Mannose Transporter Research Articles

Transcriptional regulation of cellobiose utilization by PRD-domain containing Sigma54-dependent transcriptional activator (CelR) and catabolite control protein A (CcpA) in Bacillus thuringiensis.

Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin resulting from the enzymatic hydrolysis of cellulose. It is a major source of carbon for soil bacteria. In bacteria, the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), encoded by the cel operon, is responsible for the transport and utilization of cellobiose. In this study, we analyzed the transcription and regulation of the cel operon in Bacillus thuringiensis (Bt). The cel operon is composed of five genes forming one transcription unit. β-Galactosidase assays revealed that cel operon transcription is induced by cellobiose, controlled by Sigma54, and positively regulated by CelR. The HTH-AAA+ domain of CelR recognized and specifically bound to three possible binding sites in the celA promoter region. CelR contains two PTS regulation domains (PRD1 and PRD2), which are separated by two PTS-like domains-the mannose transporter enzyme IIA component domain (EIIAMan) and the galactitol transporter enzyme IIB component domain (EIIBGat). Mutations of His-546 on the EIIAMan domain and Cys-682 on the EIIBGat domain resulted in decreased transcription of the cel operon, and mutations of His-839 on PRD2 increased transcription of the cel operon. Glucose repressed the transcription of the cel operon and catabolite control protein A (CcpA) positively regulated this process by binding the cel promoter. In the celABCDE and celR mutants, PTS activities were decreased, and cellobiose utilization was abolished, suggesting that the cel operon is essential for cellobiose utilization. Bt has been widely used as a biological pesticide. The metabolic properties of Bt are critical for fermentation. Nutrient utilization is also essential for the environmental adaptation of Bt. Glucose is the preferred energy source for many bacteria, and the presence of the phosphotransferase system allows bacteria to utilize other sugars in addition to glucose. Cellobiose utilization pathways have been of particular interest owing to their potential for developing alternative energy sources for bacteria. The data presented in this study improve our understanding of the transcription patterns of cel gene clusters. This will further help us to better understand how cellobiose is utilized for bacterial growth.

Plasticity of growth laws tunes resource allocation strategies in bacteria.

Bacteria like E. coli grow at vastly different rates on different substrates, however, the precise reason for this variability is poorly understood. Different growth rates have been attributed to 'nutrient quality', a key parameter in bacterial growth laws. However, it remains unclear to what extent nutrient quality is rooted in fundamental biochemical constraints like the energy content of nutrients, the protein cost required for their uptake and catabolism, or the capacity of the plasma membrane for nutrient transporters. Here, we show that while nutrient quality is indeed reflected in protein investment in substrate-specific transporters and enzymes, this is not a fundamental limitation on growth rate, at least for certain 'poor' substrates. We show that it is possible to turn mannose, one of the 'poorest' substrates of E. coli, into one of the 'best' substrates by reengineering chromosomal promoters of the mannose transporter and metabolic enzymes required for mannose degradation. This result falls in line with previous observations of more subtle growth rate improvement for many other carbon sources. However, we show that this faster growth rate comes at the cost of diverse cellular capabilities, reflected in longer lag phases, worse starvation survival and lower motility. We show that addition of cAMP to the medium can rescue these phenotypes but imposes a corresponding growth cost. Based on these data, we propose that nutrient quality is largely a self-determined, plastic property that can be modulated by the fraction of proteomic resources devoted to a specific substrate in the much larger proteome sector of catabolically activated genes. Rather than a fundamental biochemical limitation, nutrient quality reflects resource allocation decisions that are shaped by evolution in specific ecological niches and can be quickly adapted if necessary.

Synthesis of Neuraminidase-Resistant Sialyllactose Mimetics from N-Acyl Mannosamines using Metabolically Engineered Escherichia coli.

