Drawing up a collaborative contract: Amino acid cross-feeding between interspecies bacterial pairs.

Synthetic microbial communities have the potential to enable new platforms for bioproduction of biofuels and biopharmaceuticals. However, using engineered communities is often assumed to be difficult because of anticipated challenges in establishing and controlling community composition. Cross-feeding between microbial auxotrophs has the potential to facilitate coculture growth and stability through a mutualistic ecological interaction. We assessed cross-feeding between 13 Escherichia coli amino acid auxotrophs paired with a leucine auxotroph of Bacillus megaterium. We developed a minimal medium capable of supporting the growth of both bacteria and used the media to study coculture growth of the 13 interspecies pairs of auxotrophs in batch and continuous culture, as well as on semi-solid media. In batch culture, 8 of 13 pairs of auxotrophs were observed to grow in coculture. We developed a new metric to quantify the impact of cross-feeding on coculture growth. Six pairs also showed long-term stability in continuous culture, where coculture growth at different dilution rates highlighted differences in cross-feeding amongst the pairs. Finally, we found that cross-feeding-dependent growth on semi-solid media is highly stringent and enables identification of the most efficient pairs. These results demonstrate that cross-feeding is a viable approach for controlling community composition within diverse synthetic communities.

Influence of bacterial culture medium on peptidoglycan binding of cell wall lytic enzymes.

The bacteriolysin lysostaphin (Lst) and endolysin PlyPH are potent modular lytic enzymes with activity against clinically-relevant Gram-positive Staphylococcus aureus and Bacillus cereus, respectively. Both enzymes possess an N-terminal catalytic domain and C-terminal binding domain, with the latter conferring significant enzyme specificity. Lst and PlyPH show reduced activity in the presence of bacterial growth-supporting conditions, such as complex media. Here, we hypothesize that Lst and PlyPH bind poorly to their targets in growth media, which may influence their use in antimicrobial applications in the food industry, as therapeutics, and for control of microbial communities. To this end, binding of isolated Lst and PlyPH binding domains to target bacteria was quantified in the presence of three increasingly complex media - phosphate buffered saline (PBS), defined growth medium (AAM) and undefined complex medium (TSB) by surface plasmon resonance (SPR) and flow cytometry. Evaluation of binding kinetics by SPR demonstrated that PlyPH binding was particularly sensitive to medium composition, with 8-fold lower association and 3.4-fold lower dissociation rate constants to B. cereus in TSB compared to PBS. Flow cytometry studies indicated a decrease in the binding-dependent fluorescent populations of S. aureus and B. cereus, for lysostaphin binding domain and PlyPH binding domain, respectively, in TSB compared to PBS. Enzyme binding behavior was consistent with the enzymes' catalytic activity in the three media, thereby suggesting that compromised enzyme binding could be responsible for poor activity in more complex growth media.

Opportunities for broadening the application of cell wall lytic enzymes.

In light of emerging antibiotic resistance, bacterial cell wall lytic enzymes are promising antimicrobial agents that degrade bacterial peptidoglycan while specifically recognizing the target bacterium. The efficacy of lytic enzymes against several multi-drug-resistant pathogens infecting humans has led to many efforts focused on in vivo therapeutic applications. However, the potential for lytic enzymes to combat bacterial contamination in environments outside the human body is underexplored. The persistence of pathogenic bacteria, in either planktonic or biofilm states and on various surfaces, has facilitated the spread of bacterial infections, necessitating the development of robust strategies for detecting and killing resistant bacteria in diverse environments. Here, we present an overview of the current state-of-the-art of exploiting lytic enzymes for non-therapeutic applications including pathogen decontamination in social infrastructures and food decontamination, as well as pathogen detection. KEY POINTS: • Lytic enzymes are effective antimicrobial, antibiofilm, and sporicidal agents. • Pathogen detection using lytic enzyme-binding domains is rapid and highly sensitive. • Domain engineering is required for enhanced enzyme activity in complex environments.

Metabolic engineering of Bacillus megaterium for heparosan biosynthesis using Pasteurella multocida heparosan synthase, PmHS2.

