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Bacterial microcompartments (BMCs) are protein-based organelles found across the bacterial tree of life. They consist of a shell, made of proteins that oligomerize into hexagonally and pentagonally shaped building blocks, that surrounds enzymes constituting a segment of a metabolic pathway. The proteins of the shell are unique to BMCs. They also provide selective permeability; this selectivity is dictated by the requirements of their cargo enzymes. We have recently surveyed the wealth of different BMC types and their occurrence in all available genome sequence data by analyzing and categorizing their components found in chromosomal loci using HMM (Hidden Markov Model) protein profiles. To make this a “do-it yourself” analysis for the public we have devised a webserver, BMC Caller (https://bmc-caller.prl.msu.edu), that compares user input sequences to our HMM profiles, creates a BMC locus visualization, and defines the functional type of BMC, if known. Shell proteins in the input sequence data are also classified according to our function-agnostic naming system and there are links to similar proteins in our database as well as an external link to a structure prediction website to easily generate structural models of the shell proteins, which facilitates understanding permeability properties of the shell. Additionally, the BMC Caller website contains a wealth of information on previously analyzed BMC loci with links to detailed data for each BMC protein and phylogenetic information on the BMC shell proteins. Our tools greatly facilitate BMC type identification to provide the user information about the associated organism’s metabolism and enable discovery of new BMC types by providing a reference database of all currently known examples.

Bacterial microcompartments (BMCs) are proteinaceous prokaryotic organelles that enable the utilization of substrates such as 1,2-propanediol and ethanolamine. BMCs are mostly linked to the survival of particular pathogenic bacteria by providing a growth advantage through utilization of 1,2-propanediol and ethanolamine which are abundantly present in the human gut. Although a 1,2-propanediol utilization cluster was found in the probiotic bacteriumPropionibacterium freudenreichii, BMC-mediated metabolism of 1,2-propanediol has not been demonstrated experimentally inP. freudenreichii. In this study we show thatP. freudenreichiiDSM 20271 metabolizes 1,2-propanediol in anaerobic conditions to propionate and 1-propanol. Furthermore, 1,2-propanediol induced the formation of BMCs, which were visualized by transmission electron microscopy and resembled BMCs found in other bacteria. Proteomic analysis of 1,2-propanediol grown cells compared toL-lactate grown cells showed significant upregulation of proteins involved in propanediol-utilization (pdu-cluster), DNA repair mechanisms and BMC shell proteins while proteins involved in oxidative phosphorylation were down-regulated. 1,2-Propanediol utilizing cells actively produced vitamin B12(cobalamin) in similar amounts as cells growing onL-lactate. The ability to metabolize 1,2-propanediol may have implications for human gut colonization and modulation, and can potentially aid in delivering propionate and vitamin B12in situ.

Bacterial microcompartments (BMCs) are proteinaceous organelles that optimize specific metabolic pathways referred to as metabolosomes involving transient production of toxic volatile metabolites such as aldehydes. Previous bioinformatics analysis predicted the presence of BMCs in 23 bacterial phyla including foodborne pathogens and a link with gene clusters for the utilization of host-derived substrates such as 1,2-propanediol utilization, i.e., the Pdu cluster. Although, transcriptional regulation of the Pdu cluster and its role in Listeria monocytogenes virulence in animal models have recently been reported, the experimental identification and the physiological role of BMCs in L. monocytogenes is still unexplored. Here, we ask whether BMCs could enable utilization of 1,2-propanediol (Pd) in L. monocytogenes under anaerobic conditions. Using L. monocytogenes EGDe as a model strain, we could demonstrate efficient utilization of Pd with concomitant production of 1-propanol and propionate after 24 h of anaerobic growth, while the utilization was significantly reduced in aerobic conditions. In line with this, expression of genes encoding predicted shell proteins and the signature enzyme propanediol dehydratase is upregulated more than 20-fold in cells anaerobically grown in Pdu-induced versus non-induced control conditions. Additional proteomics analysis confirmed the presence of BMC shell proteins and Pdu enzymes in cells that show active degradation of Pd. Furthermore, using transmission electron microscopy, BMC structures have been detected in these cells linking gene expression, protein composition, and BMCs to activation of the Pdu cluster in anaerobic growth of L. monocytogenes. Studies in defined minimal medium with Pd as an energy source showed a significant increase in cell numbers, indicating that Pdu and the predicted generation of ATP in the conversion of propionyl-phosphate to the end product propionate can support anaerobic growth of L. monocytogenes. Our findings may suggest a role for BMC-dependent utilization of Pd in L. monocytogenes growth, transmission, and interaction with the human host.

