The agonistic and sexual functions of vocalizations of male brown-headed cowbirds,Molothrus ater

Functional analyses of three vocalizations of male brown-headed cowbirds, Molothrus ater, are presented and it is shown that two of these function as songs. Perched song (PS) is given by stationary birds and is used over short distances in male-male aggression and female courtship. The ‘flight whistle’ (FW) is a multisyllabic, usually pure tone, vocalization and the ‘single syllable flight call’ (SS) a monosyllabic pure tone. FWs and SSs are given occasionally by perched males and repetitively by flying males. Field playbacks of FWs showed that male cowbirds responded in the agonistic way most passerines respond to playback of conspecific song. Playbacks of the female's chatter call demonstrated that males of all three subspecies use FWs and SSs, but almost never PSs, to communicate with a female as they approach her. Thus FWs and SSs are used to communicate over long distances with both males and females. FWs are also used in one critical short-distance context as they are significantly more likely than PSs to be given just before copulations in nature, at least in our western study area. By contrast, studies of captive eastern cowbirds showed that PSs regularly precede copulations whereas FWs rarely do so. These different results for western and eastern birds may be an artefact of captivity and/or a result of geographical variation in behaviour.     

Identification of the Docking Site between a Type III Secretion System ATPase and a Chaperone for Effector Cargo

A number of Gram-negative pathogens utilize type III secretion systems (T3SSs) to inject bacterial effector proteins into the host. An important component of T3SSs is a conserved ATPase that captures chaperone-effector complexes and energizes their dissociation to facilitate effector translocation. To date, there has been limited work characterizing the chaperone-T3SS ATPase interaction despite it being a fundamental aspect of T3SS function. In this study, we present the 2.1 Å resolution crystal structure of the Salmonella enterica SPI-2-encoded ATPase, SsaN. Our structure revealed a local and functionally important novel feature in helix 10 that we used to define the interaction domain relevant to chaperone binding. We modeled the interaction between the multicargo chaperone, SrcA, and SsaN and validated this model using mutagenesis to identify the residues on both the chaperone and ATPase that mediate the interaction. Finally, we quantified the benefit of this molecular interaction on bacterial fitness in vivo using chromosomal exchange of wild-type ssaN with mutants that retain ATPase activity but no longer capture the chaperone. Our findings provide insight into chaperone recognition by T3SS ATPases and demonstrate the importance of the chaperone-T3SS ATPase interaction for the pathogenesis of Salmonella.     

PrgB promotes aggregation,biofilm formation,and conjugation through DNA binding and compaction

Gram‐positive bacteria deploy type IV secretion systems (T4SSs) to facilitate horizontal gene transfer. The T4SSs of Gram‐positive bacteria rely on surface adhesins as opposed to conjugative pili to facilitate mating. Enterococcus faecalis PrgB is a surface adhesin that promotes mating pair formation and robust biofilm development in an extracellular DNA (eDNA) dependent manner. Here, we report the structure of the adhesin domain of PrgB. The adhesin domain binds and compacts DNA in vitro. In vivo PrgB deleted of its adhesin domain does not support cellular aggregation, biofilm development and conjugative DNA transfer. PrgB also binds lipoteichoic acid (LTA), which competes with DNA binding. We propose that PrgB binding and compaction of eDNA facilitates cell aggregation and plays an important role in establishment of early biofilms in mono‐ or polyspecies settings. Within these biofilms, PrgB mediates formation and stabilization of direct cell‐cell contacts through alternative binding of cell‐bound LTA, which in turn promotes establishment of productive mating junctions and efficient intra‐ or inter‐species T4SS‐mediated gene transfer.     