BACKGROUND: Heparosan is the unsulfated precursor of heparin and heparan sulfate and its synthesis is typically the first step in the production of bioengineered heparin. In addition to its utility as the starting material for this important anticoagulant and anti-inflammatory drug, heparosan is a versatile compound that possesses suitable chemical and physical properties for making a variety of high-quality tissue engineering biomaterials, gels and scaffolds, as well as serving as a drug delivery vehicle. The selected production host was the Gram-positive bacterium Bacillus megaterium, which represents an increasingly used choice for high-yield production of intra- and extracellular biomolecules for scientific and industrial applications. RESULTS: We have engineered the metabolism of B. megaterium to produce heparosan, using a T7 RNA polymerase (T7 RNAP) expression system. This system, which allows tightly regulated and efficient induction of genes of interest, has been co-opted for control of Pasteurella multocida heparosan synthase (PmHS2). Specifically, we show that B. megaterium MS941 cells co-transformed with pT7-RNAP and pPT7_PmHS2 plasmids are capable of producing heparosan upon induction with xylose, providing an alternate, safe source of heparosan. Productivities of ~ 250 mg/L of heparosan in shake flasks and ~ 2.74 g/L in fed-batch cultivation were reached. The polydisperse Pasteurella heparosan synthase products from B. megaterium primarily consisted of a relatively high molecular weight (MW) heparosan (~ 200-300 kD) that may be appropriate for producing certain biomaterials; while the less abundant lower MW heparosan fractions (~ 10-40 kD) can be a suitable starting material for heparin synthesis. CONCLUSION: We have successfully engineered an asporogenic and non-pathogenic B. megaterium host strain to produce heparosan for various applications, through a combination of genetic manipulation and growth optimization strategies. The heparosan products from B. megaterium display a different range of MW products than traditional E. coli K5 products, diversifying its potential applications and facilitating increased product utility.

Selective antimicrobial activity of cell lytic enzymes in a bacterial consortium.

The role that the complex microbial communities play in human and environmental health cannot be understated. The increased information about community complexity, as well as the overuse of broad-spectrum antibiotics, suggest that new approaches to target specific organisms within a community context are essential towards new antimicrobial therapies. Here, we have assessed the activity and selectivity of two cell wall lytic enzymes, lysostaphin (Lst) and PlyPH, in the presence of multiple bacteria and under varied media conditions. Lst and PlyPH target the clinically relevant pathogens Staphylococcus aureus and Bacillus cereus, respectively. Lst was effective under all conditions resulting in ~ 4-log and ~ 3-log reduction at 100 µg/mL in actively growing monoculture and co-culture, respectively. PlyPH was also selective but less active and more susceptible to media and cell population changes. Lst and PlyPH activities could be increased in supernatants from actively growing cultures in the presence of a protease inhibitor cocktail, suggesting a possible role played by proteases secreted during cell growth in reducing lytic enzyme activity. This work demonstrates the utility of cell wall lytic enzymes for targeted pathogen killing or microbial community remodeling.

Engineering Bacillus megaterium Strains To Secrete Cellulases for Synergistic Cellulose Degradation in a Microbial Community.

Recent environmental concerns have intensified the need to develop systems to degrade waste biomass for use as an inexpensive carbon source for microbial chemical production. Current approaches to biomass utilization rely on pretreatment processes that include expensive enzymatic purification steps for the requisite cellulases. We aimed to engineer a synthetic microbial community to synergistically degrade cellulose by compartmentalizing the system with multiple specialized Bacillus megaterium strains. EGI1, an endoglucanase, and Cel9AT, a multimodular cellulase, were targeted for secretion from B. megaterium. A small library of signal peptides (SPs) with five amino acid linkers was selected to tag each cellulase for secretion from B. megaterium. Cellulase activity against amorphous cellulose was confirmed through a series of bioassays, and the most active SP constructs were identified as EGI1 with the LipA SP and Cel9AT with the YngK SP. The activity of the optimized cellulase secretion strains was characterized individually and in tandem to assess synergistic cellulolytic activity. The combination of EGI1 and Cel9AT yielded higher activity than either single cellulase. A coculture of EGI1 and Cel9AT secreting B. megaterium strains demonstrated synergistic behavior with higher activity than either monoculture. This cellulose degradation module can be further integrated with bioproduct synthesis modules to build complex systems for the production of high value molecules.

Transcriptional control of motility enables directional movement of Escherichia coli in a signal gradient.

