Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize the transport reaction. Binding of ATP to the NBD monomer results in formation of a NBD dimer. Hydrolysis of the ATP drives the dissociation of the dimer. The thermodynamics of distinct steps in the ATPase cycle of GlcV, the NBD of the glucose ABC transporter of the extreme thermoacidophile Sulfolobus solfataricus, were studied by isothermal titration calorimetry using the wild-type protein and two mutants, which are arrested at different steps in the ATP hydrolytic cycle. The G144A mutant is unable to dimerize, while the E166A mutant is defective in dimer dissociation. The ATP, ADP, and AMP-PNP binding affinities, stoichiometries, and enthalpies of binding were determined at different temperatures. From these data, the thermodynamic parameters of nucleotide binding, NBD dimerization, and ATP hydrolysis were calculated. The data demonstrate that the ATP hydrolysis cycle of isolated NBDs consists of consecutive steps where only the final step of ADP release is energetically unfavorable.

Characterization of the ATPase FlaI of the motor complex of the Pyrococcus furiosus archaellum and its interactions between the ATP-binding protein FlaH

The archaellum, the rotating motility structure of archaea, is best studied in the crenarchaeon Sulfolobus acidocaldarius. To better understand how assembly and rotation of this structure is driven, two ATP-binding proteins, FlaI and FlaH of the motor complex of the archaellum of the euryarchaeon Pyrococcus furiosus, were overexpressed, purified and studied. Contrary to the FlaI ATPase of S. acidocaldarius, which only forms a hexamer after binding of nucleotides, FlaI of P. furiosus formed a hexamer in a nucleotide independent manner. In this hexamer only 2 of the ATP binding sites were available for binding of the fluorescent ATP-analog MANT-ATP, suggesting a twofold symmetry in the hexamer. P. furiosus FlaI showed a 250-fold higher ATPase activity than S. acidocaldarius FlaI. Interaction studies between the isolated N- and C-terminal domains of FlaI showed interactions between the N- and C-terminal domains and strong interactions between the N-terminal domains not previously observed for ATPases involved in archaellum assembly. These interactions played a role in oligomerization and activity, suggesting a conformational state of the hexamer not observed before. Further interaction studies show that the C-terminal domain of PfFlaI interacts with the nucleotide binding protein FlaH. This interaction stimulates the ATPase activity of FlaI optimally at a 1:1 stoichiometry, suggesting that hexameric PfFlaI interacts with hexameric PfFlaH. These data help to further understand the complex interactions that are required to energize the archaellar motor

Individual variability in heart rate recovery after standardized submaximal exercise

To optimize performance, coaches and athletes are always looking for the right balance between training load and recovery. Therefore, closely monitoring of athletes is important. Heart rate recovery (HRR) after standardized sub maximal exercise has been proposed as a useful variable to monitor (Lamberts et al., 2004). However, it is well known that heart rate, next to biological variability, is influenced by several factors such as training load and psychosocial stress. So, the purpose was to look at individual variability in HRR from one week to another using the heart rate interval monitoring system (HIMS). Methods Eight elite Dutch female indoor hockey players (age: 23.9±3.91yr, length: 155.0±7.01cm, weight: 56.6±6.16kg) completed the HIMS two weeks in a row (Lamberts et al., 2004). The heart rate at the end of the last stage (HRend) was determined and the HRR was calculated one minute after the end of the last stage. Furthermore, training load and psychosocial stress and recovery were monitored using the Foster-method (1998) and the RESTQ-Sport (Nederhof et al., 2008), respectively. Results A strong correlation was found between the HRend from one week to the other (r=0.984 p

Structural Characterization of Outer Membrane Components of the Type IV Pili System in Pathogenic Neisseria

