Cookie cravings - Examining the impact of sugar content information on Christmas treat preferences via mobile eye-tracking.

BACKGROUND: Diets high in added sugar can promote the development of overweight. Especially during the Holiday season, when high-sugar food is abundant, people overeat and gain more weight than during other times of the year. The present study with mobile eye-tracking glasses (Pupil Labs Invisible) investigated how sugar content information affects food preference (liking/wanting) and visual attention (where and how long one is looking) in a buffet-like situation. METHODS: Fifty-eight participants who were well acquainted with the local Christmas traditions and foods (38 female, 19 male, one diverse; mean age = 25 years, SD = 6.3 years; mean body mass index = 22.2 kg/m2, SD = 3.2 kg/m2) were presented with four cookies and two non-food items (wrapped presents) in a free viewing task. Two of the displayed cookies were 'Christmas cookies' (cookies that are traditionally eaten only during the Holiday season) and two cookies had no Christmas association. The cookies were either labeled as cookies made with or without sugar, resulting in a 3 (Category: cookies with sugar, cookies without sugar, non-food) by 2 (Christmas association: yes, no) repeated-measures design. RESULTS: Analyses of variance indicated that participants reported higher wanting and liking for cookies with sugar, particularly Christmas cookies (interaction effect for wanting: p = .047, ηp2 = .059; interaction effect for liking: p = .017, ηp2 = .084). Sugar-free cookies were fixated more often (p = .028; d = 0.35) and shorter (p < .001; d = 0.64) than sugar cookies. CONCLUSION: Assuming that cookies are sugar-free reduced the reported preference for this product, which was associated with a more detail-oriented (critical) viewing pattern. The study's findings have potential implications for public health and can aid in developing targeted interventions to promote healthier food choices during festive periods. The new strategies should not focus on the sugar content of foods.

Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data.

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, limitations, and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for continuous extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies, while the containers and reproducible workflows could easily be deployed and extended to evaluate new methods or data sets.

Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data.

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, and limitations and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for seamless extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies. Furthermore, the containers and reproducible workflows generated in the course of this project can be seamlessly deployed and extended in the future to evaluate new methods or datasets.

CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis.

The mammalian cleavage factor I (CFIm) has been implicated in alternative polyadenylation (APA) in a broad range of contexts, from cancers to learning deficits and parasite infections. To determine how the CFIm expression levels are translated into these diverse phenotypes, we carried out a multi-omics analysis of cell lines in which the CFIm25 (NUDT21) or CFIm68 (CPSF6) subunits were either repressed by siRNA-mediated knockdown or over-expressed from stably integrated constructs. We established that >800 genes undergo coherent APA in response to changes in CFIm levels, and they cluster in distinct functional classes related to protein metabolism. The activity of the ERK pathway traces the CFIm concentration, and explains some of the fluctuations in cell growth and metabolism that are observed upon CFIm perturbations. Furthermore, multiple transcripts encoding proteins from the miRNA pathway are targets of CFIm-dependent APA. This leads to an increased biogenesis and repressive activity of miRNAs at the same time as some 3' UTRs become shorter and presumably less sensitive to miRNA-mediated repression. Our study provides a first systematic assessment of a core set of APA targets that respond coherently to changes in CFIm protein subunit levels (CFIm25/CFIm68). We describe the elicited signaling pathways downstream of CFIm, which improve our understanding of the key role of CFIm in integrating RNA processing with other cellular activities.

Overcoming barriers to the adoption of locating technologies in dementia care: a multi-stakeholder focus group study.