Herein, we describe the efficient gram-scale synthesis of α2,3- and α2,6-sialyllactose oligosaccharides as well as mimetics from N-acyl mannosamines and lactose in metabolically engineered bacterial cells grown at high cell density. We designed new Escherichia coli strains co-expressing sialic acid synthase and N-acylneuraminate cytidylyltransferase from Campylobacter jejuni together with the α2,3-sialyltransferase from Neisseria meningitidis or the α2,6-sialyltransferase from Photobacterium sp. JT-ISH-224. Using their mannose transporter, these new strains actively internalized N-acetylmannosamine (ManNAc) and its N-propanoyl (N-Prop), N-butanoyl (N-But) and N-phenylacetyl (N-PhAc) analogs and converted them into the corresponding sialylated oligosaccharides, with overall yields between 10 % and 39 % (200-700 mg.L-1 of culture). The three α2,6-sialyllactose analogs showed similar binding affinity for Sambucus nigra SNA-I lectin as for the natural oligosaccharide. They also proved to be stable competitive inhibitors of Vibrio cholerae neuraminidase. These N-acyl sialosides therefore hold promise for the development of anti-adhesion therapy against influenza viral infections.

Antimicrobial activity and mechanism of Magnolia officinalis root extract against methicillin-resistant Staphylococcus aureus based on mannose transporter

Magnolia officinalis root extract was obtained by high-pressure-assisted extraction, and the yield of honokiol (HNK) was 86.97 ± 1.05 mg/g dry power. The purified extracts and HNK inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA). Assays for alkaline phosphatases, proteins, nucleic acids leakage, and electrical conductivity, along with scanning and transmission electron microscopy measurements, confirmed the damaging effects of HNK on the cell wall and membrane of MRSA. Intake of sugar, protein production, molecular docking, and dynamic simulations revealed that HNK interfered with the transport of mannose-based carbohydrates by binding to VAL196, ILE291, ALA293, PRO294, and MET214 on the mannose transporter (ManP) in the phosphotransferase system, and thereby inhibited the carbohydrate metabolism of MRSA. These results indicated that HNK prevented MRSA from using carbohydrates by cutting off its nutritional process, and ultimately caused bacterial rupture and lysis. Therefore, ManP may be a potential target for HNK to combat MRSA infections.

The beneficial effects of polysaccharide obtained from persimmon (Diospyros kaki L.) on the proliferation of Lactobacillus and gut microbiota

The objective of this study was to investigate the effect of polysaccharide extracts from persimmon (PPE) on the proliferation of Lactobacillus and the gut microbiota of mice. Lactobacillus strains were cultured in medium containing PPE, and differential gene expression was evaluated using transcriptomics. In addition, 16S rDNA was employed to analyze the abundance and diversity of fecal colonies in mice, and the influence of PPE on the intestinal flora in mice was further examined. The results showed that Lactobacillus acidophilus NCFM and Lactobacillus acidophilus CICC 6075 could proliferate in PPE medium. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway analysis indicated that glucose metabolism-related genes, such as phosphoyruvate hydratase (eno) and PTS mannose transporter subunit IIAB (manX), were up-regulated. The metabolic pathways of fructose and mannose were also significantly up-regulated. After gavage of mice with PPE, 16S rDNA sequencing of mouse feces indicated that the beneficial bacteria in the intestines proliferated and the abundance of harmful bacteria was reduced. PPE can maintain the balance of intestinal microorganisms in mice. Therefore, PPE has a significant positive effect on both Lactobacillus proliferation and gut microbiota of mice.

Molecular docking and in silico studies of the physicochemical properties of potential inhibitors for the phosphotransferase system of Streptococcus mutans

