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Bacterial microcompartments (MCPs) are widespread protein-based organelles composed of metabolic enzymes encapsulated within a protein shell. The function of MCPs is to optimize metabolic pathways by confining toxic and/or volatile pathway intermediates. A major class of MCPs known as glycyl radical MCPs has only been partially characterized. Here, we show that uropathogenic Escherichia coli CFT073 uses a glycyl radical MCP for 1,2-propanediol (1,2-PD) fermentation. Bioinformatic analyses identified a large gene cluster (named grp for glycyl radical propanediol) that encodes homologs of a glycyl radical diol dehydratase, other 1,2-PD catabolic enzymes, and MCP shell proteins. Growth studies showed that E. coli CFT073 grows on 1,2-PD under anaerobic conditions but not under aerobic conditions. All 19 grp genes were individually deleted, and 8/19 were required for 1,2-PD fermentation. Electron microscopy and genetic studies showed that a bacterial MCP is involved. Bioinformatics combined with genetic analyses support a proposed pathway of 1,2-PD degradation and suggest that enzymatic cofactors are recycled internally within the Grp MCP. A two-component system (grpP and grpQ) is shown to mediate induction of the grp locus by 1,2-PD. Tests of the E. coli Reference (ECOR) collection indicate that >10% of E. coli strains ferment 1,2-PD using a glycyl radical MCP. In contrast to other MCP systems, individual deletions of MCP shell genes (grpE, grpH, and grpI) eliminated 1,2-PD catabolism, suggesting significant functional differences with known MCPs. Overall, the studies presented here are the first comprehensive genetic analysis of a Grp-type MCP. IMPORTANCE Bacterial MCPs have a number of potential biotechnology applications and have been linked to bacterial pathogenesis, cancer, and heart disease. Glycyl radical MCPs are a large but understudied class of bacterial MCPs. Here, we show that uropathogenic E. coli CFT073 uses a glycyl radical MCP for 1,2-PD fermentation, and we conduct a comprehensive genetic analysis of the genes involved. Studies suggest significant functional differences between the glycyl radical MCP of E. coli CFT073 and better-studied MCPs. They also provide a foundation for building a deeper general understanding of glycyl radical MCPs in an organism where sophisticated genetic methods are available.

Bacterial microcompartments (MCPs) are protein-based organelles that consist of metabolic enzymes encapsulated within a protein shell. The function of MCPs is to optimize metabolic pathways by increasing reaction rates and sequestering toxic pathway intermediates. A substantial amount of effort has been directed toward engineering synthetic MCPs as intracellular nanoreactors for the improved production of renewable chemicals. A key challenge in this area is engineering protein shells that allow the entry of desired substrates. In this study, we used site-directed mutagenesis of the PduT shell protein to remove its central iron–sulfur cluster and create openings (pores) in the shell of the Pdu MCP that have varied chemical properties. Subsequently, in vivo and in vitro studies were used to show that PduT-C38S and PduT-C38A variants increased the diffusion of 1,2-propanediol, propionaldehyde, NAD(+) and NADH across the shell of the MCP. In contrast, PduT-C38I and PduT-C38W eliminated the iron–sulfur cluster without altering the permeability of the Pdu MCP, suggesting that the side-chains of C38I and C38W occluded the opening formed by removal of the iron–sulfur cluster. Thus, genetic modification offers an approach to engineering the movement of larger molecules (such as NAD/H) across MCP shells, as well as a method for blocking transport through trimeric bacterial microcompartment (BMC) domain shell proteins.

Bacterial microcompartments (MCPs) are complex organelles that consist of metabolic enzymes encapsulated within a protein shell. In this study, we investigate the function of the PduJ MCP shell protein. PduJ is 80% identical in amino acid sequence to PduA and both are major shell proteins of the 1,2-propanediol (1,2-PD) utilization (Pdu) MCP of Salmonella. Prior studies showed that PduA mediates the transport of 1,2-PD (the substrate) into the Pdu MCP. Surprisingly, however, results presented here establish that PduJ has no role 1,2-PD transport. The crystal structure revealed that PduJ was nearly identical to that of PduA and, hence, offered no explanation for their differential functions. Interestingly, however, when a pduJ gene was placed at the pduA chromosomal locus, the PduJ protein acquired a new function, the ability to mediate 1,2-PD transport into the Pdu MCP. To our knowledge, these are the first studies to show that that gene location can determine the function of a MCP shell protein. We propose that gene location dictates protein-protein interactions essential to the function of the MCP shell.

