Emerging strategies for engineering microbial communities

From biosynthesis to bioremediation, microbes have been engineered to address a variety of biotechnological applications. A promising direction in these endeavors is harnessing the power of designer microbial consortia that consist of multiple populations with well-defined interactions. Consortia can accomplish tasks that are difficult or potentially impossible to achieve using monocultures. Despite their potential, the rules underlying microbial community maintenance and function (i.e. the task the consortium is engineered to carry out) are not well defined, though rapid progress is being made. This limited understanding is in part due to the greater challenges associated with increased complexity when dealing with multi-population interactions. Here, we review key features and design strategies that emerge from the analysis of both natural and engineered microbial communities. These strategies can provide new insights into natural consortia and expand the toolbox available to engineers working to develop novel synthetic consortia.     

Quelling the red menace: haem capture by bacteria

Haem is an important bacterial nutrient. As a prosthetic group of several proteins, haem functions as a cofactor mediating oxygen transport, energy generation, and mixed-function oxidation. In addition, the iron chelated in the porphyrin ring may serve as an iron substrate for growth. However, because of its propensity for oxidizing cellular constituents, haem is always associated with proteins. Therefore, the uptake and transit of haem across bacterial membranes requires the participation of protein escorts. Bacteria have evolved a diverse array of surface-exposed receptors dedicated to binding haem and haem-proteins. Following this selective recognition at the bacterial cell surface, haem is transported across the outer membrane via a TonB-dependent process. The control of receptor expression appears to be multifactorial, probably involving a number of global regulators. A model integrating this information is presented.     

Role of haem oxygenase-1 in microbial host defence

Haem oxygenase (HO)-1 is a cytoprotective enzyme that plays a critical role in defending the body against oxidant-induced injury during inflammatory processes. HO catalydes the degradation of haem to carbon monoxide (CO), biliverdin and ferrous iron. Biliverdin is converted to bilirubin, a potent endogenous antioxidant. CO has a number of biological functions, including anti-inflammatory properties. In various models of disease, HO-1 is known to play a critical role by ameliorating the pathological consequences of injury. In many of these models, the beneficial effects of HO-1 and its products of haem catabolism are by suppressing an inflammatory response. However, when investigating diseases due to microbial infections, inhibition of the inflammatory response could disrupt the ability of the immune system to eradicate an invading pathogen. Thus, questions remain regarding the role of HO-1 in microbial host defence. This microreview will address our present understanding of HO-1 and its functional significance in a variety of microbial infections.     

Purification strategies for microbial lipases

Microbial lipases today occupy a place of prominence among biocatalysts owing to their ability to catalyze a wide variety of reactions in aqueous and non-aqueous media. The chemo-, regio- and enantio-specific behaviour of these enzymes has caused tremendous interest among scientists and industrialists. Lipases from a large number of bacterial, fungal and a few plant and animal sources have been purified to homogeneity. This has enabled their successful sequence determination and their three-dimensional structure leading to a better understanding of their unique structure-function relationships during various hydrolytic and synthetic reactions. This article presents a critical review of different strategies which have been employed for the purification of bacterial, yeast and fungal lipases. Since protein purification is normally done in a series of sequential steps involving a combination of different techniques, the effect of sequence of steps and the number of times each step is used is analyzed. This will prove to be of immense help while planning lipase purification. Novel purification technologies now available in this field are also reviewed.     

Data collection and capture systems for microbial modeling

Summary Microbial modeling experiments require an integrated and efficient design to overcome constraints on time and human resources. The choice of an experimental system is effected by first determining the goals and scope of the model to be constructed. Kinetic studies, for example, require a different approach from single end-point models, such as time to toxin detection or growth probability. Studies have been conducted in liquid broth tubes or batch culture, agar plates, and food matrices. These traditional systems are labor intensive, however, which constrains experimental size, and thus, a model's scope and validity. To maximize experimental size, experimental systems should be automated and linked to electronic data manipulation, analysis, and presentation. Microbial modelers should also consider the relationship between the experimental environmental factors, such as pH,a _w, or temperature, and their impact on growth, virulence or toxigenesis determinants. Attaining these goals will increase the probability that the model will accurately predict microbial responses in food systems.     

