Protein mass is a major constituent of bacterial cell dry weight. An exponentially growing cell diverts most of its nutrient uptake to protein synthesis. The optimal allocation of a cell's total proteins, or proteome, to various cellular functions is a major concern for the cell growing in different growth conditions. Obligatory relations between ribosomal proteome fraction and growth rate impose further constraints on proteome resources allocation. In this dissertation, I am interested in understanding the global strategy of proteome resources allocation and exploring its physiological consequences in E. coli.

To understand the global strategy of proteome allocation, we apply a series of metabolic challenges to probe the responses of the proteome of exponentially growing E. coli using quantitative mass spectrometry. Despite the enormous complexity in the details of the underlying regulatory network, the proteome partitions into several coarse-grained sectors whose total abundances exhibit linear relations with the growth rate. These growth-rate dependent proteome fractions comprise about half of the proteome by mass, and their mutual dependencies can be characterized quantitatively by a simple proteome-based flux model involving only two effective parameters. The success and apparent generality of this simple model is due to the tight coordination between proteome partition and metabolism, suggesting a principle for resource-allocation in proteome economy. This general strategy of global gene regulation should serve as a basis for future studies on gene expression and constructing synthetic biological circuits.

We also show that proteome resources allocation has important consequences in cell physiology. An important yet mysterious phenomenon in bacterial physiology is the overflow metabolism, where the cell excretes acetate in the case of E. coli. We observed a set linear relations between acetate excretion rate and growth rate under different modes of growth limitation. By including both the carbon and proteome resources in a simple resource allocation model, we can quantitatively describe the observed linear relations. Key model parameters of protein costs were determined directly using flux analysis and protein mass spectrometry measurements. These results suggest optimal allocation of carbon and proteome resources as a possible driving force for the occurrence of overflow metabolism.

Growth and reproduction are essential requirements of living systems. Previous studies of bacterial physiology focused largely on bacteria growing exponentially in fixed environments. But such growth conditions are exception rather than the norm in nature. Bacteria in the wild must cope with environments that vary temporally and spatially. To gain better understanding of bacterial physiology, I studied growth of E. coli population in 1) dynamic nutrient environment and 2) spatially extended environment.

Rapid adaptation is a crucial determinant of bacterial fitness under changing environments. We studied adaptation of E. coli culture to abrupt change of nutrient from preferred sugars (e.g., glucose) to a metabolic waste product (e.g., acetate) that is excreted during the growth on preferred substrates. In such transitions, E. coli suffers long, multi-hour periods of growth arrest before resuming growth. By characterizing the period of growth arrest (lag time) for many pairs of carbon sources, we discovered that cells growing fast before the shift exhibit longer lag time, resulting in a remarkable trade- off relationship between fast growth and adaptability. We establish that this tradeoff results from switching the direction of central carbon metabolism from glycolysis to gluconeogenesis. The existence of tradeoff limits fitness benefit gained from fast growth and provides a criterion for selecting growth rates based on the expected abundances of substrates in the environment.

In spatially open habitats, effective colonization provides a selective advantage for bacterial populations. We next studied the growth of E. coli population that expands by chemotaxis in nutrient-replete soft-agar plates. We show the speed of population expansion, as exemplified by the propagation of chemotactic rings, increases steadily with improved growth conditions. The expansion process is shown to couple intricately with colonization, with the propagating cells leave behind a trail of offspring that slowly proliferate over time. We also show costly expression of motility apparatus is necessary to maintain the fraction of motile cells and support faster population expansion. The results provide a link from gene expression through cellular motility all the way to population dynamics, elucidating a foresighted strategy bacteria use to colonize virgin territories well before local nutrient supply runs out.

