Search

Catabolite repression is a mechanism that enables bacteria to control carbon utilization. As part of this global regulatory network, components of the phosphoenolpyruvate:carbohydrate phosphotransferase system inhibit the uptake of less favorable sugars when a preferred carbon source such as glucose is available. This process is termed inducer exclusion. In bacteria belonging to the phylum Firmicutes, HPr, phosphorylated at serine 46 (P-Ser46-HPr) is the key player but its mode of action is elusive. To address this question at the level of purified protein components, we have chosen a homolog of the Escherichia coli maltose/maltodextrin ATP-binding cassette transporter from Lactobacillus casei (MalE1-MalF1G1K12) as a model system. We show that the solute binding protein, MalE1, binds linear and cyclic maltodextrins but not maltose. Crystal structures of MalE1 complexed with these sugars provide a clue why maltose is not a substrate. P-Ser46-HPr inhibited MalE1/maltotetraose-stimulated ATPase activity of the transporter incorporated in proteoliposomes. Furthermore, cross-linking experiments revealed that P-Ser46-HPr contacts the nucleotide-binding subunit, MalK1, in proximity to the Walker A motif. However, P-Ser46-HPr did not block binding of ATP to MalK1. Together, our findings provide first biochemical evidence that P-Ser-HPr arrests the transport cycle by preventing ATP hydrolysis at the MalK1 subunits of the transporter. [ABSTRACT FROM AUTHOR]

The human gut microbiota endows the host with a wealth of metabolic functions central to health, one of which is the degradation and fermentation of complex carbohydrates. The Bacteroidetes are one of the dominant bacterial phyla of this community and possess an expanded capacity for glycan utilization. This is mediated via the coordinated expression of discrete polysaccharide utilization loci (PUL) that invariantly encode a TonB-dependent transporter (SusC) that works with a glycan-capturing lipoprotein (SusD). More broadly within Gram-negative bacteria, TonB-dependent transporters (TBDTs) are deployed for the uptake of not only sugars, but also more often for essential nutrients such as iron and vitamins. Here, we provide a comprehensive look at the repertoire of TBDTs found in the model gut symbiont Bacteroides thetaiotaomicron and the range of predicted functional domains associated with these transporters and SusD proteins for the uptake of both glycans and other nutrients. This atlas of the B. thetaiotaomicron TBDTs reveals that there are at least three distinct subtypes of these transporters encoded within its genome that are presumably regulated in different ways to tune nutrient uptake., (© 2021 John Wiley & Sons Ltd.)

MalFGK2 is an ATP-binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose-binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide-binding subunits (MalK dimer). This MBP-stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent.We found that full engagement of both lobes of maltose-bound MBP unto MalFGK2 is facilitated by nucleotides and stabilizes a semi-open MalK dimer. Maltose-bound MBP promotes the transition to the semi-open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi-open MalK2 conformation by maltose-bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi-open conformation, from which it can proceed to hydrolyze ATP. [ABSTRACT FROM AUTHOR]

Signal transduction ATPases with numerous domains ( STAND) are widespread proteins, whose activation involves inducer-dependent conversion of resting ADP-bound monomers into active ATP-bound multimers. This process notably comprises opening of the nucleotide-binding oligomerization domain ( NOD), nucleotide exchange and NOD-mediated multimerization. How inducer binding to the sensor domain, whose structure is not conserved throughout the STAND family, causes protein activation remains unclear. We used MalT, an Escherichia coli transcription factor, as a STAND model system, to address this question by dissecting the signalling pathway in vitro. We have found that inducer binding to the sensor is the first step of the activation pathway. It both triggers opening of the NOD and makes the MalT multimer competent for binding promoter MalT sites via its DNA-binding domains. Based on available data, we proposed that inducer trigger of NOD opening is a conserved STAND feature, irrespective of the sensor structure. As discussed, an additional role for the inducer, as found for MalT, might pertain to other types of STANDs. [ABSTRACT FROM AUTHOR]

The poles of bacteria exhibit several specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, we developed procedures for the tagging of mature bacteriophages with quantum dots. Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. The infecting phages colocalized with the polar protein marker IcsA–GFP. ManY, an E. coli protein that is required for phage λ DNA injection, was found to localize to the bacterial poles as well. Furthermore, labelling of λ DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to λ's developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation. [ABSTRACT FROM AUTHOR]