Manipulation of cellular motility using a target signal can facilitate the development of biosensors or microbe-powered biorobots. Here, we engineered signal-dependent motility in Escherichia coli via the transcriptional control of a key motility gene. Without manipulating chemotaxis, signal-dependent switching of motility, either on or off, led to population-level directional movement of cells up or down a signal gradient. We developed a mathematical model that captures the behaviour of the cells, enables identification of key parameters controlling system behaviour, and facilitates predictive-design of motility-based pattern formation. We demonstrated that motility of the receiver strains could be controlled by a sender strain generating a signal gradient. The modular quorum sensing-dependent architecture for interfacing different senders with receivers enabled a broad range of systems-level behaviours. The directional control of motility, especially combined with the potential to incorporate tuneable sensors and more complex sensing-logic, may lead to tools for novel biosensing and targeted-delivery applications.

Towards cell-free isobutanol production: Development of a novel immobilized enzyme system.

Producing fuels and chemical intermediates with cell cultures is severely limited by low product concentrations (≤0.2%(v/v)) due to feedback inhibition, cell instability, and lack of economical product recovery processes. We have developed an alternate simplified production scheme based on a cell-free immobilized enzyme system. Two immobilized enzymes (keto-acid decarboxylase (KdcA) and alcohol dehydrogenase (ADH)) and one enzyme in solution (formate dehydrogenase (FDH) for NADH recycle) produced isobutanol titers 8 to 20 times higher than the highest reported titers with S. cerevisiae on a mol/mol basis. These high conversion rates and low protein leaching were achieved by covalent immobilization of enzymes (ADH) and enzyme fusions (fKdcA) on methacrylate resin. The new enzyme system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.135 (mole isobutanol produced for each mole ketoisovaleric acid consumed). Further increasing titer will require continuous removal of the isobutanol using an in situ recovery system.

Synthetic Quorum Sensing and Cell-Cell Communication in Gram-Positive Bacillus megaterium.

The components of natural quorum-sensing (QS) systems can be used to engineer synthetic communication systems that regulate gene expression in response to chemical signals. We have used the machinery from the peptide-based agr QS system from Staphylococcus aureus to engineer a synthetic QS system in Bacillus megaterium to enable autoinduction of a target gene at high cell densities. Growth and gene expression from these synthetic QS cells were characterized in both complex and minimal media. We also split the signal production and sensing components between two strains of B. megaterium to produce sender and receiver cells and characterized the resulting communication in liquid media and on semisolid agar. The system described in this work represents the first synthetic QS and cell-cell communication system that has been engineered to function in a Gram-positive host, and it has the potential to enable the generation of dynamic gene regulatory networks in B. megaterium and other Gram-positive organisms.

Men as caregivers of the elderly: support for the contributions of sons.

Emerging practice research on filial sources of health care support has indicated that there is a growing trend for sons to assume some responsibility for the health care needs of their aging parents. The purpose of this work is to propose that outcomes observed through a secondary analysis of data from a previous mixed methods research project, conducted with a sample of 60 elderly women residing in independent living centers, supports this concept in elder care. The present study is a retrospective interpretation utilizing the original database to examine the new question, "What specific roles do sons play in caregiving of their elderly mothers?" While daughters presently continue to emerge in existing health care studies as the primary care provider, there is a significant pattern in these data for older patients to depend upon sons for a variety of instrumental activities of daily living. As the baby-boomers age, there is more of cohort trend for their families to be smaller, adult daughters to be employed, and for adult children to be more geographically mobile. These factors may combine to make health care support networks more limited for the current aging population, challenging the elderly and their health care providers to revisit the cultural gender norms that are used to identify caregivers.

Impact of external flow on the dynamics of swimming microorganisms near surfaces.

Swimming microorganisms have been previously observed to accumulate along walls in confined systems both experimentally and in computer simulations. Here, we use computer simulations of dilute populations for a simplified model of an organism to calculate the dynamics of swimmers between two walls with an external fluid flow. Simulations with and without hydrodynamic interactions (HIs) are used to quantify their influence on surface accumulation. We found that the accumulation of organisms at the wall is larger when HIs are included. An external fluid flow orients the organisms parallel to the fluid flow, which reduces the accumulation at the walls. The effect of the flow on the orientations is quantified and compared to previous work on upstream swimming of organisms and alignment of passive rods in flow. In pressure-driven flow, the zero shear rate at the channel center leads to a dip in the concentration of organisms in the center. The curvature of this dip is quantified as a function of the flow rate. The fluid flow also affects the transport of organisms across the channel from one wall to the other.

