A review on recent developments in mass spectrometry instrumentation and quantitative tools advancing bacterial proteomics

Proteomics has evolved substantially since its early days, some 20 years ago. In this mini-review, we aim to provide an overview of general methodologies and more recent developments in mass spectrometric approaches used for relative and absolute quantitation of proteins. Enhancement of sensitivity of the mass spectrometers as well as improved sample preparation and protein fractionation methods are resulting in a more comprehensive analysis of proteomes. We also document some upcoming trends for quantitative proteomics such as the use of label-free quantification methods. Hopefully, microbiologists will continue to explore proteomics as a tool in their research to understand the adaptation of microorganisms to their ever changing environment. We encourage them to incorporate some of the described new developments in mass spectrometry to facilitate their analyses and improve the general knowledge of the fascinating world of microorganisms.     

Paternity in mallards: effects of sperm quality and female sperm selection for inbreeding avoidance

Postcopulatory processes might play an important role in sexualselection. In theory, fertilization success could be controlledby females via selection of particular sperm within their reproductivetract, or it could be determined by sperm competition per se.In practice, these two mechanisms are difficult to disentangle.To assess the relative importance of both mechanisms we usedartificial insemination in combination with measurements ofsperm quality (swimming speed and motility) in mallards. Inthis species, females often lack behavioral control over copulationsand hence may use postcopulatory mechanisms to optimize theirreproductive output. One important factor affecting female fitnessmay be selection of genetically compatible males. To investigatethe influence of sperm quality and parental relatedness on paternitywe inseminated 12 groups of related females with a sperm mixturecontaining equal numbers of sperm from a brother and from anunrelated male. Paternity was independent of the relatednessof the siring male to the female but was significantly affectedby long-term sperm swimming speed and motility. No interactionbetween relatedness and sperm quality on paternity was observed.These results suggest that female mallards are not able to selectsperm on a purely genetic basis and emphasize the importanceof sperm quality in gaining paternity.     

Above- and below-ground vertebrate herbivory may each favour a different subordinate species in an aquatic plant community

At least two distinct trade-offs are thought to facilitate higher diversity in productive plant communities under herbivory. Higher investment in defence and enhanced colonization potential may both correlate with decreased competitive ability in plants. Herbivory may thus promote coexistence of plant species exhibiting divergent life history strategies. How different seasonally tied herbivore assemblages simultaneously affect plant community composition and diversity is, however, largely unknown. Two contrasting types of herbivory can be distinguished in the aquatic vegetation of the shallow lake Lauwersmeer. In summer, predominantly above-ground tissues are eaten, whereas in winter, waterfowl forage on below-ground plant propagules. In a 4-year exclosure study we experimentally separated above-ground herbivory by waterfowl and large fish in summer from below-ground herbivory by Bewick’s swans in winter. We measured the individual and combined effects of both herbivory periods on the composition of the three-species aquatic plant community. Herbivory effect sizes varied considerably from year to year. In 2 years herbivore exclusion in summer reinforced dominance of Potamogeton pectinatus with a concomitant decrease in Potamogeton pusillus, whereas no strong, unequivocal effect was observed in the other 2 years. Winter exclusion, on the other hand, had a negative effect on Zannichellia palustris, but the effect size differed considerably between years. We suggest that the colonization ability of Z. palustris may have enabled this species to be more abundant after reduction of P. pectinatus tuber densities by swans. Evenness decreased due to herbivore exclusion in summer. We conclude that seasonally tied above- and below-ground herbivory may each stimulate different components of a macrophyte community as they each favoured a different subordinate plant species.     

Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha i subunits and regulates Galpha i-mediated cell signaling

Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein alpha subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Galpha(z) subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Galpha(z,) but also with Galpha(i), as supported by in vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind (35)S-labeled Galpha(i1) protein in an AlF(4)(-)dependent manner. The interaction between RGSZ1 and Galpha(i) was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Galpha(i1) in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Galpha(i)-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an alpha(2)-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Galpha(i) and regulates Galpha(i)-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.     

Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis

Background

F-spondin is a multi-domain extracellular matrix (ECM) protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin_N domain and the F-spondin domain (FS domain) comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin.