This study identified potential inhibitory compounds of the phosphoenolpyruvate-sugar.Phosphotransferase system of S. mutans, specifically enzyme II mannose transporter (EIIMan) in its subunits IIA, IIB and IIC by means of a selection protocol and in silico molecular analysis.Intervening the phosphotransferase system would compromise the physiological behavior and the pathogenic expression of S. mutans, and possibly other acidogenic bacteria that use phosphotransferases in their metabolism—making the phosphotransferase system a therapeutic target for the selective control of acidogenic microorganisms in caries control.Several computational techniques were used to evaluate molecular, physicochemical, and toxicological aspects of various compounds. Molecular docking was used to calculate the binding potential (ΔG) between receptor protein subunits and more than 836,000 different chemical compounds from the ZINC database. Physicochemical parameters related to the compounds' pharmacokinetic and pharmacodynamic indicators were evaluated, including absorption, distribution, metabolism, excretion, and toxicity (ADMET), and chemical analysis characterized the compounds structures.Thirteen compounds with EII binding potential of the phosphotransferase system of S. mutans and favorable ADMET properties were identified. Six spirooxindoles and three pyrrolidones stand out from the found compounds; unique structural characteristics of spirooxindoles and pyrrolidones associated with various reported biological activities like anti-microbial, antiinflammatory, anticancer, nootropic, neuroprotective and antiepileptic effects, among other pharmacological effects with surprising differences in terms of mechanisms of action.Following studies will provide more evidence of the action of these compounds on the phosphotransferase system of S. mutans, and its possible applications.

Characterization of a novel sugar transporter involved in sugarcane bagasse degradation in Trichoderma reesei

BackgroundTrichoderma reesei is a saprophytic fungus implicated in the degradation of polysaccharides present in the cell wall of plants. T. reesei has been recognized as the most important industrial fungus that secretes and produces cellulase enzymes that are employed in the production of second generation bioethanol. A few of the molecular mechanisms involved in the process of biomass deconstruction by T. reesei; in particular, the effect of sugar transporters and induction of xylanases and cellulases expression are yet to be known.ResultsIn our study, we characterized a novel sugar transporter, which was previously identified by our group through in silico analysis of RNA-seq data. The novel T. reesei 69957-sugar transport system (Tr69957) is capable of transporting xylose, mannose, and cellobiose using a T. reesei 69957-sugar transport system in Saccharomyces cerevisiae. The deletion of Tr69957 in T. reesei affected the fungal growth and biomass accumulation, and the sugar uptake in the presence of mannose, cellobiose, and xylose. Molecular docking studies revealed that Tr69957 shows reduced protein–ligand binding energy for interactions towards disaccharides in comparison with monosaccharides. Furthermore, the deletion of Tr69957 affected the gene expression of cellobiohydrolases (cel7a and cel6a), β-glucosidases (cel3a and cel1a), and xylanases (xyn1 and xyn2) in the cultures of parental and mutant strains in the presence of cellobiose and sugarcane bagasse (SCB).ConclusionThe transporter Tr69957 of T. reesei can transport cellobiose, xylose, and mannose, and can affect the expression of a few genes encoding enzymes, such as cellulases and xylanases, in the presence of SCB. We showed for the first time that a filamentous fungus (T. reesei) contains a potential mannose transporter that may be involved in the degradation of cellulose.

Translational regulation by bacterial small RNAs via an unusual Hfq-dependent mechanism.

In bacteria, the canonical mechanism of translational repression by small RNAs (sRNAs) involves sRNA-mRNA base pairing that occludes the ribosome binding site (RBS), directly preventing translation. In this mechanism, the sRNA is the direct regulator, while the RNA chaperone Hfq plays a supporting role by stabilizing the sRNA. There are a few examples where the sRNA does not directly interfere with ribosome binding, yet translation of the target mRNA is still inhibited. Mechanistically, this non-canonical regulation by sRNAs is poorly understood. Our previous work demonstrated repression of the mannose transporter manX mRNA by the sRNA SgrS, but the regulatory mechanism was unknown. Here, we report that manX translation is controlled by a molecular role-reversal mechanism where Hfq, not the sRNA, is the direct repressor. Hfq binding adjacent to the manX RBS is required for sRNA-mediated translational repression. Translation of manX is also regulated by another sRNA, DicF, via the same non-canonical Hfq-dependent mechanism. Our results suggest that the sRNAs recruit Hfq to its binding site or stabilize the mRNA-Hfq complex. This work adds to the growing number of examples of diverse mechanisms of translational regulation by sRNAs in bacteria.