Quorum sensing-modulated AND-gate promoters control gene expression in response to a combination of endogenous and exogenous signals.

We have constructed and characterized two synthetic AND-gate promoters that require both a quorum-sensing (QS) signal and an exogenously added inducer to turn on gene expression. The engineered promoters, LEE and TTE, contain binding sites for the QS-dependent repressor, EsaR, and either LacI or TetR, and they are induced by an acyl-homoserine lactone (AHL) signal and IPTG or aTc. Although repression of both LEE and TTE by wild-type EsaR was observed, induction of gene expression at physiologically relevant concentrations of AHL required the use of an EsaR variant with higher signal sensitivity. Gene expression from both LEE and TTE was shown to require both signal molecules, and gene expression above background levels was not observed with either signal alone. We added endogenous production of AHL to evaluate the ability of the promoters to function in a QS-dependent manner and observed that gene expression increased as a function of cell density only in the presence of exogenously added IPTG or aTc. Cell-cell communication-dependent AND-gate behaviors were demonstrated using an agar plate assay, where cells containing the engineered promoters were shown to respond to AHL produced by a second E. coli strain only in the presence of exogenously added IPTG or aTc. The promoters described in this work demonstrate that EsaR and its target DNA sequence can be used to engineer new promoters to respond to cell density or cell-cell communication. Further, the AND-gate promoters described here may serve as a template for new regulatory systems that integrate QS and the presence of key metabolites or other environmental cues to enable dynamic changes in gene expression for metabolic engineering applications.

Effect of spaceflight on Pseudomonas aeruginosa final cell density is modulated by nutrient and oxygen availability.

BACKGROUND: Abundant populations of bacteria have been observed on Mir and the International Space Station. While some experiments have shown that bacteria cultured during spaceflight exhibit a range of potentially troublesome characteristics, including increases in growth, antibiotic resistance and virulence, other studies have shown minimal differences when cells were cultured during spaceflight or on Earth. Although the final cell density of bacteria grown during spaceflight has been reported for several species, we are not yet able to predict how different microorganisms will respond to the microgravity environment. In order to build our understanding of how spaceflight affects bacterial final cell densities, additional studies are needed to determine whether the observed differences are due to varied methods, experimental conditions, or organism specific responses. RESULTS: Here, we have explored how phosphate concentration, carbon source, oxygen availability, and motility affect the growth of Pseudomonas aeruginosa in modified artificial urine media during spaceflight. We observed that P. aeruginosa grown during spaceflight exhibited increased final cell density relative to normal gravity controls when low concentrations of phosphate in the media were combined with decreased oxygen availability. In contrast, when the availability of either phosphate or oxygen was increased, no difference in final cell density was observed between spaceflight and normal gravity. Because motility has been suggested to affect how microbes respond to microgravity, we compared the growth of wild-type P. aeruginosa to a ΔmotABCD mutant deficient in swimming motility. However, the final cell densities observed with the motility mutant were consistent with those observed with wild type for all conditions tested. CONCLUSIONS: These results indicate that differences in bacterial final cell densities observed between spaceflight and normal gravity are due to an interplay between microgravity conditions and the availability of substrates essential for growth. Further, our results suggest that microbes grown under nutrient-limiting conditions are likely to reach higher cell densities under microgravity conditions than they would on Earth. Considering that the majority of bacteria inhabiting spacecrafts and space stations are likely to live under nutrient limitations, our findings highlight the need to explore the impact microgravity and other aspects of the spaceflight environment have on microbial growth and physiology.

Engineering the esaR promoter for tunable quorum sensing- dependent gene expression.

Quorum sensing (QS) systems enable bacteria to coordinate their behavior as a function of local population density and are often used in synthetic systems that require cell−cell communication. We have engineered the esaR promoter, P(esaR), which is repressed by the QS regulator E(saR). E(saR)-dependent gene expression from P(esaR) is induced by 3-oxo-hexanoyl-homoserine lactone (3OC6HSL). Here, we report a set of modified P(esaR) promoters that contain a second E(saR) binding site. We observed changes in gene expression levels, regulatory range, 3OC6HSL sensitivity, and the regulatory role of E(saR) that are dependent on the position of the second binding site. Combining the new promoters with endogenous 3OC6HSL production led to QS-dependent systems that exhibit a range of expression levels and timing. These promoters represent a new set of tools for modulating QS-dependent gene expression and may be used to tune the regulation of multiple genes in response to a single QS signal.