Structures of the type IV pili secretin complexes from Neisseria gonorrhoeae and Neisseria meningitidis, embedded in outer membranes were investigated by transmission electron microscopy. Single particle averaging revealed additional domains not observed previously. Secretin complexes of N. gonorrhoeae showed a double ring structure with a 14-15-fold symmetry in the central ring, and a 14-fold symmetry of the peripheral ring with 7 spikes protruding. In secretin complexes of N. meningitidis, the spikes were absent and the peripheral ring was partly or completely lacking. When present, it had a 19-fold symmetry. The structures of the complexes in several pil mutants were determined. Structures obtained from the pilC1/C2 adhesin and the pilW minor pilin deletion strains were similar to wild-type, whereas deletion of the homologue of N. meningitidis PilW resulted in the absence of secretin structures. Remarkably, the pilE pilin subunit and pilP lipoprotein deletion mutants showed a change in the symmetry of the peripheral ring from 14 to 19 and loss of spikes. The pilF ATPase mutant also lost the spikes, but maintained 14-fold symmetry. These results show that secretin complexes contain previously unidentified large and flexible extra domains with a probable role in stabilization or assembly of type IV pili

Effect of UV irradiation on Sulfolobus acidocaldarius and involvement of the general transcription factor TFB3 in the early UV response

Schult F, Lee TN, Albersmeier A, et al. Effect of UV irradiation on Sulfolobus acidocaldarius and involvement of the general transcription factor TFB3 in the early UV response. NUCLEIC ACIDS RESEARCH. 2018;46(14):7179-7192.Exposure to UV light can result in severe DNA damage. The alternative general transcription factor (GTF) TFB3 has been proposed to play a key role in the UV stress response in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Reporter gene assays confirmed that tfb3 is upregulated 90-180 min after UV treatment. In vivo tagging and immunodetection of TFB3 confirmed the induced expression at 90 min. Analysis of a tfb3 insertion mutant showed that genes encoding proteins of the Ups pili and the Ced DNA importer are no longer induced in a tfb3 insertion mutant after UV treatment, which was confirmed by aggregation assays. Thus, TFB3 plays a crucial role in the activation of these genes. Genome wide transcriptome analysis allowed a differentiation between a TFB3-dependent and a TFB3-independent early UV response. The TFB3-dependent UV response is characterized by the early induction of TFB3, followed by TFB3-dependent expression of genes involved in e.g. Ups pili formation and the Ced DNA importer. Many genes were downregulated in the tfb3 insertion mutant confirming the hypothesis that TFB3 acts as an activator of transcription. The TFB3-independent UV response includes the repression of nucleotide metabolism, replication and cell cycle progression in order to allow DNA repair

Characterization of the Single Stranded DNA Binding Protein SsbB Encoded in the Gonoccocal Genetic Island

Background: Most strains of Neisseria gonorrhoeae carry a Gonococcal Genetic Island which encodes a type IV secretion system involved in the secretion of ssDNA. We characterize the GGI-encoded ssDNA binding protein, SsbB. Close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA-processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase, and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins. Methodology/Principal Findings: In contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB forms a stable tetramer. Electrophoretic mobility shift assays and fluorescence titration assays, as well as atomic force microscopy demonstrate that SsbB binds ssDNA specifically with high affinity. SsbB binds single-stranded DNA with minimal binding frames for one or two SsbB tetramers of 15 and 70 nucleotides. The binding mode was independent of increasing Mg 2+ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity

Conjugative Plasmids of Neisseria gonorrhoeae

Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1α, β, γ, δ and ε subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between N. gonorrhoeae strains, but did not enhance transfer of a genetic marker

The potential for immunoglobulins and host defense peptides (HDPs) to reduce the use of antibiotics in animal production

Abstract Innate defense mechanisms are aimed at quickly containing and removing infectious microorganisms and involve local stromal and immune cell activation, neutrophil recruitment and activation and the induction of host defense peptides (defensins and cathelicidins), acute phase proteins and complement activation. As an alternative to antibiotics, innate immune mechanisms are highly relevant as they offer rapid general ways to, at least partially, protect against infections and enable the build-up of a sufficient adaptive immune response. This review describes two classes of promising alternatives to antibiotics based on components of the innate host defense. First we describe immunoglobulins applied to mimic the way in which they work in the newborn as locally acting broadly active defense molecules enforcing innate immunity barriers. Secondly, the potential of host defense peptides with different modes of action, used directly, induced in situ or used as vaccine adjuvants is described