Continual renewal and replication of persistent Leishmania major parasites in concomitantly immune hosts

In most natural infections or after recovery, small numbers of Leishmania parasites remain indefinitely in the host. Persistent parasites play a vital role in protective immunity against disease pathology upon reinfection through the process of concomitant immunity, as well as in transmission and reactivation, yet are poorly understood. A key question is whether persistent parasites undergo replication, and we devised several approaches to probe the small numbers in persistent infections. We find two populations of persistent Leishmania major: one rapidly replicating, similar to parasites in acute infections, and another showing little evidence of replication. Persistent Leishmania were not found in "safe" immunoprivileged cell types, instead residing in macrophages and DCs, ∼60% of which expressed inducible nitric oxide synthase (iNOS). Remarkably, parasites within iNOS+ cells showed normal morphology and genome integrity and labeled comparably with BrdU to parasites within iNOS- cells, suggesting that these parasites may be unexpectedly resistant to NO. Nonetheless, because persistent parasite numbers remain roughly constant over time, their replication implies that ongoing destruction likewise occurs. Similar results were obtained with the attenuated lpg2- mutant, a convenient model that rapidly enters a persistent state without inducing pathology due to loss of the Golgi GDP mannose transporter. These data shed light on Leishmania persistence and concomitant immunity, suggesting a model wherein a parasite reservoir repopulates itself indefinitely, whereas some progeny are terminated in antigen-presenting cells, thereby stimulating immunity. This model may be relevant to understanding immunity to other persistent pathogen infections.

SLC2A11 (GLUT11) as mannose preferential transporter (607.15)

Most studies of hexose transporters (GLUT family) focus on glucose and fructose import for energy generation. Few studies examine their role in delivering sugars for biosynthetic pathways. Exogenous mannose can directly contribute nearly 50% of the mannose found in glycoproteins, even though its extracellular concentration (50 µM) is 100‐fold lower than physiological glucose (5mM). To explain this preference, we hypothesized the existence of a high affinity mannose‐preferential hexose transporter. We identified a patient with damaging mutation in SLC2A11 (Glucose transporter, GLUT11) and applied our new GC‐MS method to determine the contributions of 13C‐glucose and 13C‐mannose to N‐glycans. Patient fibroblasts showed reduced direct contribution of mannose, prompting us to investigate GLUT 11 as potential mannose transporter. RNAi knockdown of GLUT11 confirmed that it directly contributes mannose to N‐glycans , especially at the physiological range (50 µM) suggesting that GLUT11 has low Kuptake for mannose. The reported Km for glucose is 160µM. Preliminary results show that knockdown of GLUT 1 has no effect on mannose flux into N‐glycans. We suggest that GLUT11 is the first mannose‐preferential transporter for N‐glycan synthesis. Other mannose preferential transporters may exist.Grant Funding Source: R01DK55615 and The Rocket Fund

Solution Structure of the IIAChitobiose-HPr Complex of the N,N′-Diacetylchitobiose Branch of the Escherichia coli Phosphotransferase System

The solution structure of the complex of enzyme IIA of the N,N'-diacetylchitobiose (Chb) transporter with the histidine phosphocarrier protein HPr has been solved by NMR. The IIA(Chb)-HPr complex completes the structure elucidation of representative cytoplasmic complexes for all four sugar branches of the bacterial phosphoryl transfer system (PTS). The active site His-89 of IIA(Chb) was mutated to Glu to mimic the phosphorylated state. IIA(Chb)(H89E) and HPr form a weak complex with a K(D) of ~0.7 mM. The interacting binding surfaces, concave for IIA(Chb) and convex for HPr, complement each other in terms of shape, residue type, and charge distribution, with predominantly hydrophobic residues, interspersed by some uncharged polar residues, located centrally, and polar and charged residues at the periphery. The active site histidine of HPr, His-15, is buried within the active site cleft of IIA(Chb) formed at the interface of two adjacent subunits of the IIA(Chb) trimer, thereby coming into close proximity with the active site residue, H89E, of IIA(Chb). A His89-P-His-15 pentacoordinate phosphoryl transition state can readily be modeled without necessitating any significant conformational changes, thereby facilitating rapid phosphoryl transfer. Comparison of the IIA(Chb)-HPr complex with the IIA(Chb)-IIB(Chb) complex, as well as with other cytoplasmic complexes of the PTS, highlights a unifying mechanism for recognition of structurally diverse partners. This involves generating similar binding surfaces from entirely different underlying structural elements, large interaction surfaces coupled with extensive redundancy, and side chain conformational plasticity to optimize diverse sets of intermolecular interactions.

