NMR assignment of the Rhodobacter sphaeroides fasciclin-1 domain protein (Fdp)

We report the almost complete assignment of ¹H, ¹³C and ¹⁵N nuclei in the 137-residue his-tagged fasciclin domain protein (Fdp) from Rhodobacter sphaeroides. Fdp is homologous to fasciclin I domains, including Drosophila FAS1 and M. tuberculosis MPB70 and plays a role in cell adhesion.     

Fitness increases with partner and neighbour allopreening

Altruism and selfishness are fundamental characteristics of human and animal societies. Among colonial biparental species, breeding outcome depends on interactions between mates and neighbours. However, the relationships between cooperation within and among partnerships and fitness have not been fully investigated. We show that in the highly colonial common guillemot (Uria aalge), altruistic behaviour (allopreening) towards a mate was positively related to long-term fitness, whereas allopreening a neighbour was related to current fitness. Turnover is much lower within than between pairs, so our results suggest that allopreening within pairs generates fitness returns at longer timescales than between pairs. Allopreening not only removes ectoparasites and maintains plumage condition, but may also have important social functions. We found a negative relationship between fight rate and allopreen rate between breeding neighbours, with nests exhibiting low breeding success having a higher frequency of fights with neighbours. We also found evidence for reciprocity in allopreening. Thus, allopreening may function as a reciprocal stress reducer, to decrease the likelihood of fights and associated breeding failure. We suggest that altruistic behaviour has long-term benefits for the survival of the offspring when living in a crowded neighbourhood.     

Eat now,pay later? Evidence of deferred food-processing costs in diving seals

Seals may delay costly physiological processes (e.g. digestion) that are incompatible with the physiological adjustments to diving until after periods of active foraging. We present unusual profiles of metabolic rate (MR) in grey seals measured during long-term simulation of foraging trips (4-5 days) that provide evidence for this. We measured extremely high MRs (up to almost seven times the baseline levels) and high heart rates during extended surface intervals, where the seals were motionless at the surface. These occurred most often during the night and occurred frequently many hours after the end of feeding bouts. The duration and amount of oxygen consumed above baseline levels during these events was correlated with the amount of food eaten, confirming that these metabolic peaks were related to the processing of food eaten during foraging periods earlier in the day. We suggest that these periods of high MR represent a payback of costs deferred during foraging.     

Structure-templated predictions of novel protein interactions from sequence information

The multitude of functions performed in the cell are largely controlled by a set of carefully orchestrated protein interactions often facilitated by specific binding of conserved domains in the interacting proteins. Interacting domains commonly exhibit distinct binding specificity to short and conserved recognition peptides called binding profiles. Although many conserved domains are known in nature, only a few have well-characterized binding profiles. Here, we describe a novel predictive method known as domain–motif interactions from structural topology (D-MIST) for elucidating the binding profiles of interacting domains. A set of domains and their corresponding binding profiles were derived from extant protein structures and protein interaction data and then used to predict novel protein interactions in yeast. A number of the predicted interactions were verified experimentally, including new interactions of the mitotic exit network, RNA polymerases, nucleotide metabolism enzymes, and the chaperone complex. These results demonstrate that new protein interactions can be predicted exclusively from sequence information.     

Sequence analysis of the conserved protamine gene cluster shows that it contains a fourth expressed gene

Structural data are presented on the protamine gene cluster (PGC) of human, mouse, rat, and bull. By restriction mapping we demonstrate that the organization of the protamine cluster is conserved throughout all four species, i.e., the genes are situated in a head to tail arrangement in the order: protamine l-protamine 2-transition protein 2. Further, we established the nucleotide sequence of the entire human PGC (25 kb in total) and the 3′ portion of the rat protamine cluster (PRM2 and TNP2 genes and intergenic region). In addition, a 1 kb fragment of the bovine and murine protamine cluster, situated between PRM2 and TNP2, was sequenced. This fragment is conserved regarding sequence, position, and orientation in all species examined, and was classified as likely coding region by gene recognition program GRAIL. Using the rat fragment as a probe in RNA blots, we detected a testis-specific signal of about 0.5 kb. Finally, we demonstrate a high density of Alu elements, both full and fragmented copies, in the human PGC and discuss their localization with respect to evolutionary and functional aspects. © 1996 Wiley-Liss, Inc.     

Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.Subject terms: Environmental microbiology, Sequencing, Microbiology     

Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex.

We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promoter and the SV40 intron/polyadenylation site. Transgenes were prepared for cloning into this vector by introduction of compatible restriction sites by PCR. A vector expressing rat granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed using DNA-protein complex as well as by traditional recombination techniques. The efficacy of our adenoviral cloning system utilizing DNA-protein complex was two logs higher than that seen using homologous recombination. All viruses generated by directional ligation of the insert into the vector DNA-protein complexes contained the desired transgene in the correct orientation. This technique greatly simplifies and accelerates the generation of recombinant adenoviral vectors.     

Identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses

Ebola virus causes lethal hemorrhagic fever in humans, but currently there are no effective vaccines or antiviral compounds for this infectious disease. Passive transfer of monoclonal antibodies (MAbs) protects mice from lethal Ebola virus infection (J. A. Wilson, M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart, Science 287:1664-1666, 2000). However, the epitopes responsible for neutralization have been only partially characterized because some of the MAbs do not recognize the short synthetic peptides used for epitope mapping. To identify the amino acids recognized by neutralizing and protective antibodies, we generated a recombinant vesicular stomatitis virus (VSV) containing the Ebola virus glycoprotein-encoding gene instead of the VSV G protein-encoding gene and used it to select escape variants by growing it in the presence of a MAb (133/3.16 or 226/8.1) that neutralizes the infectivity of the virus. All three variants selected by MAb 133/3.16 contained a single amino acid substitution at amino acid position 549 in the GP2 subunit. By contrast, MAb 226/8.1 selected three different variants containing substitutions at positions 134, 194, and 199 in the GP1 subunit, suggesting that this antibody recognized a conformational epitope. Passive transfer of each of these MAbs completely protected mice from a lethal Ebola virus infection. These data indicate that neutralizing antibody cocktails for passive prophylaxis and therapy of Ebola hemorrhagic fever can reduce the possibility of the emergence of antigenic variants in infected individuals.