Peptide-based communication system enables Escherichia coli to Bacillus megaterium interspecies signaling.

The use of mixtures of microorganisms, or microbial consortia, has the potential to improve the productivity and efficiency of increasingly complex bioprocesses. However, the use of microbial consortia has been limited by our ability to control and coordinate the behaviors of microorganisms in synthetic communities. Synthetic biologists have previously engineered cell-cell communication systems that employ machinery from bacterial quorum-sensing (QS) networks to enable population-level control of gene expression. However, additional communication systems, such as those that enable communication between different species of bacteria, are needed to enable the use of diverse species in microbial consortia for bioprocessing. Here, we use the agr QS system from Staphylococcus aureus to generate an orthogonal synthetic communication system between Gram-negative Escherichia coli and Gram-positive Bacillus megaterium that is based on the production and recognition of autoinducing peptides (AIPs). We describe the construction and characterization of two types of B. megaterium "receiver" cells, capable of AIP-dependent gene expression in response to AIPs that differ by a single amino acid. Further, we observed interspecies communication when these receiver cells were co-cultured with AIP-producing E. coli. We show that the two AIP-based systems exhibit differences in sensitivity and specificity that may be advantageous in tuning communication-dependent networks in synthetic consortia. These peptide-based communication systems will enable the coordination of gene expression, metabolic pathways and growth between diverse microbial species, and represent a key step towards the use of microbial consortia in bioprocessing and biomanufacturing.

Spaceflight promotes biofilm formation by Pseudomonas aeruginosa.

Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight.

Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity.

The use of cell-cell communication or "quorum sensing (QS)" elements from Gram-negative Proteobacteria has enabled synthetic biologists to begin engineering systems composed of multiple interacting organisms. However, additional tools are necessary if we are to progress toward synthetic microbial consortia that exhibit more complex, dynamic behaviors. EsaR from Pantoea stewartii subsp. stewartii is a QS regulator that binds to DNA as an apoprotein and releases the DNA when it binds to its cognate signal molecule, 3-oxohexanoyl-homoserine lactone (3OC6HSL). In the absence of 3OC6HSL, EsaR binds to DNA and can act as either an activator or a repressor of transcription. Gene expression from P(esaR), which is repressed by wild-type EsaR, requires 100- to 1000-fold higher concentrations of signal than commonly used QS activators, such as LuxR and LasR. Here we have identified EsaR variants with increased sensitivity to 3OC6HSL using directed evolution and a dual ON/OFF screening strategy. Although we targeted EsaR-dependent derepression of P(esaR), our EsaR variants also showed increased 3OC6HSL sensitivity at a second promoter, P(esaS), which is activated by EsaR in the absence of 3OC6HSL. Here, the increase in AHL sensitivity led to gene expression being turned off at lower concentrations of 3OC6HSL. Overall, we have increased the signal sensitivity of EsaR more than 70-fold and generated a set of EsaR variants that recognize 3OC6HSL concentrations ranging over 4 orders of magnitude. QS-dependent transcriptional regulators that bind to DNA and are active in the absence of a QS signal represent a new set of tools for engineering cell-cell communication-dependent gene expression.

Modular optimization of multi-gene pathways for fatty acids production in E. coli.

Microbial fatty acid-derived fuels have emerged as promising alternatives to petroleum-based transportation fuels. Here we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titre improvements in a multi-gene fatty acid metabolic pathway. On the basis of central pathway architecture, E. coli fatty acid biosynthesis was re-cast into three modules: the upstream acetyl coenzyme A formation module; the intermediary acetyl-CoA activation module; and the downstream fatty acid synthase module. Combinatorial optimization of transcriptional levels of these three modules led to the identification of conditions that balance the supply of acetyl-CoA and consumption of malonyl-CoA/ACP. Refining protein translation efficiency by customizing ribosome binding sites for both the upstream acetyl coenzyme A formation and fatty acid synthase modules enabled further production improvement. Fed-batch cultivation of the engineered strain resulted in a final fatty acid production of 8.6 g l(-1). The modular engineering strategies demonstrate a generalized approach to engineering cell factories for valuable metabolites production.