Functional characterisation of human SGLT-5 as a novel kidney-specific sodium-dependent sugar transporter

Sodium glucose cotransporters (SGLT) actively catalyse carbohydrate transport across cellular membranes. Six of the 12 known SGLT family members have the capacity to bind and/or transport monosaccharides (SGLT-1 to 6); of these, all but SGLT-5 have been characterised. Here we demonstrate that human SGLT-5 is exclusively expressed in the kidney. Four splice variants were detected and the most abundant SGLT-5-mRNA was functionally characterised. SGLT-5 mediates sodium-dependent [14C]-α-methyl-d-glucose (AMG) transport that can be inhibited by mannose, fructose, glucose, and galactose. Uptake studies using demonstrated high capacity transport for mannose and fructose and, to a lesser extent, glucose, AMG, and galactose. SGLT-5 mediated mannose, fructose and AMG transport was weakly (μM potency) inhibited by SGLT-2 inhibitors. In summary, we have characterised SGLT-5 as a kidney mannose transporter. Further studies are warranted to explore the physiological role of SGLT-5.

The small RNA SgrS controls sugar–phosphate accumulation by regulating multiple PTS genes

A number of bacterial small RNAs (sRNAs) act as global regulators of stress responses by controlling expression of multiple genes. The sRNA SgrS is expressed in response to glucose–phosphate stress, a condition associated with disruption of glycolytic flux and accumulation of sugar–phosphates. SgrS has been shown to stimulate degradation of the ptsG mRNA, encoding the major glucose transporter. This study demonstrates that SgrS regulates the genes encoding the mannose and secondary glucose transporter, manXYZ. Analysis of manXYZ mRNA stability and translation in the presence and absence of SgrS indicate that manXYZ is regulated by SgrS under stress conditions and when SgrS is ectopically expressed. In vitro footprinting and in vivo mutational analyses showed that SgrS base pairs with manXYZ within the manX coding sequence to prevent manX translation. Regulation of manX did not require the RNase E degradosome complex, suggesting that the primary mechanism of regulation is translational. An Escherichia coli ptsG mutant strain that is manXYZ+ experiences stress when exposed to the glucose analogs α-methyl glucoside or 2-deoxyglucose. A ptsG manXYZ double mutant is resistant to the stress, indicating that PTS transporters encoded by both SgrS targets are involved in taking up substrates that cause stress.