The ATP binding cassette (ABC-) transporter mediating the uptake of maltose/maltodextrins in Escherichia coli/Salmonella enterica serovar Typhimurium is one of the best characterized systems and serves as a model for studying the molecular mechanism by which ABC importers exert their functions. The transporter is composed of a periplasmic maltose binding protein (MalE), and a membrane-bound complex (MalFGK2), comprising the pore-forming hydrophobic subunits, MalF and MalG, and two copies of the ABC subunit, MalK. We report on the isolation of suppressor mutations within malFG that partially restore transport of a maltose-negative mutant carrying the malK809 allele (MalKQ140K). The mutation affects the conserved LSGGQ motif that is involved in ATP binding. Three out of four suppressor mutations map in periplasmic loops P2 and P1 respectively of MalFG. Cross-linking data revealed proximity of these regions to MalE. In particular, as demonstrated in vitro and in vivo, Gly-13 of substrate-free and substrate-loaded MalE is in close contact to Pro-78 of MalG. These data suggest that MalE is permanently in close contact to MalG-P1 via its N-terminal domain. Together, our results are interpreted in favour of the notion that substrate availability is communicated from MalE to the MalK dimer via extracytoplasmic loops of MalFG, and are discussed with respect to a current transport model. [ABSTRACT FROM AUTHOR]

To examine the relationship between folding and aggregation in the periplasm of Escherichia coli, we have analysed the cellular fates of exported proteins fused to either the wild-type maltose-binding protein (MalE) or the aggregation-prone variant MalE31. The propensity of fusion proteins to aggregate in the periplasm was determined by the intrinsic folding characteristics of the upstream protein. When β-lactamase or alkaline phosphatase was linked to the C-terminus of MalE31, the resultant fusion proteins accumulated in an insoluble form, but retained their catalytic activity. In addition, these protein aggregates induced an extracytoplasmic stress response, similar to unfused MalE31. However, using a fluorescent substrate, we found that alkaline phosphatase activity was present inside periplasmic aggregates. These results suggest that periplasmic inclusion body formation may result in intermolecular interactions between participating proteins without loss of function of the fused enzymes. [ABSTRACT FROM AUTHOR]

Mannose-binding type 1 pili are important virulence factors for the establishment ofEscherichia coliurinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenicE. colito uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butylα-d-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (Kd = 0.15 µM) than mannose (Kd = 2.3 µM). Exploration of the binding affinities ofα-d-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliatedE. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti-adhesive drugs to prevent anticipated and recurrent UTIs. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette (ABC) transporter superfamily is one of the most widespread of all gene families and currently has in excess of 1100 members in organisms ranging from the Archaea to man. The movement of the diverse solutes of ABC transporters has been accepted as being strictly unidirectional, with recent models indicating that they are irreversible. However, contrary to this paradigm, we show that three solute-binding protein-dependent (SBP) ABC transporters of amino acids, i.e. the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra) of Rhizobium leguminosarum and the histidine permease (His) of Salmonella typhimurium , are bidirectional, being responsible for efflux in addition to the uptake of solutes. The net solute movement measured for an ABC transporter depends on the rates of uptake and efflux, which are independent; a plateau is reached when both are saturated. SBP ABC transporters promote active uptake because, although the V max values for uptake and efflux are not significantly different, there is a 103 -104 higher affinity for uptake of solute compared with efflux. Therefore, the SBP ABC transporters are able to support a substantial concentration gradient and provide a net uptake of solutes into bacterial cells. [ABSTRACT FROM AUTHOR]

Lamb of Escherichia coli K12, also called maltoporin, is an outer membrane protein, which specifically facilitates the diffusion of maltose and maltodextrin through the bacterial outer membrane. Each monomer is composed of an 18-stranded antiparallel β-barrel. In the present work, on the basis of the known X-ray structure of LamB, the effects of modifications of the β-barrel domain of maltoporin were studied in vivo and in vitro. We show that: (i) the substitution of the pair of strands β13-β14 of the E. coli maltoporin with the corresponding pair of strands from the functionally related maltoporin of Salmonella typhimurium yielded a protein active in vivo and in vitro; and (ii) the removal of one pair of β-strands (deletion β13-β14) from the E. coli maltoporin, or its replacement by a pair of strands from the general porin OmpF of E. coli, leads to recombinant proteins that lost in vivo maltoporin activities but still kept channel formation and carbohydrate binding in vitro. We also inserted into deletion β13-β14 the portion of the E. coli Lamb protein comprising strands β13 to β16. This resulted in a protein expected to have 20 β-strands and which completely lost all LamB-specific activities in vivo and in vitro. [ABSTRACT FROM AUTHOR]