Quantitative studies of bacterial growth have historically focused on planktonic cells consuming dissolved substrates. In this work, we extend such studies to another important class of nutrients ubiquitous in nature: particulate substrates, not dissolvable in water. These substrates are the major nutrient sources in a variety of important natural ecosystems such as the ocean, soils, and guts, as well as synthetic ones, e.g., plastics. Understanding the interactions and growth of bacteria with such substrates is therefore vital, as it will allow to predict and manipulate the roles bacteria play in these important environments.Using chitin as a model structured substrate, we study its degradation by Vibrio sp. 1A01 and report a number of surprising findings. When culturing 1A01 with chitin flakes, we find that although chitin was the only source of nutrients in the cultures, the population had a high propensity for detachment and a majority of cells lived in the planktonic state. Despite this high detachment rate, the population was able to maintain an exponential rate of growth. Quantitative proteomic studies help elucidate how these cells manage to support high rates of dispersal while still replicating on the particles in spite of their low level of chitinase expression: secreted chitinases accumulate on particles, supporting the growth of the resident population of cells, irrespective of how many cells detached. This molecular “trick” enables the population to overcome the colonization/dispersal tradeoff often associated with growth on particles. Next, we formulated a spatiotemporal microscopic model of bacteria-chitin interactions constrained by our experimental data. Our model replicated experimental findings and provided rigorous definitions of the spatially separated subpopulations characterized above. In addition, we examined how microscopic parameters governing the local interaction of cells with particles translated to macroscopic features of the population such as growth rate and spatial extent. Finally, our model allowed us to probe regimes not accessible experimentally, particularly related to cultures starting from low-density initial conditions. We uncovered a 2-dimensional phase transition in the space of initial conditions where a critical number of both cells and enzymes is necessary for enabling growth. This allowed us to identify the mechanistic basis for the Allee effect in such systems. We discuss various mechanisms that constrain these initial conditions. Our results call for further studies of the physiology of starving cells and their transition to regrowth. Taken together our work is an experimental and theoretical attempt to tame the complexity of the various mechanisms governing growth in structured environments.

Microscopic cells and debris combine to form large, sinking aggregates in the upper ocean, contributing to carbon sequestration and food web supply via the biological carbon pump. Bacteria are implicated in marine aggregate formation, but we lack a detailed understanding of their role. Certain taxonomic groups, such as Alteromonas spp., appear to be specialized for growth in aggregates (AKA particle-associated), but we do not understand the details of their physiology and ecology that support this specialization. Using laboratory cultivation of natural isolates, I have undertaken a broad survey of aggregate formation by marine bacteria, followed by in-depth investigation of isolates from Alteromonas spp. I successfully identified specific compounds eliciting aggregation (“chemoaggregants”), revealed a genetic requirement impacting multiple aggregation-associated phenotypes, and elaborated a previously unrecognized connection between aggregation and proteolytic growth. These findings advance our understanding of the eco-physiological capabilities of Alteromonas spp., demonstrating their selective aggregation in response to certain nutrients and providing a rationale for this response in the context of organic matter degradation and growth.

In different environments, bacteria are known to allocate their proteome differently and achieve different growth rates. Extensive quantitative studies on proteomic allocations in steady states have been done. Yet it’s still not clear how bacteria manage to adjust their proteome responding to different environments through thousands of reactions and eventually reach different growth rates. In order to gain better understanding on the adaptation strategy and mechanism, in my dissertation, I studied the kinetic behavior of E.coli during various environmental changes. Mathematical modeling were used to quantitatively capture the proteomic re-allocation and growth rate adaptation during transitions. Further studies on the kinetics of Guanosine tetraphosphate (ppGpp) and translational elongation rate during growth transition revealed the molecular basis of well-known ribosomal ‘growth law’. In Chapter 2 and 3, we studied the growth transitions from ‘rich media’, a condition with ample amino acids or/and other nutrient supplies. We started with the case of methionine depletion in Chapter 2. MetE were found to be the major bottleneck in met downshift. By adapting the flux-controlled regulatory model established for carbon shift, we quantitatively captured the relationship between pre-shift MetE reserves and lag time before growth recovery. In Chapter 3, we studied the proteomic allocation during all AAs depletion. Quantitative proteomic analysis revealed a linear relationship between the onset time of enzymes across AAs biosynthesis pathways and their fractional reserve in pre-shift condition, indicating a ‘as-needed’ gene expression strategy during all AAs downshift. Combining flux-controlled global regulation and pathway specific end-production inhibition, we successfully captured the proteome recovery kinetics using only data collected in the pre- and post-shift steady-states. Chapter 4 is focused on bacterial kinetic response to sub-lethal chloramphenicol (an antibiotic inhibiting translation) treatment. By using translational elongation rate as a flux sensor and the key signaling variable, we accurately predicted the kinetics of biomass accumulation and gene expressions. In Chapter 5, we show that translational elongation rate is inversely proportional to ppGpp, an essential molecule regulating translational machinery, during transient and steady-states. We established ppGpp rate-sensing strategy and thus closed a key regulatory circuit linking ppGpp, growth rate, ribosomal content and translational rate together.