Bacterial choline degradation in the human gut has been associated with cancer and heart disease. In addition, recent studies found that a bacterial microcompartment is involved in choline utilization by Proteus and Desulfovibrio species. However, many aspects of this process have not been fully defined. Here, we investigate choline degradation by the uropathogen Escherichia coli 536. Growth studies indicated E. coli 536 degrades choline primarily by fermentation. Electron microscopy indicated that a bacterial microcompartment was used for this process. Bioinformatic analyses suggested that the choline utilization ( cut ) gene cluster of E. coli 536 includes two operons, one containing three genes and a main operon of 13 genes. Regulatory studies indicate that the cutX gene encodes a positive transcriptional regulator required for induction of the main cut operon in response to choline supplementation. Each of the 16 genes in the cut cluster was individually deleted, and phenotypes were examined. The cutX , cutY , cutF , cutO , cutC , cutD , cutU , and cutV genes were required for choline degradation, but the remaining genes of the cut cluster were not essential under the conditions used. The reasons for these varied phenotypes are discussed. IMPORTANCE Here, we investigate choline degradation in E. coli 536. These studies provide a basis for understanding a new type of bacterial microcompartment and may provide deeper insight into the link between choline degradation in the human gut and cancer and heart disease. These are also the first studies of choline degradation in E. coli 536, an organism for which sophisticated genetic analysis methods are available. In addition, the cut gene cluster of E. coli 536 is located in pathogenicity island II (PAI-II 536 ) and hence might contribute to pathogenesis.

DD-carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the dd-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (ΔdacAdacC) of E. coli, strengthening its physiology as a dd-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours dd-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities.

SUMMARY Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles.

Outer membrane vesicles (OMVs) are released from Pseudomonas syringae pv. tomato T1 (Pst T1) during their normal growth. These extracellular compartments are comprised of a complete set of biological macromolecules that includes proteins, lipids, lipopolysaccharides, etc. It is evident from proteomics analyses the OMVs of Pst T1 contain membrane- and virulence-associated proteins. In addition, OMVs of this organism are also associated with phytotoxin, coronatine. Therefore, OMVs of Pst T1 must play a significant role during pathogenicity to host plant. However, further studies are required whether these structures can serve as "vehicles" for the transport of virulence factors into the host membrane.

Bacterial microcompartments (MCPs) are extremely large proteinaceous organelles that consist of an enzymatic core encapsulated within a complex protein shell. A key question in MCP biology is the nature of the interactions that guide the assembly of thousands of protein subunits into a well-ordered metabolic compartment. In this report, we show that the N-terminal 37 amino acids of the PduB protein have a critical role in binding the shell of the 1,2-propanediol utilization (Pdu) microcompartment to its enzymatic core. Several mutations were constructed that deleted short regions of the N terminus of PduB. Growth tests indicated that three of these deletions were impaired MCP assembly. Attempts to purify MCPs from these mutants, followed by gel electrophoresis and enzyme assays, indicated that the protein complexes isolated consisted of MCP shells depleted of core enzymes. Electron microscopy substantiated these findings by identifying apparently empty MCP shells but not intact MCPs. Analyses of 13 site-directed mutants indicated that the key region of the N terminus of PduB required for MCP assembly is a putative helix spanning residues 6 to 18. Considering the findings presented here together with prior work, we propose a new model for MCP assembly. IMPORTANCE Bacterial microcompartments consist of metabolic enzymes encapsulated within a protein shell and are widely used to optimize metabolic process. Here, we show that the N-terminal 37 amino acids of the PduB shell protein are essential for assembly of the 1,2-propanediol utilization microcompartment. The results indicate that it plays a key role in binding the outer shell to the enzymatic core. We propose that this interaction might be used to define the relative orientation of the shell with respect to the core. This finding is of fundamental importance to our understanding of microcompartment assembly and may have application to engineering microcompartments as nanobioreactors for chemical production.