Systematic Regional Variations in Purkinje Cell Spiking Patterns

In contrast to the uniform anatomy of the cerebellar cortex, molecular and physiological studies indicate that significant differences exist between cortical regions, suggesting that the spiking activity of Purkinje cells (PCs) in different regions could also show distinct characteristics. To investigate this possibility we obtained extracellular recordings from PCs in different zebrin bands in crus IIa and vermis lobules VIII and IX in anesthetized rats in order to compare PC firing characteristics between zebrin positive (Z+) and negative (Z−) bands. In addition, we analyzed recordings from PCs in the A2 and C1 zones of several lobules in the posterior lobe, which largely contain Z+ and Z− PCs, respectively. In both datasets significant differences in simple spike (SS) activity were observed between cortical regions. Specifically, Z− and C1 PCs had higher SS firing rates than Z+ and A2 PCs, respectively. The irregularity of SS firing (as assessed by measures of interspike interval distribution) was greater in Z+ bands in both absolute and relative terms. The results regarding systematic variations in complex spike (CS) activity were less consistent, suggesting that while real differences can exist, they may be sensitive to other factors than the cortical location of the PC. However, differences in the interactions between SSs and CSs, including the post-CS pause in SSs and post-pause modulation of SSs, were also consistently observed between bands. Similar, though less strong trends were observed in the zonal recordings. These systematic variations in spontaneous firing characteristics of PCs between zebrin bands in vivo, raises the possibility that fundamental differences in information encoding exist between cerebellar cortical regions.     

Purification and characterization of two sucrose synthase isoforms from Japanese pear fruit

Two isoforms (SS I and SS II) of sucrose synthase (SS; EC 2.54.1.13) were purified from Japanese pear fruit and their properties were compared. SS I mainly appeared in young fruit and SS II mainly in mature fruit. SS I and SS II were purified to the specific activity of 3.37 and 4.26 (units (mg protein)(-1)), respectively. The MW of native and subunit proteins of SS I and SS II were almost the same and both SSs seemed to be a tetramer composed of an 83 kDa polypeptide. However, the ionic charges of the native proteins and the kinetic parameters of SSs were different. Specifically, the Km value for UDP-glucose in SS I was the same as that for UDP, while the Km value for UDP-glucose in SS II was less than that for UDP. SS II easily reacted for sucrose synthesis than sucrose cleavage compared with SS I. Therefore, it is considered that SS I and SS II play different roles in the utilization of carbohydrate in young and mature fruit, respectively.     

Maintenance of variation in sexually selected traits in females: a case study using intrasexual aggression in tree swallows Tachycineta bicolor

A fundamental question in evolutionary biology is how phenotypic variation is maintained in the face of selection that ought to deplete that variation. Much research has investigated this question in traits favored via sexual selection in males, with a common solution implicating the condition dependence of sexually selected phenotypes. Despite growing interest in sexual selection on females, it is not clear if the same mechanisms maintain variation in female ornaments, weaponry or other female behaviors targeted by sexual selection. An important step in testing condition dependence in females is thus to identify whether sexually selected female phenotypes are associated with condition and also with potential costs. Here, I examine these two components of condition dependence for a sexually selected behavior, intrasexual aggression, in female tree swallows Tachycineta bicolor. I asked whether high levels of intrasexual aggression map onto natural variation in female condition and whether aggression is associated with one potential behavioral cost: performance in a vertically challenging test of flight. More aggressive females were heavier for their body size, heavier for their wing size and showed decreased flight ability, relative to less aggressive females. These findings are consistent with condition dependence, where only females in better condition are able to be highly aggressive. The association between high aggression and reduced flight ability may result from the additional lift required to power these relatively heavier birds. These associations between natural variation in aggressive behavior, morphology and flight ability are consistent with condition dependence because they confirm two basic assumptions of condition dependence: a link between aggression and condition, and a link between aggression and a behavioral cost, the speed of escape flight. As the first study to examine these assumptions for a conspicuous behavior favored by intrasexual selection in females, this study suggests broad relevance of condition dependence.     