BACKGROUND: Locating technologies are a subtype of assistive technology that aim to support persons with dementia by helping manage spatial orientation impairments and provide aid to care partners by intervening when necessary. Although a variety of locating devices are commercially available, their adoption has remained low in the past years. Several studies have explored barriers to the adoption of assistive technologies from the perspective of professional stakeholders, but in-depth explorations for locating technologies are sparse. Additionally, the inputs of business professionals are lacking. The aim of this study was to expand knowledge on barriers to the adoption of locating technologies from a multi-stakeholder professional perspective, and to explore strategies to optimize adoption. METHODS: In total, 22 professionals working in business (n = 7), healthcare (n = 6) and research (n = 9) fields related to gerontology and gerontechnology participated in our focus group study. Perceptions on the value of using locating technologies for dementia care, barriers to their adoption, as well as salient services and information dissemination strategies were explored. After verbatim transcription, transcripts were analysed following an inductive data-driven content analysis approach in MAXQDA. RESULTS: Six key adoption barriers centering on: (1) awareness-, (2) technological-, (3) product characteristic- and (4) capital investment-based limitations, (5) unclear benefits, as well as (6) ethical concerns emerged. The interplay between barriers was high. Five core themes on services and information dissemination strategies centering on: (1) digital autonomy support, (2) emergency support, (3) information dissemination actors, (4) product acquisition, and (5) product advertising were extracted. CONCLUSIONS: Our study with interdisciplinary stakeholders expands knowledge on barriers to the adoption of locating technologies for dementia care, and reinforces recommendations that an interdisciplinary strategy is needed to optimize adoption. Also, our findings show that focusing on services to increase digital autonomy and on information dissemination strategies has been largely overlooked and may be particularly effective.

PolyASite 2.0: a consolidated atlas of polyadenylation sites from 3' end sequencing.

Generated by 3' end cleavage and polyadenylation at alternative polyadenylation (poly(A)) sites, alternative terminal exons account for much of the variation between human transcript isoforms. More than a dozen protocols have been developed so far for capturing and sequencing RNA 3' ends from a variety of cell types and species. In previous studies, we have used these data to uncover novel regulatory signals and cell type-specific isoforms. Here we present an update of the PolyASite (https://polyasite.unibas.ch) resource of poly(A) sites, constructed from publicly available human, mouse and worm 3' end sequencing datasets by enforcing uniform quality measures, including the flagging of putative internal priming sites. Through integrated processing of all data, we identified and clustered sites that are closely spaced and share polyadenylation signals, as these are likely the result of stochastic variations in processing. For each cluster, we identified the representative - most frequently processed - site and estimated the relative use in the transcriptome across all samples. We have established a modern web portal for efficient finding, exploration and export of data. Database generation is fully automated, greatly facilitating incorporation of new datasets and the updating of underlying genome resources.

Version 4.0 of PaxDb: Protein abundance data, integrated across model organisms, tissues, and cell-lines.

Protein quantification at proteome-wide scale is an important aim, enabling insights into fundamental cellular biology and serving to constrain experiments and theoretical models. While proteome-wide quantification is not yet fully routine, many datasets approaching proteome-wide coverage are becoming available through biophysical and MS techniques. Data of this type can be accessed via a variety of sources, including publication supplements and online data repositories. However, access to the data is still fragmentary, and comparisons across experiments and organisms are not straightforward. Here, we describe recent updates to our database resource "PaxDb" (Protein Abundances Across Organisms). PaxDb focuses on protein abundance information at proteome-wide scope, irrespective of the underlying measurement technique. Quantification data is reprocessed, unified, and quality-scored, and then integrated to build a meta-resource. PaxDb also allows evolutionary comparisons through precomputed gene orthology relations. Recently, we have expanded the scope of the database to include cell-line samples, and more systematically scan the literature for suitable datasets. We report that a significant fraction of published experiments cannot readily be accessed and/or parsed for quantitative information, requiring additional steps and efforts. The current update brings PaxDb to 414 datasets in 53 organisms, with (semi-) quantitative abundance information covering more than 300,000 proteins.

A widespread glutamine-sensing mechanism in the plant kingdom.