Urinary tract infections (UTIs) are common and in general are caused by intestinal uropathogenic Escherichia coli (UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels the competitive overgrowth of intestinal E. coli, both pathogenic enterohemorrhagic E. coli and commensal strains. During a UTI, urease-negative E. coli bacteria thrive, despite the comparative nutrient limitation in urine., Urinary tract infections (UTIs) are common and in general are caused by intestinal uropathogenic Escherichia coli (UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels the competitive overgrowth of intestinal E. coli, both pathogenic enterohemorrhagic E. coli and commensal strains. During a UTI, urease-negative E. coli bacteria thrive, despite the comparative nutrient limitation in urine. The role of ethanolamine as a potential nutrient source during UTIs is understudied. We evaluated the role of the metabolism of ethanolamine as a potential nitrogen and carbon source for UPEC in the urinary tract. We analyzed infected urine samples by culture, high-performance liquid chromatography, reverse transcription-quantitative PCR, and genomic sequencing. The ethanolamine concentration in urine was comparable to the concentration of the most abundant reported urinary amino acid, d-serine. Transcription of the eut operon was detected in the majority of urine samples containing E. coli screened. All sequenced UPEC strains had conserved eut operons, while metabolic genotypes previously associated with UTI (dsdCXA, metE) were mainly limited to phylogroup B2. In vitro ethanolamine was found to be utilized as a sole source of nitrogen by UPEC strains. The metabolism of ethanolamine in artificial urine medium (AUM) induced metabolosome formation and provided a growth advantage at the physiological levels found in urine. Interestingly, eutE (which encodes acetaldehyde dehydrogenase) was required for UPEC strains to utilize ethanolamine to gain a growth advantage in AUM, suggesting that ethanolamine is also utilized as a carbon source. These data suggest that urinary ethanolamine is a significant additional carbon and nitrogen source for infecting E. coli strains.

Bacterial microcompartments are self-assembling subcellular structures surrounded by a semipermeable protein shell and found only in bacteria, but not archaea or eukaryotes. The general functions of the bacterial microcompartments are to concentrate enzymes, metabolites, and cofactors for multistep pathways; maintain the cofactor ratio; protect the cell from toxic metabolic intermediates; and protect the encapsulated pathway from unwanted side reactions. The bacterial microcompartments were suggested to play a significant role in organisms of the human gut microbiome, especially for various pathogens. Here, we used a comparative genomics approach to analyze the bacterial microcompartments in 646 individual genomes of organisms commonly found in the human gut microbiome. The bacterial microcompartments were found in 150 (23.2%) analyzed genomes. These microcompartments include previously known ones for the utilization of ethanolamine, 1,2-propanediol, choline, and fucose/rhamnose. Moreover, we reconstructed two novel pathways associated with the bacterial microcompartments. These pathways are catabolic pathways for the utilization of 1-amino-2-propanol/1-amino-2-propanone and xanthine. Remarkably, the xanthine utilization pathway does not demonstrate similarity to previously known microcompartment-associated pathways. Thus, we describe a novel type of bacterial microcompartment.