The trimeric protein LamB of Escherichia coli K-12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane. Each monomer consists of an 18-stranded antiparallel β-barrel with nine surface loops (L1 to L9). The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro. In vivo, single-, ΔL4, ΔL5, ΔL6, and double-loop deletions, ΔL4 + ΔL5 and ΔL5 + ΔL6, abolished maltoporin functions, but not the double deletion ΔL4 + ΔL6 and the triple deletion ΔL4 + ΔL5 + ΔL6. While deletion of the central variable portion of loop L9 (ΔL9v) affected maltoporin function only moderately, the combination of ΔL9v with the double deletion of loops L4 and L6 (triple deletion ΔL4 + ΔL6 + ΔL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro. In vitro, the carbohydrate-binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes. The deletion of loop L9v alone (LamBΔL9v), of two loops L4 and L6 (LamBΔL4 + ΔL6), of three loops L4, L5 and L6 (LamBΔL4 + ΔL5 + ΔL6) or of L4, L6 and L9v (LamBΔL4 + ΔL6 + ΔL9v) had relatively little influence on the carbohydrate-binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side. The deletion of one of the loops L4 (LamBΔL4) or L6 (LamBΔL6) resulted in an asymmetric carbohydrate binding. The in vivo and in vitro results, together with those of the purification across the starch column,... [ABSTRACT FROM AUTHOR]

The outer membrane of Vibrio cholerae contains a maltose-inducible major protein, OmpS (43 kDa), that is common to different isolates. Nucleotide sequence analysis of the corresponding structural gene, ompS, revealed an open reading frame encoding a 412-amino-acid polypeptide. The amino acid sequence of OmpS is similar to that of LamB, the Escherichia coli maltoporin, and to ScrY of Kiebsiella pneumoniae, although the antigenic determinants of these proteins are different. The cloned ompS gene complemented an ompS mutation of V. cholerae and the corresponding polypeptide could function as a maltoporin in a LamB- mutant of E. coli The promoter region of ompS is highly homologous to the malK-lamB promoter of E. coli and the ompS gene is controlled by MalT in E. coli This indicates that the same kind of regulatory mechanism is used to activate the ompS expression in V. cholerae and malK-lamB expression in E. coli An ompS-lacZ transcriptional fusion was used to demonstrate a dual control in ompS expression; the ompS gene is responsive to the inducers maltose and trehalose but in their absence it is also expressed in response to growth-phase. These different modes of induction might be of importance during different stages of V. cholerae infection. [ABSTRACT FROM AUTHOR]

Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes. REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria. These REP sequences may participate in the folding of the bacterial chromosome. In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular integration host factor (IHF) recognition sequence. We term these sequences RIP (for repetitive IHF-binding palindromic) elements and demonstrate that IHF binds to them specifically. It is estimated that there are about 70 RIP elements in E. coli. Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome. The possible function of the RIP element is discussed. [ABSTRACT FROM AUTHOR]

The maIG gene encodes a hydrophobic cytoplasmic membrane protein which is required for the energy-dependent transport of maltose and maltodextrins in Escherichia coli. The MaIG protein, together with MalF and MalK proteins, forms a multimeric complex in the membrane consisting of two MalK subunits for each MalF and MaIG subunit. Fifteen mutations have been isolated in maIG by random linker insertion mutagenesis. Two regions essential for maltose transport have been identified. In particular, a hydrophjiic region containing the peptidic motif EAA-G---I-LP, highly conserved among inner membrane proteins from binding protein-dependent transport systems, is essential for maltose transport. The results also show that several regions of MaIG are not essential for function. A region (residues 30-50) encompassing the first predicted transmembrane segment and the first periplasmic loop in MaIG may be modified extensively with little effect on maltose transport and no effect on the stability and the localization of the protein. A region located at the middle of the protein (residues 153-157) is not essential for the function of the protein. A region, essential for maltodextrin utilization but not for maltose transport, has been identified near the C-terminus of the protein. [ABSTRACT FROM AUTHOR]

