixed two-state transition. Accurate thermodynamic parameters are needed to describe the complicated free energy landscape of CVN, and we provide updated values for CVN unfolding.

Interpretation of p-cyanophenylalanine fluorescence in proteins in terms of solvent exposure and contribution of side-chain quenchers: a combined fluorescence, IR and molecular dynamics study.

The use of noncoded amino acids as spectroscopic probes of protein folding and function is growing rapidly, in large part because of advances in the methodology for their incorporation. Recently p-cyanophenylalanine has been employed as a fluorescence and IR probe, as well as a FRET probe to study protein folding, protein-membrane interactions, protein-protein interactions and amyloid formation. The probe has been shown to be exquisitely sensitive to hydrogen bonding interactions involving the cyano group, and its fluorescence quantum yield increases dramatically when it is hydrogen bonded. However, a detailed understanding of the factors which influence its fluorescence is required to be able to use this popular probe accurately. Here we demonstrate the recombinant incorporation of p-cyanophenylalanine in the N-terminal domain of the ribosomal protein L9. Native state fluorescence is very low, which suggests that the group is sequestered from solvent; however, IR measurements and molecular dynamics simulations show that the cyano group is exposed to solvent and forms hydrogen bonds to water. Analysis of mutant proteins and model peptides demonstrates that the reduced native state fluorescence is caused by the effective quenching of p-cyanophenylalanine fluorescence via FRET to tyrosine side-chains. The implications for the interpretation of p-cyanophenylalanine fluorescence measurements and FRET studies are discussed.

Computational models explain the oligosaccharide specificity of cyanovirin-N.

The prokaryotic lectin cyanovirin-N (CV-N) is a potent inhibitor of HIV envelope-mediated cell entry, and thus is a leading candidate among a new class of potential anti-HIV microbicides. The activity of CV-N is a result of interactions with the D1 arm of high-mannose oligosaccharides on the viral glycoprotein gp120. Here, we present computationally refined models of CV-N recognition of the di- and trisaccharides that represent the terminal three sugars of the D1 arm by each CV-N binding site. These models complement existing structural data, both from NMR spectroscopy and X-ray crystallography. When used with a molecular dynamics/continuum electrostatic (MD/PBSA) approach to compute binding free energies, these models explain the relative affinity of each site for the two saccharides. This work presents the first validation of the application of continuum electrostatic models to carbohydrate-protein association. Taken as a whole, the results both provide models of CV-N sugar recognition and demonstrate the utility of these computational methods for the study of carbohydrate-binding proteins.