Identification of the Soluble Starch Synthase Activities of Maize Endosperm

This study identified the complement of soluble starch synthases (SSs) present in developing maize (Zea mays) endosperm. The product of the du1 gene, DU1, was shown to be one of the two major soluble SSs. The C-terminal 450 residues of DU1 comprise eight sequence blocks conserved in 28 known or predicted glucan synthases. This region of DU1 was expressed in Escherichia coli and shown to possess SS activity. DU1-specific antisera detected a soluble endosperm protein of more than 200 kD that was lacking in du1- mutants. These antisera eliminated 20% to 30% of the soluble SS activity from kernel extracts. Antiserum against the isozyme zSSI eliminated approximately 60% of the total soluble SS, and immunodepletion of du1- mutant extracts with this antiserum nearly eliminated SS activity. Two soluble SS activities were identified by electrophoretic fractionation, each of which correlated specifically with zSSI or DU1. Thus, DU1 and zSSI accounted for the great majority of soluble SS activity present in developing endosperm. The relative activity of the two isozymes did not change significantly during the starch biosynthetic period. DU1 and zSSI may be interdependent, because mutant extracts lacking DU1 exhibited a significant stimulation of the remaining SS activity.     

A Distinct and Divergent Lineage of Genomic Island-Associated Type IV Secretion Systems in Legionella

Legionella encodes multiple classes of Type IV Secretion Systems (T4SSs), including the Dot/Icm protein secretion system that is essential for intracellular multiplication in amoebal and human hosts. Other T4SSs not essential for virulence are thought to facilitate the acquisition of niche-specific adaptation genes including the numerous effector genes that are a hallmark of this genus. Previously, we identified two novel gene clusters in the draft genome of Legionella pneumophila strain 130b that encode homologues of a subtype of T4SS, the genomic island-associated T4SS (GI-T4SS), usually associated with integrative and conjugative elements (ICE). In this study, we performed genomic analyses of 14 homologous GI-T4SS clusters found in eight publicly available Legionella genomes and show that this cluster is unusually well conserved in a region of high plasticity. Phylogenetic analyses show that Legionella GI-T4SSs are substantially divergent from other members of this subtype of T4SS and represent a novel clade of GI-T4SSs only found in this genus. The GI-T4SS was found to be under purifying selection, suggesting it is functional and may play an important role in the evolution and adaptation of Legionella. Like other GI-T4SSs, the Legionella clusters are also associated with ICEs, but lack the typical integration and replication modules of related ICEs. The absence of complete replication and DNA pre-processing modules, together with the presence of Legionella-specific regulatory elements, suggest the Legionella GI-T4SS-associated ICE is unique and may employ novel mechanisms of regulation, maintenance and excision. The Legionella GI-T4SS cluster was found to be associated with several cargo genes, including numerous antibiotic resistance and virulence factors, which may confer a fitness benefit to the organism. The in-silico characterisation of this new T4SS furthers our understanding of the diversity of secretion systems involved in the frequent horizontal gene transfers that allow Legionella to adapt to and exploit diverse environmental niches.     

Cloning,characterisation and comparative analysis of a starch synthase IV gene in wheat: functional and evolutionary implications

Background  

Starch is of great importance to humans as a food and biomaterial, and the amount and structure of starch made in plants is determined in part by starch synthase (SS) activity. Five SS isoforms, SSI, II, III, IV and Granule Bound SSI, have been identified, each with a unique catalytic role in starch synthesis. The basic mode of action of SSs is known; however our knowledge of several aspects of SS enzymology at the structural and mechanistic level is incomplete. To gain a better understanding of the differences in SS sequences that underscore their specificity, the previously uncharacterised SSIVb from wheat was cloned and extensive bioinformatics analyses of this and other SSs sequences were done.     

Molecular architecture of bacterial type IV secretion systems

Bacterial type IV secretion systems (T4SSs) are a versatile group of nanomachines that can horizontally transfer DNA through conjugation and deliver effector proteins into a wide range of target cells. The components of T4SSs in gram-negative bacteria are organized into several large subassemblies: an inner membrane complex, an outer membrane core complex, and, in some species, an extracellular pilus. Cryo-electron tomography has been used to define the structures of T4SSs in intact bacteria, and high-resolution structural models are now available for isolated core complexes from conjugation systems, the Xanthomonas citri T4SS, the Helicobacter pylori Cag T4SS, and the Legionella pneumophila Dot/Icm T4SS. In this review, we compare the molecular architectures of these T4SSs, focusing especially on the structures of core complexes. We discuss structural features that are shared by multiple T4SSs as well as evolutionary strategies used for T4SS diversification. Finally, we discuss how structural variations among T4SSs may confer specialized functional properties.     