Of the five dd-carboxypeptidases in Escherichia coli, only PBP5 demonstrates its physiological significance by maintaining cell shape and intrinsic beta-lactam resistance. DacD can partially compensate for the lost beta-lactam resistance in PBP5 mutant, although its biochemical reason is unclear. To understand the mechanism(s) underlying such behaviour, we constructed soluble DacD (sDacD) and compared its biophysical and biochemical properties with those of sPBP5, in vitro. Unlike sPBP6, sDacD can deacylate Bocillin significantly, which is very similar to sPBP5. sDacD shows weak dd-carboxypeptidase activity, although lower than that of sPBP5. Bioinformatics analyses reveal a similar architecture of sPBP5 and sDacD. Therefore, based on the obtained results we can infer that biochemically DacD and PBP5 are more closely related to each other than to PBP6, enabling DacD and PBP5 to play a nearly similar physiological function in terms of recovering the lost beta-lactam resistance.

Out of the four DD-carboxypeptidases (DD-CPases) in Escherichia coli, only penicillin-binding protein (PBP) 5 performs physiological functions such as maintaining cell shape; its nearest homolog, PBP6, cannot perform such functions. Moreover, unlike PBP6, PBP5 efficiently processes both beta-lactam, and peptide substrates. The crystal structure of PBP5 reveals strong inter-residue hydrogen-bonding interactions around the active site, which favor its catalytic activity. However, the recently solved crystal structure of PBP6 cannot explain the reason for the observed functional discrepancies between the two proteins. Enzymatic analyses indicate that moving the morphology maintenance domain from one protein to another can alter the affinities and activities of PBP5 and 6 toward their substrates. To determine why the activities of these enzymes differ, we used molecular modeling, and docking analyses with substrate-mimetic ligands to estimate how amino-acid alterations in the morphology maintenance domain would affect the structure of PBP and hence its substrate specificity. The results obtained from kinetic analyses were directly correlated to the three-dimensional structures of the PBPs determined through in silico analyses, indicating a change in the active-site microarchitectures of PBP5 and 6 as a plausible cause of the difference in their biochemical behaviors.

Penicillin-binding protein (PBP) 5 plays a critical role in maintaining normal cellular morphology in mutants of Escherichia coli lacking multiple PBPs. The most closely related homologue, PBP 6, is 65% identical to PBP 5, but is unable to substitute for PBP 5 in returning these mutants to their wild-type shape. The relevant differences between PBPs 5 and 6 are localized in a 20-amino acid stretch of domain I in these proteins, which includes the canonical KTG motif at the active site. We determined how these differences affected the enzymatic properties of PBPs 5 and 6 toward beta-lactam binding and the binding and hydrolysis of two peptide substrates. We also investigated the enzymatic properties of recombinant fusion proteins in which active site segments were swapped between PBPs 5 and 6. The results suggest that the in vivo physiological role of PBP 5 is distinguished from PBP 6 by the higher degree of DD-carboxypeptidase activity of the former.

Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.

Antarctic bacteria are adapted to the extremely low temperature. The transcriptional and translational machineries of these bacteria are adapted to the sub-zero degrees of temperature. Studies directed towards identifying the changes in the protein profiles during changes in the growth temperatures of an Antarctic bacterium Pseudomonas syringae Lz4W may help in understanding the molecular basis of cold adaptation. In this study, subcellular fractionation methods of proteins were used for the enrichment and identification of proteins including low abundance proteins. The membrane proteins of the bacterium P. syringae Lz4W were prepared employing sucrose density gradient method. The proteins were separated through 2D gel-electrophoresis with the pH ranges 3-10, 4-7 and 5-8 using the detergent, amidosulfobetaine (ASB-14). The proteins separated on the 1D SDS PAGE and 2D gels were identified with the help of LC-ESI MS/MS and MALDI TOF TOF using bioinformatic programs MASCOT and SEQUEST. Since the genome sequence of P. syringae Lz4W is not available, the proteins are identified by using the genome database of the Pseudomonas sp. available at NCBI. The present studies focus on identifying temperature dependent expression of proteins by employing LC-MS/MS method and the functional significance of these proteins is discussed.