Characterization of product capture resin during microbial cultivations

Various bioactive small molecules produced by microbial cultivation are degraded in the culture broth or may repress the formation of additional product. The inclusion of hydrophobic adsorber resin beads to capture these products in situ and remove them from the culture broth can reduce or prevent this degradation and repression. These product capture beads are often subjected to a dynamic and stressful microenvironment for a long cultivation time, affecting their physical structure and performance. Impact and collision forces can result in the fracturing of these beads into smaller pieces, which are difficult to recover at the end of a cultivation run. Various contaminating compounds may also bind in a non-specific manner to these beads, reducing the binding capacity of the resin for the product of interest (fouling). This study characterizes resin bead binding capacity (to monitor bead fouling), and resin bead volume distributions (to monitor bead fracture) for an XAD-16 adsorber resin used to capture epothilone produced during myxobacterial cultivations. Resin fouling was found to reduce the product binding capacity of the adsorber resin by 25–50%. Additionally, the degree of resin bead fracture was found to be dependent on the cultivation length and the impeller rotation rate. Microbial cultivations and harvesting processes should be designed in such a way to minimize bead fragmentation and fouling during cultivation to maximize the amount of resin and associated product harvested at the end of a run.     

Emerging strategies of bone and joint repair

The advances in biomedicine over the past decade have provided revolutionary insights into molecules that mediate cell proliferation and differentiation. Findings on the complex interplay of cells, growth factors, matrix molecules and cell adhesion molecules in the process of tissue patterning have vitalized the revolutionary approach of bioregenerative medicine and tissue engineering. Here we review the impact of recent work in this interdisciplinary field on the treatment of musculoskeletal disorders. This novel concept combines the transplantation of pluripotent stem cells, and the use of specifically tailored biomaterials, arrays of bioactive molecules and gene transfer technologies to direct the regeneration of pathologically altered musculoskeletal tissues.     

Emerging roles of Abl family tyrosine kinases in microbial pathogenesis

Abl family kinases are central regulators of multiple cellular processes controlling actin dynamics, proliferation and differentiation. Recent studies indicate that different pathogens highjack Abl kinase signalling to reorganize the host actin cytoskeleton and promote the tyrosine phosphorylation of four known bacterial and viral effector proteins. Abl signalling is implicated in such diverse processes as microbial invasion, viral release from host cells, actin-based motility, actin-rich pedestal formation and cell scattering. Thus, Abl kinases are emerging as crucial regulators of multiple pathological signalling cascades during infection. Therapeutic intervention against Abl kinase activity might be an effective and novel strategy to combat serious microbial diseases.     

Highlights in haem biosynthesis

The haem biosynthesis pathway continues to provide surprises, from the first enzyme, 5-aminolaevulinic acid synthase, the mRNA of which contains an iron-responsive element, to the last, ferrochelatase, that contains an iron sulphur cluster. 5-Aminolaevulinate dehydratases from animals are zinc-dependent enzymes while those from plants require magnesium. The first X-ray structure of a haem synthesis enzyme, porphobilinogen deaminase, has not only yielded clues about the mechanism of tetrapyrrole assembly but has also provided insight into the molecular basis of the human disease acute intermittent porphyria. Evidence is growing to suggest that a previously unsuspected alternative haem pathway may exist.     

Emerging strategies for expanding the toolbox of enzymes in biocatalysis

Expanding the repertoire of reactions available to enzymes is an enduring challenge in biocatalysis. Owing to the synthetic versatility of transition metals, metalloenzymes have been favored targets for achieving new catalytic functions. Although less well explored, enzymes lacking metal centers can also be effective catalysts for non-natural reactions, providing access to reaction modalities that compliment those available to metals. By understanding how these activation modes can reveal new functions, strategies can be developed to access novel biocatalytic reactions. This review will cover discoveries in the last two years which access catalytic reactions that go beyond the native repertoire of metal-free biocatalysts.     