Characterization of a Mannose Utilization System in Bacillus subtilis

The mannose operon of Bacillus subtilis consists of three genes, manP, manA, and yjdF, which are responsible for the transport and utilization of mannose. Upstream and in the same orientation as the mannose operon a regulatory gene, manR, codes for a transcription activator of the mannose operon, as shown in this study. Both mannose operon transcription and manR transcription are inducible by mannose. The presence of mannose resulted in a 4- to 7-fold increase in expression of lacZ from the manP promoter (P(manP)) and in a 3-fold increase in expression of lacZ from the manR promoter (P(manR)). The transcription start sites of manPA-yjdF and manR were determined to be a single A residue and a single G residue, respectively, preceded by -10 and -35 boxes resembling a vegetative sigma(A) promoter structure. Through deletion analysis the target sequences of ManR upstream of P(manP) and P(manR) were identified between bp -80 and -35 with respect to the transcriptional start site of both promoters. Deletion of manP (mannose transporter) resulted in constitutive expression from both the P(manP) and P(manR) promoters, indicating that the phosphotransferase system (PTS) component EII(Man) has a negative effect on regulation of the mannose operon and manR. Moreover, both P(manP) and P(manR) are subject to carbon catabolite repression (CCR). By constructing protein sequence alignments a DNA binding motif at the N-terminal end, two PTS regulation domains (PRDs), and an EIIA- and EIIB-like domain were identified in the ManR sequence, indicating that ManR is a PRD-containing transcription activator. Like findings for other PRD regulators, the phosphoenolpyruvate (PEP)-dependent phosphorylation by the histidine protein HPr via His15 plays an essential role in transcriptional activation of P(manP) and P(manR). Phosphorylation of Ser46 of HPr or of the homologous Crh protein by HPr kinase and formation of a repressor complex with CcpA are parts of the B. subtilis CCR system. Only in the double mutant with an HPr Ser46Ala mutation and a crh knockout mutation was CCR strongly reduced. In contrast, P(manR) and P(manP) were not inducible in a ccpA deletion mutant.

X-ray Structures of the Three Lactococcus lactis Dihydroxyacetone Kinase Subunits and of a Transient Intersubunit Complex

Bacterial dihydroxyacetone (Dha) kinases do not exchange the ADP for ATP but utilize a subunit of the phosphoenolpyruvate carbohydrate phosphotransferase system for in situ rephosphorylation of a permanently bound ADP-cofactor. Here we report the 2.1-angstroms crystal structure of the transient complex between the phosphotransferase subunit DhaM of the phosphotransferase system and the nucleotide binding subunit DhaL of the Dha kinase of Lactococcus lactis, the 1.1-angstroms structure of the free DhaM dimer, and the 2.5-angstroms structure of the Dha-binding DhaK subunit. Conserved salt bridges and an edge-to-plane stacking contact between two tyrosines serve to orient DhaL relative to the DhaM dimer. The distance between the imidazole Nepsilon2 of the DhaM His-10 and the beta-phosphate oxygen of ADP, between which the gamma-phosphate is transferred, is 4.9 angstroms. An invariant arginine, which is essential for activity, is appropriately positioned to stabilize the gamma-phosphate in the transition state. The (betaalpha)4alpha fold of DhaM occurs a second time as a subfold in the DhaK subunit. By docking DhaL-ADP to this subfold, the nucleotide bound to DhaL and the C1-hydroxyl of Dha bound to DhaK are positioned for in-line transfer of phosphate.

Solution NMR Structures of Productive and Non-productive Complexes between the A and B Domains of the Cytoplasmic Subunit of the Mannose Transporter of the Escherichia coli Phosphotransferase System

Solution structures of complexes between the isolated A (IIA(Man)) and B (IIB(Man)) domains of the cytoplasmic component of the mannose transporter of Escherichia coli have been solved by NMR. The complex of wild-type IIA(Man) and IIB(Man) is a mixture of two species comprising a productive, phosphoryl transfer competent complex and a non-productive complex with the two active site histidines, His-10 of IIA(Man) and His-175 of IIB(Man), separated by approximately 25A. Mutation of the active site histidine, His-10, of IIA(Man) to a glutamate, to mimic phosphorylation, results in the formation of a single productive complex. The apparent equilibrium dissociation constants for the binding of both wild-type and H10E IIA(Man) to IIB(Man) are approximately the same (K(D) approximately 0.5 mM). The productive complex can readily accommodate a transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the Nepsilon2 atom of His-10 of IIA(Man) and the Ndelta1 atom of His-175 of IIB(Man) with negligible (<0.2A) local backbone conformational changes in the immediate vicinity of the active site. The non-productive complex is related to the productive one by a approximately 90 degrees rotation and approximately 37A translation of IIB(Man) relative to IIA(Man), leaving the active site His-175 of IIB(Man) fully exposed to solvent in the non-productive complex. The interaction surface on IIA(Man) for the non-productive complex comprises a subset of residues used in the productive complex and in both cases involves both subunits of IIA(Man). The selection of the productive complex by IIA(Man)(H10E) can be attributed to neutralization of the positively charged Arg-172 of IIB(Man) at the center of the interface. The non-productive IIA(Man)-IIB(Man) complex may possibly be relevant to subsequent phosphoryl transfer from His-175 of IIB(Man) to the incoming sugar located on the transmembrane IIC(Man)-IID(Man) complex.