Escherichia coli PBP5, PBP6 and DacD, encoded by dacA, dacC and dacD, respectively, share substantial amino acid identity and together constitute ~50 % of the total penicillin-binding proteins of E. coli. PBP5 helps maintain intrinsic β-lactam resistance within the cell. To test if PBP6 and DacD play simlar roles, we deleted dacC and dacD individually, and dacC in combination with dacA, from E. coli 2443 and compared β-lactam sensitivity of the mutants and the parent strain. β-Lactam resistance was complemented by wild-type, but not dd-carboxypeptidase-deficient PBP5, confirming that enzymic activity of PBP5 is essential for β-lactam resistance. Deletion of dacC and expression of PBP6 during exponential or stationary phase did not alter β-lactam resistance of a dacA mutant. Expression of DacD during mid-exponential phase partially restored β-lactam resistance of the dacA mutant. Therefore, PBP5 dd-carboxypeptidase activity is essential for intrinsic β-lactam resistance of E. coli and DacD can partially compensate for PBP5 in this capacity, whereas PBP6 cannot.

Escherichia coli penicillin-binding protein 5 (PBP5), a dd-carboxypeptidase encoded by the dacA gene, plays a key role in the maintenance of cell shape. Although PBP5 shares one of the highest copy numbers among the PBPs, it is not essential for cell survival. To determine the effect of this redundant PBP on beta-lactam antibiotic susceptibility, PBP5 was deleted from O-antigen-negative E. coli K-12 (CS109) and O8-antigen-positive E. coli 2443, thus creating strains AM15-1 and AG1O5-1, respectively. Compared with the parent strains, both mutants were four- to eight-fold more susceptible to all the beta-lactam antibiotics tested. Reversion to beta-lactam resistance was observed in the mutants upon complementing with cloned PBP5, indicating the involvement of PBP5 in maintaining an O-antigen-independent intrinsic beta-lactam resistance in E. coli cells. To check whether other dacA homologues were able to substitute this behaviour of E. coli PBP5, AG1O5-1 was complemented with its nearest dacA homologues (Salmonella enterica serovar Typhimurium LT2, Vibrio cholerae and Haemophilus influenzae). All of the cloned homologues were capable of restoring the lost beta-lactam resistance in AG1O5-1, either completely or at least partially. Therefore, apart from maintaining cell shape, involvement of PBP5 in maintaining intrinsic beta-lactam resistance is an important physiological observation and we speculate that such a strategy of deleting PBP5 may be helpful to introduce beta-lactam susceptibility in the laboratory.

Bacterial cell shape is, in part, mediated by the peptidoglycan (murein) sacculus. Penicillin-binding proteins (PBPs) catalyze the final stages of murein biogenesis and are the targets of beta-lactam antibiotics. Several low molecular mass PBPs including PBP4, PBP5, PBP6 and DacD seem to possess DD-carboxypeptidase (DD-CPase) activity, but these proteins are dispensable for survival in laboratory culture. The physiological functions of DD-CPases in vivo are unresolved and it is unclear why bacteria retain these seemingly non-essential and enzymatically redundant enzymes. However, PBP5 clearly contributes to maintenance of cell shape in some PBP mutant backgrounds. In this review, we focus on recent findings concerning the physiological functions of the DD-CPases in vivo, identify gaps in the current knowledge of these proteins and suggest some possible courses for future study that might help reconcile current models of bacterial cell morphology.

Escherichia coli (E. coli) has four known DD-carboxypeptidases (DD-CPases), namely penicillin-binding protein (PBP) 4, 5, 6, and DacD, and are thought to restrict the unwanted crosslink formation between the nascent peptidoglycan strands. So far, the in vivo functions of these enzymes are mostly unknown, except for PBP 5, which helps maintain cell shape. In spite of high degree of identity, PBP 6 is unable to imitate the shape maintaining activity of PBP 5. A 20 amino acids (aa) segment near to the KTG motif forms the morphology maintenance domain (MMD) in PBP 5 and the replacement of the corresponding amino acids of PBP 6 with that of PBP 5 MMD restores normal morphology in E. coli.