Glutamine is the primary metabolite of nitrogen assimilation from inorganic nitrogen sources in microorganisms and plants. The ability to monitor cellular nitrogen status is pivotal for maintaining metabolic homeostasis and sustaining growth. The present study identifies a glutamine-sensing mechanism common in the entire plant kingdom except Brassicaceae. The plastid-localized PII signaling protein controls, in a glutamine-dependent manner, the key enzyme of the ornithine synthesis pathway, N-acetyl-l-glutamate kinase (NAGK), that leads to arginine and polyamine formation. Crystal structures reveal that the plant-specific C-terminal extension of PII, which we term the Q loop, forms a low-affinity glutamine-binding site. Glutamine binding alters PII conformation, promoting interaction and activation of NAGK. The binding motif is highly conserved in plants except Brassicaceae. A functional Q loop restores glutamine sensing in a recombinant Arabidopsis thaliana PII protein, demonstrating the modular concept of the glutamine-sensing mechanism adopted by PII proteins during the evolution of plant chloroplasts.

An in vivo EGF receptor localization screen in C. elegans Identifies the Ezrin homolog ERM-1 as a temporal regulator of signaling.

The subcellular localization of the epidermal growth factor receptor (EGFR) in polarized epithelial cells profoundly affects the activity of the intracellular signaling pathways activated after EGF ligand binding. Therefore, changes in EGFR localization and signaling are implicated in various human diseases, including different types of cancer. We have performed the first in vivo EGFR localization screen in an animal model by observing the expression of the EGFR ortholog LET-23 in the vulval epithelium of live C. elegans larvae. After systematically testing all genes known to produce an aberrant vulval phenotype, we have identified 81 genes regulating various aspects of EGFR localization and expression. In particular, we have found that ERM-1, the sole C. elegans Ezrin/Radixin/Moesin homolog, regulates EGFR localization and signaling in the vulval cells. ERM-1 interacts with the EGFR at the basolateral plasma membrane in a complex distinct from the previously identified LIN-2/LIN-7/LIN-10 receptor localization complex. We propose that ERM-1 binds to and sequesters basolateral LET-23 EGFR in an actin-rich inactive membrane compartment to restrict receptor mobility and signaling. In this manner, ERM-1 prevents the immediate activation of the entire pool of LET-23 EGFR and permits the generation of a long-lasting inductive signal. The regulation of receptor localization thus serves to fine-tune the temporal activation of intracellular signaling pathways.

Sequential chemotherapies for advanced gastric cancer: a retrospective analysis of 111 patients.

BACKGROUND: The role of second-line chemotherapy in advanced gastric cancer is not yet fully established. PATIENTS AND METHODS: We analysed 111 patients with advanced gastric cancer treated at the University Hospital Heidelberg (51) and the private oncology practice Bottrop/Dorsten (60) between 2001 and 2011, comparing the outcome of patients with first-line chemotherapy and those who received second-line chemotherapy. RESULTS: Thirty-six patients were treated with one chemotherapy regimen, 75 patients received at least two different chemotherapies. Patients who received one chemotherapy regimen were older (median age 69 years) and had a shorter overall survival (6 months) than patients receiving sequential chemotherapies [median age 61 years, p = 0.009, overall survival 14 months (2-42), p = 0.001]. Under second-line chemotherapy, partial response was observed in 25 patients (33%) and stable disease for ≥3 months in 26 patients (35%). Patients treated before 2005 had a slightly better overall survival than patients treated in or after 2005. Survival was not influenced by the treatment centre (primary or tertiary), but was influenced by former surgery. CONCLUSION: The prognosis of advanced gastric cancer is still poor. Selected patients may benefit from individualized salvage chemotherapy after failure of first-line chemotherapy.

Signal-transduction protein P(II) from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.