The use of bacterial production could improve commercial feasibility for biochemicals such as pharmaceuticals and natural products. However, many non-native reactions in bacterial hosts have unacceptably low yields due to pathway toxicity or competition with other cellular processes. Compartmentalization is a potential solution to these problems, but compartmentalized organelles were long thought to be found only in eukaryotic cells. Remarkably, bacterial microcompartments (MCPs) are protein-based organelles that perform this sequestration function. We aim to apply these MCPs for metabolic engineering and the enhancement of biosynthesis pathways that do not function optimally in the cytosol. MCPs may also serve as a scaffold for application outside of the cell. To achieve this goal, we characterized the stability of MCPs when exposed to a range of pH and temperature to define the limitations of this complex structure. We next demonstrated that it is possible to form chimeric MCPs by combining shell proteins from different systems in nature. Importantly, we found that these changes to shell protein structure can enhance the function of the encapsulated pathway. We discovered that pathway performance can also be improved by a single point mutation to the pore of a shell protein, likely altering small molecule diffusion across the shell. We are investigating the role of shell protein pore chemistry in tuning small molecule diffusion. Finally, we test signal sequence peptides at the C-terminus of proteins and find that variation of signal peptide sequence results in altered expression level and loading within MCPs. In total, these tools improve our ability to design custom bacterial organelles to control the partitioning of chemical reactions within cells.

A certain type of bacterial inclusion, known as a bacterial microcompartment, was recently identified and imaged through cryo-electron tomography. A reconstructed 3D object from single-axis limited angle tilt-series cryo-electron tomography contains missing regions and this problem is known as the missing wedge problem. Due to missing regions on the reconstructed images, analyzing their 3D structures is a challenging problem. The existing methods overcome this problem by aligning and averaging several similar shaped objects. These schemes work well if the objects are symmetric and several objects with almost similar shapes and sizes are available. Since the bacterial inclusions studied here are not symmetric, are deformed, and show a wide range of shapes and sizes, the existing approaches are not appropriate. This research develops new statistical methods for analyzing geometric properties, such as volume, symmetry, aspect ratio, polyhedral structures etc., of these bacterial inclusions in presence of missing data. These methods work with deformed and non-symmetric varied shaped objects and do not necessitate multiple objects for handling the missing wedge problem. The developed methods and contributions include: (a) an improved method for manual image segmentation, (b) a new approach to 'complete' the segmented and reconstructed incomplete 3D images, (c) a polyhedral structural distance model to predict the polyhedral shapes of these microstructures, (d) a new shape descriptor for polyhedral shapes, named as polyhedron profile statistic, and (e) the Bayes classifier, linear discriminant analysis and support vector machine based classifiers for supervised incomplete polyhedral shape classification. Finally, the predicted 3D shapes for these bacterial microstructures belong to the Johnson solids family, and these shapes along with their other geometric properties are important for better understanding of their chemical and biological characteristics.

Cytochrome P450 (CYP) 3A4 is a membrane protein that catalyzes hydroxylation of endogenous and exogenous substrates. Protein-protein interaction is a crucial factor that regulates the function of enzymes. However, protein-protein interactions involving human CYPs have not been fully understood. In this study, extensive protein-protein interactions involving CYP3A4 were determined by a shotgun analysis of immunoprecipitate utilizing anti-CYP3A4 antibody. Our shotgun analysis revealed that 149 proteins were immunoprecipitated with anti-CYP3A4 antibody in human liver microsomes. We further determined that 51 proteins of 149 proteins were specifically immunoprecipitated with the anti-CYP3A4 antibody. Our analysis demonstrated that other CYP isoforms are interacting with CYP3A4, which is in agreement with previous findings. Based on our current and previous findings, we propose that drug-metabolizing enzymes such as CYP3A4 and UDP-glucuronosyltransferase 2B7 form a metabolosome, which is a functional unit of metabolism consisting of multiple metabolism-related proteins.

Ethanolamine is used as an energy source by phylogenetically diverse bacteria including pathogens, by the concerted action of proteins from the eut-operon. Previous studies have revealed the presence of eutBC genes encoding ethanolamine-ammonia lyase, a key enzyme that breaks ethanolamine into acetaldehyde and ammonia, in about 100 bacterial genomes including members of gamma-proteobacteria. However, ethanolamine utilization has not been reported for any member of the Vibrio genus. Our comparative genomics study reveals the presence of genes that are involved in ethanolamine utilization in several Vibrio species. Using Vibrio alginolyticus as a model system we demonstrate that ethanolamine is better utilized as a nitrogen source than as a carbon source. Reviewers This article was reviewed by Dr. Lakshminarayan Iyer and Dr. Vivek Anantharaman (nominated by Dr. L Aravind).