Structural biology of type VI secretion systems

Type VI secretion systems (T6SSs) are transenvelope complexes specialized in the transport of proteins or domains directly into target cells. These systems are versatile as they can target either eukaryotic host cells and therefore modulate the bacteria-host interaction and pathogenesis or bacterial cells and therefore facilitate access to a specific niche. These molecular machines comprise at least 13 proteins. Although recent years have witnessed advances in the role and function of these secretion systems, little is known about how these complexes assemble in the cell envelope. Interestingly, the current information converges to the idea that T6SSs are composed of two subassemblies, one resembling the contractile bacteriophage tail, whereas the other subunits are embedded in the inner and outer membranes and anchor the bacteriophage-like structure to the cell envelope. In this review, we summarize recent structural information on individual T6SS components emphasizing the fact that T6SSs are composite systems, adapting subunits from various origins.     

Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals

Splice sites (SSs)—short nucleotide sequences flanking introns—are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints.     

Identification of activated cryptic 5' splice sites using structure profiles and odds measure

The activation of cryptic 5' splice sites (5' SSs) is often related to human hereditary diseases. The DNA-based mutation screening strategies are commonly used to recognize the cryptic 5' SSs, because features of the local DNA sequence can influence the choice of cryptic 5' SSs. To improve the identification of the cryptic 5' SSs, we developed a structure-based method, named SPO (structure profiles and odds measure), which combines two parameters, the structural feature derived from hydroxyl radical cleavage pattern and odds measure, to assess the likelihood of a cryptic 5' SS activation in competing with its paired authentic 5' SS. Compared to the current tools for identifying activated cryptic 5' SSs, the SPO algorithm achieves higher prediction accuracy than the other methods, including MaxEnt, MDD, Markov model, weight matrix model, Shapiro and Senapathy matrix, R(i) and ΔG. In addition, the predicted ΔSPO scores from the SPO algorithm exhibited a greater degree of correlation with the strength of cryptic 5' SS activation than that measured from the other seven methods. In conclusion, the SPO algorithm provides an optimal identification of cryptic 5' SSs, can be applied in designing mutagenesis experiments for various splicing events and may be helpful to investigate the relationship between structural variants and human hereditary diseases.     

The archetype Pseudomonas aeruginosa proteins TssB and TagJ form a novel subcomplex in the bacterial type VI secretion system

In Pseudomonas aeruginosa three type VI secretion systems (T6SSs) coexist, called H1‐ to H3‐T6SSs. Several T6SS components are proposed to be part of a macromolecular complex resembling the bacteriophage tail. The T6SS protein HsiE1 (TagJ) is unique to the H1‐T6SS and absent from the H2‐ and H3‐T6SSs. We demonstrate that HsiE1 interacts with a predicted N‐terminal α‐helix in HsiB1 (TssB) thus forming a novel subcomplex of the T6SS. HsiB1 is homologous to the Vibrio cholerae VipA component, which contributes to the formation of a bacteriophage tail sheath‐like structure. We show that the interaction between HsiE1 and HsiB1 is specific and does not occur between HsiE1 and HsiB2. Proteins of the TssB family encoded in T6SS clusters lacking a gene encoding a TagJ‐like component are often devoid of the predicted N‐terminal helical region, which suggests co‐evolution. We observe that a synthetic peptide corresponding to the N‐terminal 20 amino acids of HsiB1 interacts with purified HsiE1 protein. This interaction is a common feature to other bacterial T6SSs that display a TagJ homologue as shown here with Serratia marcescens. We further show that hsiE1 is a non‐essential gene for the T6SS and suggest that HsiE1 may modulate incorporation of HsiB1 into the T6SS.     