P(II) proteins belong to a family of highly conserved signal-transduction proteins that occurs widely in bacteria, archaea and plants. They respond to the central metabolites ATP, ADP and 2-OG (2-oxoglutarate), and control enzymes, transcription factors and transport proteins involved in nitrogen metabolism. In the present study, we examined the effect of ADP on in vitro P(II)-signalling properties for the cyanobacterium Synechococcus elongatus, a model for oxygenic phototrophic organisms. Different ADP/ATP ratios strongly affected the properties of P(II) signalling. Increasing ADP antagonized the binding of 2-OG and directly affected the interactions of P(II) with its target proteins. The resulting P(II)-signalling properties indicate that, in mixtures of ADP and ATP, P(II) trimers are occupied by mixtures of adenylate nucleotides. Binding and kinetic activation of NAGK (N-acetyl-L-glutamate kinase), the controlling enzyme of arginine biosynthesis, by P(II) was weakened by ADP, but relief from arginine inhibition remained unaffected. On the other hand, ADP enhanced the binding of P(II) to PipX, a co-activator of the transcription factor NtcA and, furthermore, antagonized the inhibitory effect of 2-OG on P(II)-PipX interaction. These results indicate that S. elongatus P(II) directly senses the adenylate energy charge, resulting in target-dependent differential modification of the P(II)-signalling properties.

Dissipation and sequestration of the veterinary antibiotic sulfadiazine and its metabolites under field conditions.

Veterinary antibiotics introduced into the environment may change the composition and functioning of soil microbial communities and promote the spreading of antibiotic resistance. Actual risks depend on the antibiotic's persistence and (bio)accessibility, which may differ between laboratory and field conditions. We examined the dissipation and sequestration of sulfadiazine (SDZ) and its main metabolites in soil under field conditions and how it was influenced by temperature, soil moisture, plant roots, and soil aggregation compared to controlled laboratory experiments. A sequential extraction accounted for easily extractable (CaCl2-extractable) and sequestered (microwave-extractable, residual) SDZ fractions. Dissipation from both fractions was largely temperature-dependent and could be well predicted from laboratory data recorded at different temperatures. Soil moisture additionally seemed to control sequestration, being accelerated in dry soil. Sequestration, as indicated by increasing apparent distribution coefficients and decreasing rates of kinetic release into CaCl2, governed the antibiotic's long-term fate in soil. Besides, we observed spatial gradients of antibiotic concentrations across soil aggregates and in the vicinity of roots. The former were short-lived and equilibrated due to aggregate reorganization, while dissipation of the easily extractable fraction was accelerated near roots throughout the growth period. There was little if any impact of the plants on residual SDZ concentrations.

N-acetyl-L-glutamate kinase (NAGK) from oxygenic phototrophs: P(II) signal transduction across domains of life reveals novel insights in NAGK control.

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the first committed step in arginine biosynthesis in organisms that perform the cyclic pathway of ornithine synthesis. In eukaryotic and bacterial oxygenic phototrophs, the activity of NAGK is controlled by the P(II) signal transduction protein. Recent X-ray analysis of NAGK-P(II) complexes from a higher plant (Arabidopsis thaliana) and a cyanobacterium (Synechococcus elongatus) revealed that despite several differences, the overall structure of the complex is highly similar. The present study analyzes the functional conservation of P(II)-mediated NAGK regulation in plants and cyanobacteria to distinguish between universal properties and those that are specific for the different phylogenetic lineages. This study shows that plant and cyanobacterial P(II) proteins can mutually regulate the NAGK enzymes across the domains of life, implying a high selective pressure to conserve P(II)-NAGK interaction over more than 1.2 billion years of separate evolution. The non-conserved C-terminus of S. elongatus NAGK was identified as an element, which strongly enhances arginine inhibition and is responsible for most of the differences between S. elongatus and A. thaliana NAGK with respect to arginine sensitivity. Both P(II) proteins relieve arginine inhibition of NAGK, and in both lineages, P(II)-mediated relief from arginine inhibition is antagonized by 2-oxoglutarate. Together, these properties highlight the conserved role of P(II) as a signal integrator of the C/N balance sensed as 2-oxoglutarate to regulate arginine synthesis in oxygenic phototrophs.

Treatment with cetuximab, bevacizumab and irinotecan in heavily pretreated patients with metastasized colorectal cancer.

