TonB-dependent transporters (TBDTs) are present in all gram-negative bacteria and mediate energy-dependent uptake of molecules that are too scarce or large to be taken up efficiently by outer membrane (OM) diffusion channels. This process requires energy that is derived from the proton motive force and delivered to TBDTs by the TonB-ExbBD motor complex in the inner membrane. Together with the need to preserve the OM permeability barrier, this has led to an extremely complex and fascinating transport mechanism for which the fundamentals, despite decades of research, are still unclear. In this review, we describe our current understanding of the transport mechanism of TBDTs, their potential role in the delivery of novel antibiotics, and the important contributions made by TBDT-associated (lipo)proteins. Expected final online publication date for the Annual Review of Microbiology, Volume 77 is September 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

TonB-Dependent Transport Across the Bacterial Outer Membrane.

The cofactor challenge in synthetic methylotrophy: bioengineering and industrial applications.

Regulation of glycogen metabolism is of vital importance in organisms of all three kingdoms of life. Although the pathways involved in glycogen synthesis and degradation are well known, many regulatory aspects around the metabolism of this polysaccharide remain undeciphered. Here, we used the unicellular cyanobacterium Synechocystis as a model to investigate how glycogen metabolism is regulated in nitrogen‐starved dormant cells, which entirely rely on glycogen catabolism to resume growth upon nitrogen repletion. We identified phosphoglucomutase 1 (PGM1) as a key regulatory point in glycogen metabolism, and post‐translational modification as an essential mechanism for controlling its activity. We could show that PGM1 is phosphorylated ata residue in the regulatory latch domain (Ser 47) during nitrogen starvation, which inhibits its activity. Inactivation of PGM1 by phosphorylation at Ser 47 prevents premature degradation of the glycogen stores and appears to be essential for survival of Synechocystis in the dormant state. Remarkably, this regulatory mechanism seems to be evolutionary conserved in PGM1 enzymes, from bacteria to humans.

Regulatory phosphorylation event of phosphoglucomutase 1 tunes its activity to regulate glycogen metabolism

DNA-amplicon-based microbiota profiling can estimate species diversity and abundance but cannot resolve genetic differences within individuals of the same species. Here we report the development of modular bacterial tags (MoBacTags) encoding DNA barcodes that enable tracking of near-isogenic bacterial commensals in an array of complex microbiome communities. Chromosomally integrated DNA barcodes are then co-amplified with endogenous marker genes of the community by integrating corresponding primer binding sites into the barcode. We use this approach to assess the contributions of individual bacterial genes to Arabidopsis thaliana root microbiota establishment with synthetic communities that include MoBacTag-labelled strains of Pseudomonas capeferrum. Results show reduced root colonization for certain mutant strains with defects in gluconic-acid-mediated host immunosuppression, which would not be detected with traditional amplicon sequencing. Our work illustrates how MoBacTags can be applied to assess scaling of individual bacterial genetic determinants in the plant microbiota. 

Chromosomal barcodes for simultaneous tracking of near-isogenic bacterial strains in plant microbiota.

Regulation of glycogen metabolism is of vital importance in organisms of all three kingdoms of life. Although the pathways involved in glycogen synthesis and degradation are well known, many regulatory aspects around the metabolism of this polysaccharide remain undeciphered. Here, we used the unicellular cyanobacterium Synechocystis as a model to investigate how glycogen metabolism is regulated in dormant nitrogen-starved cells, which entirely rely on glycogen catabolism to restore growth. We found that the activity of the enzymes involved in glycogen synthesis and degradation is tightly controlled at different levels via post-translational modifications. Phosphorylation of phosphoglucomutase 1 (Pgm1) on a peripheral residue (Ser63) regulates Pgm1 activity and controls the mobilization of the glycogen stores. Inhibition of Pgm1 activity via phosphorylation on Ser63 appears essential for survival of Synechocystis in the dormant state. Remarkably, this regulatory mechanism seems to be conserved from bacteria to humans. Moreover, phosphorylation of Pgm1 influences the formation of a metabolon, which includes Pgm1, oxidative pentose phosphate cycle protein (OpcA) and glucose-6-phosphate dehydrogenase (G6PDH). Analysis of the steady-state levels of the metabolic products of glycogen degradation together with protein-protein interaction studies revealed that the activity of G6PDH and the formation of this metabolon are under additional redox control, likely to ensure metabolic channeling of glucose-6-phosphate to the required pathways for each developmental stage. Significance statement In this study, we showed that post-translational modification of phosphoglucomutase 1 (Pgm1) via phosphorylation at a peripheral residue is a key, evolutionary-conserved regulatory mechanism that controls the utilization of the glycogen reserves. We identified Pgm1 as a central metabolic valve, associating with oxidative pentose phosphate cycle protein (OpcA) and glucose-6-phosphate dehydrogenase (G6PDH) into a metabolon. This interaction is regulated by the phosphorylation state of Pgm1 and the redox state of OpcA, and probably allows direction of the carbon flux into the required metabolic pathways.