Marine bacteria are responsible for a significant portion of decomposition and recycling of the organic matter produced by primary production. Moreover, these bacteria can impact the distribution of organic matter within the water column by driving aggregation and sinking of various particles in a pathway known as the biological carbon pump. The process of aggregation can be driven by bacteria which are able to stick to one another as well as to particles. While bacteria within the ocean experience a nutrient depleted environment with occasional encounters of nutrient hotspots, we know little about how this may impact their aggregative behaviors. After examining eleven strains derived from nine species of marine bacteria cultured in minimal medium with twenty-nine distinct carbon sources, distinct patterns in aggregation and growth behaviors of these bacteria have emerged. We observed many cases in which cell-cell aggregation can be triggered during growth by the presence of certain dissolved nutrients. Each strain has a unique combination of nutrients which support growth or aggregation. We categorize the strains into three types of responses: constitutive-aggregator, selective-aggregator, and non-aggregators. Constitutive and selective aggregators appear to be metabolic generalists, while non-aggregators show a preference for growth on acids. In a few cases aggregation occurred without bacterial growth. Certain carbon sources had a bias towards triggering aggregation in strains which grew with them, while others demonstrated an even split between growth with and without aggregation. The findings indicate a trend in aggregation behaviors which appears to be nutrient and strain specific.

In this dissertation, we present a comprehensive study on two sets of phenomenological models examining bacterial population dynamics, focusing on chemotaxis-driven expansion and microbial interactions in non-steady-state environments. Our first model provides a quantitative understanding of how chemotaxis and cell growth lead to the rapid expansion of bacterial populations. We establish analytical relations that describe the dependence of expansion speed and density profile on molecular, cellular, and environmental parameters. We demonstrate that expansion speeds can significantly increase when the environmental availability of chemicals is high relative to the cellular limits of chemical sensing. Our results offer a mathematical framework for investigating the roles of taxis in diverse ecological contexts across broad parameter regimes.The second set of models explores microbial interactions in non-steady-state environments, as ecological dynamics often feature large self-generated environmental changes driving microbes through distinct physiological states. We introduce a phenomenological model to investigate the dynamic coexistence of microbes in cyclic environments. By considering growth according to a global ecological coordinate, specifically total community biomass, our model bypasses specific interactions leading to different physiological states. Our analysis provides rigorous, quantitative criteria for the dynamic coexistence of many species in terms of differential species' dominance ("growth niche") along the ecological coordinates. Our research shifts the focus of ecosystem dynamics from bottom-up studies based on inter-species interaction to top-down studies based on accessible macroscopic observables such as growth rates and total biomass. This approach allows for a quantitative examination of community-wide characteristics. In summary, this dissertation presents a detailed analytical investigation of bacterial population dynamics, shedding light on the underlying processes of chemotaxis-driven expansion and microbial interactions in changing environments. The mathematical frameworks and insights provided have broad applicability to diverse ecological contexts and open up new avenues for understanding complex spatiotemporal dynamic processes in microbial ecosystems.

Survival through periods of starvation is crucial for bacterial fitness. The role of gene expression in sustaining the viability of starving bacteria has been extensively studied, including the identification and investigation of 'stress response' proteins regulated by the general stress sigma factor RpoS in E. coli. However, less is known about the costs and benefits of metabolic processes sustaining gene expression during starvation. This thesis work describes a comprehensive physiological study of E. coli cells under glucose starvation, quantifying the temporal dynamics of resource generation and utilization, and connecting them to cell viability. We focus on a preparatory period, the stationary phase, where the proteome is remodeled after entering starvation, before the onset of significant population death. We find two proteins are degraded for every protein synthesized, and five-six proteins degraded for every protein accumulated, resulting in the turnover of over half of the proteome for a moderate gain in stress proteins. Such extensive global proteolysis is enabled by a little-known member of the RpoS regulon, YeaG, which works in concert with well-known members of the proteolytic machineries. The total amount of protein synthesis is dictated by the amount of protein degradation, reflecting a dominant, supply-driven strategy of resource allocation for starving cells. YeaG-mediated proteome remodeling accounts for a major share of the viability boost provided by RpoS. In a mutant where energy can be supplemented without cell growth, YeaG activity is largely unnecessary, suggesting that a major physiological function of proteome remodeling is to increase the energy efficiency of starving cells for survival. Such knowledge of the physiology and molecular biology of starving bacteria offers new insights on bacterial vulnerabilities and inspires new strategies for engineering efficient bacterial bio-factories.