Results

We present the crystal structure of human F-spondin FS domain at 1.95Å resolution. The structure reveals a Ca²⁺-binding C2 domain variant with an 8-stranded antiparallel β-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain.

Conclusion

The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca²⁺- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.     

Mesostigmatic mites (Acari: Mesostigmata) in nests of the Eurasian griffon vulture (<Emphasis Type="Italic">Gyps fulvus</Emphasis>) in Croatia

We examined the species composition and community structure of mites of the order Mesostigmata (Acari) in nests of the Eurasian griffon vulture (Gyps fulvus Hablizl, 1783) in Croatia. Material collected from 18 nests included 565 mites belonging to seven species. The most abundant species were Leiodinychus orbicularis (C.L. Koch, 1839) (Trematuridae) and Androlaelaps casalis (Berlese, 1887) (Laelapidae). The results were compared with the community structure and frequency of dominant species of Mesostigmata in nests of 32 other bird species. Leiodinychus orbicularis occurred in the nests of 13 species of birds. It is a typical nidicolous species which occurs most frequently in the perennial nests of birds of prey. In contrast, A. casalis rarely occurs in the nests of birds of prey.     

Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways

Newborns are colonized with an intestinal microbiota shortly after birth, but the factors governing the retention and abundance of specific microbial lineages are unknown. Nursing infants consume human milk oligosaccharides (HMOs) that pass undigested to the distal gut, where they may be digested by microbes. We determined that the prominent neonate gut residents, Bacteroides thetaiotaomicron and Bacteroides fragilis, induce the same genes during HMO consumption that are used to harvest host mucus glycans, which are structurally similar to HMOs. Lacto-N-neotetraose, a specific HMO component, selects for HMO-adapted species such as Bifidobacterium infantis, which cannot use mucus, and provides a selective advantage to B. infantis in vivo when biassociated with B. thetaiotaomicron in the gnotobiotic mouse gut. This indicates that the complex oligosaccharide mixture within HMOs attracts both mutualistic mucus-adapted species and HMO-adapted bifidobacteria to the infant intestine that likely facilitate both milk and future solid food digestion.     

Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution

Amylosucrase is a transglycosidase which belongs to family 13 of the glycoside hydrolases and transglycosidases, and catalyses the formation of amylose from sucrose. Its potential use as an industrial tool for the synthesis or modification of polysaccharides is hampered by its low catalytic efficiency on sucrose alone, its low stability and the catalysis of side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling and selective screening (directed evolution) was applied, in order to generate more efficient variants of the enzyme. This resulted in isolation of the most active amylosucrase (Asn387Asp) characterized to date, with a 60% increase in activity and a highly efficient polymerase (Glu227Gly) that produces a longer polymer than the wild-type enzyme. Furthermore, judged from the screening results, several variants are expected to be improved concerning activity and/or thermostability. Most of the amino acid substitutions observed in the totality of these improved variants are clustered around specific regions. The secondary sucrose-binding site and beta strand 7, connected to the important Asp393 residue, are found to be important for amylosucrase activity, whereas a specific loop in the B-domain is involved in amylosucrase specificity and stability.     

Different effects of carbohydrate binding modules on the viscoelasticity of nanocellulose gels

Many cellulose degrading and modifying enzymes have distinct parts called carbohydrate binding modules (CBMs). The CBMs have been shown to increase the concentration of enzymes on the insoluble substrate and thereby enhance catalytic activity. It has been suggested that CBMs also have a role in disrupting or dispersing the insoluble cellulose substrate, but dispute remains and explicit evidence of such a mechanism is lacking. We produced the isolated CBMs from two major cellulases (Cel6A and Cel7A) from Trichoderma reesei as recombinant proteins in Escherichia coli. We then studied the viscoelastic properties of native unmodified cellulose nanofibrils (CNF) in combination with the highly purified CBMs to detect possible functional effects of the CBMs on the CNF. The two CBMs showed clearly different effects on the viscoelastic properties of CNF. The difference in effects is noteworthy, yet it was not possible to conclude for example disruptive effects. We discuss here the alternative explanations for viscoelastic effects on CNF caused by CBMs, including the effect of ionic cosolutes.