BACKGROUND: Modern therapy algorithms for advanced colorectal cancer include the monoclonal antibodies bevacizumab and cetuximab. Routinely, these antibodies are given sequentially in combination with chemotherapy. The question whether a combination of bevacizumab and cetuximab is beneficial has not been answered. The results of the BOND-2 study showed that tumor drug resistance to irinotecan can be overcome by addition of both cetuximab and bevacizumab. PATIENTS AND METHODS: Here, we present the cases of five patients who were heavily pretreated and already had received cetuximab (and in two cases also bevacizumab). These patients received a chemotherapeutic regimen consisting of irinotecan, cetuximab and bevacizumab. RESULTS: The combination of these two antibodies with irinotecan surprisingly induced marked tumor response in four out of five patients. CONCLUSION: There are currently no published data concerning the question whether resistance against one monoclonal antibody can be overcome by the addition of another monoclonal antibody. These cases point to a possible novel treatment approach and provide an incentive for further experimental investigations. The treatment was well tolerated and should be considered as a further medical treatment strategy.

Docking of the periplasmic FecB binding protein to the FecCD transmembrane proteins in the ferric citrate transport system of Escherichia coli.

Citrate-mediated iron transport across the cytoplasmic membrane is catalyzed by an ABC transporter that consists of the periplasmic binding protein FecB, the transmembrane proteins FecC and FecD, and the ATPase FecE. Salt bridges between glutamate residues of the binding protein and arginine residues of the transmembrane proteins are predicted to mediate the positioning of the substrate-loaded binding protein on the transmembrane protein, based on the crystal structures of the ABC transporter for vitamin B(12), consisting of the BtuF binding protein and the BtuCD transmembrane proteins (E. L. Borths et al., Proc. Natl. Acad. Sci. USA 99:16642-16647, 2002). Here, we examined the role of the residues predicted to be involved in salt-bridge formation between FecB and FecCD by substituting these residues with alanine, cysteine, arginine, and glutamate and by analyzing the citrate-mediated iron transport of the mutants. Replacement of E93 in FecB with alanine [FecB(E93A)], cysteine, or arginine nearly abolished citrate-mediated iron transport. Mutation FecB(E222R) nearly eliminated transport, and FecB(E222A) and FecB(E222C) strongly reduced transport. FecD(R54C) and FecD(R51E) abolished transport, whereas other R-to-C mutations in putative interaction sites between FecCD and FecB substantially reduced transport. The introduced cysteine residues in FecB and FecCD also served to examine the formation of disulfide bridges in place of salt bridges between the binding protein and the transmembrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results suggest cross-linking of FecB(E93C) to FecD(R54C) and FecB(E222C) to FecC(R60C). The data are consistent with the proposal that FecB(E93) is contained in the region that binds to FecD and FecB(E222) in the region that binds to FecC.

Short time to progression under first-line chemotherapy is a negative prognostic factor for time to progression and residual survival under second-line chemotherapy in advanced pancreatic cancer.

OBJECTIVES: Second-line chemotherapy is widely used in advanced pancreatic cancer. However, only few data exist concerning the question who might benefit from second-line therapy. We intended to identify prognostic factors for time to second progression (TTP2) and residual survival following second-line chemotherapy of patients with advanced pancreatic cancer. METHODS: We performed a retrospective cohort study on 78 patients who started palliative chemotherapy at our department from January 2004 to June 2006 due to advanced adenocarcinoma of the pancreas. 46 patients who progressed following first-line therapy were analyzed. RESULTS: In multivariate analyses, time to first progression (TTP1) <6 months and elevated LDH at the time of diagnosis were shown to be strong and highly significant independent prognostic factors for TTP2 and residual survival. For patients with TTP1 <6 months, prognosis was poor with a median residual survival of 4.4 versus 7.5 months for those with TTP1 > or =6 months. CONCLUSION: In our cohort of patients with advanced pancreatic cancer treated with second-line chemotherapy, TTP1 <6 months is a strong negative prognostic factor for TTP2 and residual survival.

The genome of the novel phage Rtp, with a rosette-like tail tip, is homologous to the genome of phage T1.