13C-detected HN(CA)C and HMCMC experiments using a single methyl-reprotonated sample for unambiguous methyl resonance assignment

Methyl groups provide an important source of structural and dynamic information in NMR studies of proteins and their complexes. For this purpose sequence-specific assignments of methyl 1H and 13C resonances are required. In this paper we propose the use of 13C-detected 3D HN(CA)C and HMCMC experiments for assignment of methyl 1H and 13C resonances using a single selectively methyl protonated, perdeuterated and 13C/15N-labeled sample. The high resolution afforded in the 13C directly-detected dimension allows one to rapidly and unambiguously establish correlations between backbone HN strips from the 3D HN(CA)C spectrum and methyl group HmCm strips from the HMCMC spectrum by aligning all possible side-chain carbon chemical shifts and their multiplet splitting patterns. The applicability of these experiments for the assignment of methyl 1H and 13C resonances is demonstrated using the 18.6 kDa B domain of the Escherichia coli mannose transporter (IIBMannose).

The Mannose Transporter Complex: an Open Door for the Macromolecular Invasion of Bacteria

In this issue of the Journal of Bacteriology, Bieler et al. demonstrate (i) that the toxic activity of microcin E492 is independent of the “route of administration,” viz. whether it is added from the outside (as in nature) or expressed endogenously in the cytoplasm, and (ii) that microcin E492 requires the transporter for mannose to deploy its ion channel-forming activity. This is the strongest and most direct evidence to date that the mannose transporter is involved in microcin activity in gram-negative enterobacteria.

Molecular cloning and characterization of a Pichia pastoris ortholog of the yeast Golgi GDP-mannose transporter gene

There are two structural profiles in the yeast Golgi. The Golgi of Saccharomyces cerevisiae is composed of a number of vesicular compartments dispersed in the cytoplasm as recognized by a large number of Golgi marker proteins. In contrast, the Golgi of Pichia pastoris was reported to be organized in a small number of stacked cisternae located near the transitional endoplasmic reticulum (tER) sites by electron microscopy and immunofluorescent staining of a few marker proteins. The guanosine diphosphate (GDP)-mannose transporter (GMT) is an essential component in the yeast Golgi apparatus. We isolated an ortholog of the GMT gene of P. pastoris and visualized the gene product by epitope tagging to verify the structural characteristics of the Golgi. The tagged product in P. pastoris cell was observed in rod-like compartments in which Och1 mannosyltransferase was also found and the tER marker Sec12 and Sec13 proteins localized very close to them. The present results add further evidence of the restricted localization of the Golgi in P. pastoris cell.

EDD, a novel phosphotransferase domain common to mannose transporter EIIA, dihydroxyacetone kinase, and DegV

Using a recently developed program (SCOPmap) designed to automatically assign new protein structures to existing evolutionary-based classification schemes, we identify a evolutionarily conserved domain (EDD) common to three different folds: mannose transporter EIIA domain (EIIA-man), dihydroxyacetone kinase (Dak), and DegV. Several lines of evidence support unification of these three folds into a single superfamily: statistically significant sequence similarity detected by PSI-BLAST; "closed structural grouping" using DALI Z-scores (each protein inside a group finds all other group members with scores higher than those to proteins outside the group) that includes only these proteins sharing a unique alpha-helical hairpin at the C-terminus and excludes all other proteins with similar topology; similar domain fusions connect Dak and DegV, and genomic neighborhood organizations connect Dak and EIIA-man. Finally, both Dak and EIIA-man perform similar phosphotransfer reactions, suggesting a phosphotransferase activity for the DegV-like family of proteins, whose function other than lipid binding revealed in the crystal structure remains unknown.