Optimal resting-growth strategies of microbial populations in fluctuating environments

Bacteria spend most of their lifetime in non-growing states which allow them to survive extended periods of stress and starvation. When environments improve, they must quickly resume growth to maximize their share of limited nutrients. Cells with higher stress resistance often survive longer stress durations at the cost of needing more time to resume growth, a strong disadvantage in competitive environments. Here we analyze the basis of optimal strategies that microorganisms can use to cope with this tradeoff. We explicitly show that the prototypical inverse relation between stress resistance and growth rate can explain much of the different types of behavior observed in stressed microbial populations. Using analytical mathematical methods, we determine the environmental parameters that decide whether cells should remain vegetative upon stress exposure, downregulate their metabolism to an intermediate optimum level, or become dormant. We find that cell-cell variability, or intercellular noise, is consistently beneficial in the presence of extreme environmental fluctuations, and that it provides an efficient population-level mechanism for adaption in a deteriorating environment. Our results reveal key novel aspects of responsive phenotype switching and its role as an adaptive strategy in changing environments.     

Recent insights in microbial exopolysaccharide biosynthesis and engineering strategies

1. Download : Download high-res image (163KB)
2. Download : Download full-size image

     

Biosynthesis and functional role of haem O and haem A

Haem O and/or haem A are specifically synthesized for the haem-copper respiratory oxidases. A 17-carbon hydroxyethylfarnesyl chain at the pyrrole ring A of the haems seems essential for catalytic functions at the oxygen-reduction site. The discovery of haem O in the cytochrome bo complex from Escherichia coli was a breakthrough in the studies on haem A biosynthesis. Molecular biological and biochemical studies in the past three years demonstrated that the cyoE/ctaB/COX10 genes are indispensable for functional expression of the terminal oxidases and encode a novel enzyme haem O synthase (protohaem IX farnesyltransferase). It has recently been suggested that the ctaA gene adjacent to the ctaB-ctaCDEF gene cluster in Bacillus subtilis encodes haem A synthase (haem O monooxygenase). In this article, we review current knowledge of the genes for haem O and haem A biosyntheses, the location and regulation of haem O synthase, the possible enzymatic mechanism of farnesyl transfer to haem B and the possible roles of the farnesylated haems.     

Divergence in plant and microbial allocation strategies explains continental patterns in microbial allocation and biogeochemical fluxes

Allocation trade‐offs shape ecological and biogeochemical phenomena at local to global scale. Plant allocation strategies drive major changes in ecosystem carbon cycling. Microbial allocation to enzymes that decompose carbon vs. organic nutrients may similarly affect ecosystem carbon cycling. Current solutions to this allocation problem prioritise stoichiometric tradeoffs implemented in plant ecology. These solutions may not maximise microbial growth and fitness under all conditions, because organic nutrients are also a significant carbon resource for microbes. I created multiple allocation frameworks and simulated microbial growth using a microbial explicit biogeochemical model. I demonstrate that prioritising stoichiometric trade‐offs does not optimise microbial allocation, while exploiting organic nutrients as carbon resources does. Analysis of continental‐scale enzyme data supports the allocation patterns predicted by this framework, and modelling suggests large deviations in soil C loss based on which strategy is implemented. Therefore, understanding microbial allocation strategies will likely improve our understanding of carbon cycling and climate.     

Metaproteomic strategies and applications for gut microbial research

The human intestine hosts various complex microbial communities that are closely associated with multiple health and disease processes. Determining the composition and function of these microbial communities is critical to unveil disease mechanisms and promote human health. Recently, meta-omic strategies have been developed that use high-throughput techniques to provide a wealth of information, thus accelerating the study of gut microbes. Metaproteomics is a newly emerged analytical approach that aims to identify proteins on a large scale in complex environmental microbial communities (e.g., the gut microbiota). This review introduces the recent analytical strategies and applications of metaproteomics, with a focus on advances in gut microbiota research, including a discussion of the limitations and challenges of these approaches.