/pdf/regulatory-phosphorylation-site-tunes-phosphoglucomutase-1-1qw0466b8j.pdf

Regulatory phosphorylation site tunes Phosphoglucomutase 1 as a metabolic valve to control mobilization of glycogen stores

Glucose is taken up by Escherichia coli through the phosphotransferase system (PTS) as the preferred carbon source. PTS mutants grow with glucose as a carbon source only in the presence of pyrroloquinoline quinone (PQQ), which is needed as a redox cofactor for the glucose dehydrogenase Gcd. The membrane-anchored Gcd enzyme oxidizes glucose to gluconolactone in the periplasm. For this reaction to occur, external supply of PQQ is required as E. coli is unable to produce PQQ de novo. Growth experiments show that PqqU (YncD) is the TonB-ExbBD dependent transporter for PQQ through the outer membrane. PQQ protected the cells from the PqqU dependent phage IsaakIselin (Bas10) by competition for the receptor protein. As a high affinity uptake system PqqU allows E. coli to activate Gcd even at surrounding PQQ concentrations of about 1 nmol/l. At about 30 fold higher PQQ concentrations the activation of Gcd gets PqqU independent. Due to its small size Pqq may also pass the outer membrane through porins. The PQQ dependent production of gluconate has been demonstrated in many plant growth promoting bacteria that solubilise phosphate minerals in the soil by secreting this acid. Under Pi limiting conditions also E. coli induces the glucose dehydrogenase and secretes gluconate, even in absence of PTS, that is, even when the bacterium is unable to grow on glucose without PQQ.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/mmi.14975

The TonB dependent uptake of pyrroloquinoline-quinone (PQQ) and secretion of gluconate by Escherichia coli K-12

The reactions of α-D-phosphohexomutases (αPHM) are ubiquitous, key to primary metabolism and essential for several processes in all domains of life. The functionality of these enzymes relies on an initial auto-phosphorylation step which requires the presence of α-D-glucose-1,6-bisphosphate (Glc-1,6-BP). While well investigated in vertebrates, the origin of this activator compound in bacteria is unknown. Here we show that the Slr1334 protein from the unicellular cyanobacterium Synechocysitis sp. PCC 6803 is a Glc-1,6-BP-synthase. Biochemical analysis revealed that Slr1334 efficiently converts fructose-1,6-bisphosphate and α-D-glucose-1-phosphate/α-D-glucose-6-phosphate into Glc-1,6-BP and also catalyzes the reverse reaction. Phylogenetic analysis revealed that the slr1334 product belongs to a primordial subfamily of αPHMs that is present especially in deeply branching bacteria and also includes human commensals and pathogens. Interestingly, the homologue of Slr1334 in the human gut bacterium Bacteroides salyersiae catalyzes the same reaction, suggesting a conserved and essential role for the members of this αPHM subfamily.

https://www.biorxiv.org/content/biorxiv/early/2021/12/02/2021.12.02.470922.full.pdf

Glucose-1,6-bisphosphate, a key metabolic regulator, is synthesized by a distinct family of α-D-phosphohexomutases widely distributed in prokaryotes

α-D-Carba-glucosamine (CGlcN) is a carbocyclic analog of α-D-glucosamine that inhibits growth of Bacillus subtilis and Staphylococcus aureus. CGlcN is internalized and concomitantly phosphorylated via the phosphotransferase system yielding α-D-carba-glucosamine-6-phosphate (CGlcN6P), which interferes with expression of the glutamine-fructose-6-phosphate amidotransferase (GlmS; glucosamine synthase) by activating the glmS riboswitch. Herein, we report that CGlcN6P is efficiently metabolized to carbasugar nucleotides along the peptidoglycan biosynthetic route. Mass spectrometric analysis confirmed the occurrence of carbocyclic peptidoglycan nucleotides UDP-carba-D-N-acetyl-glucosamine (UDP-CGlcNAc) and UDP-carba-D-N-acetylmuramic acid-pentapeptide (UDP-CMurNAc-5P) in the presence of CGlcN and revealed accumulation of these carba-metabolites upon antibiotic treatment interfering with biosynthetic enzyme functions. Thus, carbocyclic carbohydrates and nucleotide analogs are generated by the promiscuous bacterial cell wall biosynthetic enzymes and act as antimetabolites, causing bacterial growth inhibition by interference with cell wall synthesis. Our findings reveal CGlcN not only as putative antibiotic molecule with previously unknown antimetabolite mode of action, but also as tool to study the bacterial cell wall metabolism, e.g., in synergy with other antibiotics.

/pdf/metabolization-of-a-d-carba-glucosamine-in-vivo-generates-1ctyngp3.pdf

Metabolization of α-D-carba-glucosamine in vivo generates antimetabolites of cell wall precursors

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The TonB dependent uptake of pyrroloquinoline-quinone (PQQ) and secretion of gluconate by Escherichia coli K-12

Glucose-1,6-bisphosphate, a key metabolic regulator, is synthesized by a distinct family of α-D-phosphohexomutases widely distributed in prokaryotes

Metabolization of α-D-carba-glucosamine in vivo generates antimetabolites of cell wall precursors