The genes coding for the binding-protein-dependent lactose transport system and β-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was Identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and β-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria. [ABSTRACT FROM AUTHOR]

Escherichia coli K-12 strain PS1-28-37 carries the multicopy plasmid pPSO28-37 containing a DNA fragment coding for two of the proteins that enable bacteria to utilize sucrose as sole carbon source. One of the different gene products of the plasmid is the outer membrane protein, ScrY. This protein was isolated and purified by chromatography across a gel filtration column. Reconstitution experiments with lipid bilayer membrane demonstrated that ScrY formed ion-permeable channels with properties very similar to those of general diffusion pores of enteric bacteria. The presence of sugars in the aqueous phase led to a dose-dependent block of ion transport through the channel, like the situation found with LamB (maltoporin) of Escherichia coli and Salmonella typhimurium. The binding constants of a variety of different sugars were determined. The stability constant for malto-oligosaccharide binding increased with increasing numbers of glucose residues. Disaccharides generally had a larger binding constant than monosaccharides. The binding of different sugars to ScrY and LamB of E. coli is discussed with respect to the kinetics of sugar movement through the channel. [ABSTRACT FROM AUTHOR]

During the molecular analysis of a plasmid-coded sucrose metabolic pathway of enteric bacteria, a gene, scrY, was found whose product, ScrY, had all the properties of a bacterial porin (Schmid et al., 1988). Loss of this protein (Mr, 58kDa), localized in the outer membrane, led, as shown here, to an increase in the apparent Km, for sucrose transport in whole cells from 10µM in wild-type cells to 300µM in mutant cells. This contrasts with the Km for sucrose phosphorylation as measured in membrane vesicles from mutant and wild-type cells, which remained unchanged at about 10 µM, and reflects the activity of the sucrose-specific Enzymell of the phosphoenolpyruvate-dependent carbohydrate:phosphotransf erase system (PTS) responsible for uptake through the inner membrane. Furthermore, the presence of ScrY restored growth on maltodextrins in cells devoid of LamB, thus complementing the lack of this maltoporin. The amino acid sequence deduced from the DNA sequence was determined for the plasmid-coded and the ScrY porin coded in the chromosome of Klebsiella pneumoniae. Both show high identity (86%) to each other, and to the channel domain of LamB, further corroborating the conclusion that they constitute porins. [ABSTRACT FROM AUTHOR]

The oprP gene encoding the Pseudomonas aeruginosa phosphate-specific outer membrane porin protein OprP was sequenced. Comparison of the derived amino acid sequence with the known sequences of other bacterial porins demonstrated that OprP could be no better aligned to these porin sequences than it could to the periplasmic phosphate-binding protein PhoS of Escherichia coli. Southern hybridization and restriction mapping of the oprP gene in 37 clinical isolates and the 17 serotype strains of P. aeruginosa revealed that restriction sites in the vicinity of the oprP gene were highly conserved. Several species from the Pseudomonas fluorescens rRNA homology group contained DNA that hybridized to an oprP gene probe. [ABSTRACT FROM AUTHOR]

The nucleotide sequence of the ugp genes of Escherichia coli K-12, which encode a phosphate-limitation inducible uptake system for sn-glycerol-3-phosphate and glycerophosphoryl diesters, was determined. The genetic organization of the operon differed from previously published results. A single promoter, containing a putative pho box, was detected by S1-nuclease mapping. The promoter is followed by four open reading frames, designated ugpB, A, E and C, which encode a periplasmic binding protein, two hydrophobic membrane proteins and a protein that is likely to couple energy to the transport system, respectively. The sequences of the proteins contain the characteristics of several other binding protein-dependent transport systems, but they seem to be particularly closely related to the maltose system. [ABSTRACT FROM AUTHOR]

A computer search for repeated sequences led us to identify five REP (repetitive extragenic palindromic) sequences in the 3-terminal region of the Escherichia coli ribonucleoside diphosphate reductase gene (nrdA). These REP sequences are located within a putative duplicated DNA region, the first of them being part of the carboxy-terminal coding region of the nrdA gene. This is the first report of a REP sequence within a structural gene and also the first example of a REP sequence apparently generated by DNA duplication. [ABSTRACT FROM AUTHOR]