A new Escherichia coli phage, named Rtp, was isolated and shown to be closely related to phage T1. Electron microscopy revealed that phage Rtp has a morphologically unique tail tip consisting of four leaf-like structures arranged in a rosette, whereas phage T1 has thinner, flexible leaves that thicken toward the ends. In contrast to T1, Rtp did not require FhuA and TonB for infection. The 46.2-kb genome of phage Rtp encodes 75 open reading frames, 47 of which are homologous to phage T1 genes. Like phage T1, phage Rtp encodes a large number of small genes at the genome termini that exhibit no sequence similarity to known genes. Six predicted genes larger than 300 nucleotides in the highly homologous region of Rtp are not found in T1. Two predicted HNH endonucleases are encoded at positions different from those in phage T1. The sequence similarity of rtp37, -38, -39, -41, -42, and -43 to equally arranged genes of lambdoid phages suggests a common tail assembly initiation complex. Protein Rtp43 is homologous to the lambda J protein, which determines lambda host specificity. Since the two proteins differ most in the C-proximal area, where the binding site to the LamB receptor resides in the J protein, we propose that Rtp43 contributes to Rtp host specificity. Lipoproteins similar to the predicted lipoprotein Rtp45 are found in a number of phages (encoded by cor genes) in which they prevent superinfection by inactivating the receptors. We propose that, similar to the proposed function of the phage T5 lipoprotein, Rtp45 prevents inactivation of Rtp by adsorption to its receptor during cells lysis. Rtp52 is a putative transcriptional regulator, for which 10 conserved inverted repeats were identified upstream of genes in the Rtp genome. In contrast, the much larger E. coli genome has only one such repeat sequence.

ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus.

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.

Point mutations in transmembrane helices 2 and 3 of ExbB and TolQ affect their activities in Escherichia coli K-12.

Replacement of glutamate 176, the only charged amino acid in the third transmembrane helix of ExbB, with alanine (E176A) abolished ExbB activity in all determined ExbB-dependent functions of Escherichia coli. Combination of the mutations T148A in the second transmembrane helix and T181A in the third transmembrane helix, proposed to form part of a proton pathway through ExbB, also resulted in inactive ExbB. E176 and T148 are strictly conserved in ExbB and TolQ proteins, and T181 is almost strictly conserved in ExbB, TolQ, and MotA.

TonB of Escherichia coli activates FhuA through interaction with the beta-barrel.

FhuA is a multifunctional protein in the outer membrane of Escherichia coli that actively transports Fe(3+)-ferrichrome and the antibiotics albomycin and rifamycin CGP 4832, and serves as a receptor for the unrelated phages T5, T1, phi80 and UC-1, colicin M and microcin J25. The energy source for active transport is the proton-motive force of the cytoplasmic membrane, which is required for all FhuA functions except infection by phage T5, and is thought to be mediated to the outer-membrane receptor FhuA by the TonB protein. The crystal structure of FhuA consists of a beta-barrel that is closed by a globular domain. The proximal region carries the TonB box (residues 7-11), for which genetic evidence exists that it interacts with the region around residue 160 of TonB. However, deletion of the TonB box along with the globular domain results in a protein, FhuAdelta5-160, that still displays TonB-dependent active ferrichrome transport across the outer membrane and confers sensitivity to the FhuA ligands. In this study synthetic nonapeptides identical in sequence to amino acids 150-158, 151-159, 152-160, 153-161 and 158-166 of TonB were shown to reduce ferrichrome transport of cells via wild-type FhuA and the corkless derivative FhuAdelta5-160, which suggests that this TonB region is involved in the interaction of TonB with the beta-barrel of FhuA. TonB missense mutants reduced the activity of FhuA and FhuAdelta5-160. TonB proteins of different Enterobacteriaceae activated FhuA and FhuAdelta5-160 to a similar degree. TonB of Pantoea agglomerans displayed low activity in an E. coli tonB mutant. Sequencing of the tonB gene of P. agglomerans revealed differences from E. coli TonB in the region around residue 160 of the deduced protein; these differences might contribute to the lower activity of the P. agglomerans TonB protein when coupled to the E. coli FhuA protein. The data support the theory that the beta-barrel receives the energy from the cytoplasmic membrane via TonB and responds to the energy input and thus represents the transporting domain of FhuA.