Flower choice in honeybees: Effects of food value and flower density

To test the effects of food value on the flower choice, individual honeybees (Apis mellifera) were offered a choice of 25 % sucrose solution (SS) and 1 of 6 different SSs, ranging from 5 % to 50 % SS, at either a low or a high flower density. Artificial flowers were filled with each SS. The honeybees showed a stronger preference for a concentrated SS to a diluted SS at a high than at a low flower density, and the degree of preference was positively correlated to the difference in the sucrose concentration between paired SSs. These foraging patterns were consistent with qualitative predictions from optimal foraging theory. Furthermore, it was found that experience in feeding on a concentrated SS lowered the foraging motivation for a diluted SS at the high flower density, but not at the low flower density. I discuss the effects of food density, food profitability and experience on the foraging behaviour of honeybees.     

Missplicing suppressor alleles of Arabidopsis PRE-MRNA PROCESSING FACTOR 8 increase splicing fidelity by reducing the use of novel splice sites

Efficient splicing requires a balance between high-fidelity splice-site (SS) selection and speed. In Saccharomyces cerevisiae, Pre-mRNA processing factor 8 (Prp8) helps to balance precise SS selection and rapid, efficient intron excision and exon joining. argonaute1-52 (ago1-52) and incurvata13 (icu13) are hypomorphic alleles of the Arabidopsis thaliana genes ARGONAUTE1 (AGO1) and AUXIN RESISTANT6 (AXR6) that harbor point mutations creating a novel 3′SS and 5′SS, respectively. The spliceosome recognizes these novel SSs, as well as the intact genuine SSs, producing a mixture of wild-type and aberrant mature mRNAs. Here, we characterized five novel mutant alleles of PRP8 (one of the two Arabidopsis co-orthologs of yeast Prp8), naming these alleles morphology of ago1-52 suppressed5 (mas5). In the mas5-1 background, the spliceosome preferentially recognizes the intact genuine 3′SS of ago1-52 and 5′SS of icu13. Since point mutations that damage genuine SSs make the spliceosome prone to recognizing cryptic SSs, we also tested alleles of four genes carrying damaged genuine SSs, finding that mas5-1 did not suppress their missplicing. The mas5-1 and mas5-3 mutations represent a novel class of missplicing suppressors that increase splicing fidelity by hampering the use of novel SSs, but do not alter general pre-mRNA splicing.     

Identification of the Vibrio parahaemolyticus type III secretion system 2-associated chaperone VocC for the T3SS2-specific effector VopC

The enteropathogen Vibrio parahaemolyticus possesses two sets of type III secretion systems, T3SS1 and T3SS2. Effector proteins secreted by these T3SSs are delivered into host cells, leading to cell death or diarrhea. However, it is not known how specific effectors are secreted through a specific T3SS when both T3SSs are expressed within bacteria. One molecule thought to determine secretion specificity is a T3SS-associated chaperone; however, no T3SS2-specific chaperone has been identified. Therefore, we screened T3SS2 chaperone candidates by a pull-down assay using T3SS2 effectors fused with glutathione-S-transferase. A secretion assay revealed that the newly identified cognate chaperone VocC for the T3SS2-specific effector VopC was required for the efficient secretion of the substrate through T3SS2. Further experiments determined the chaperone-binding domain and the amino-terminal secretion signal of the cognate effector. These findings, in addition to the previously identified T3SS1-specific chaperone, VecA, provide a strategy to clarify the specificity of effector secretion through T3SSs of V.?parahaemolyticus.     

Promiscuity drives sexual selection in a socially monogamous bird

Many socially monogamous species paradoxically show signs of strong sexual selection, suggesting cryptic sources of sexual competition among males. Darwin argued that sexual selection could operate in monogamous systems if breeding sex ratios are biased or if some males attract highly fecund females. Alternatively, sexual selection might result from promiscuous copulations outside the pair bond, although several recent studies have cast doubt on this possibility, in particular by showing that variance in apparent male reproductive success (number of social young) differs little from variance in actual male reproductive success (number of young sired). Our results from a long-term study of the socially monogamous splendid fairy-wren (Malurus splendens) demonstrate that such comparisons are misleading and do not adequately assess the effects of extra-pair paternity (EPP). By partitioning the opportunity for selection and calculating Bateman gradients, we show that EPP has a strong effect on male annual and lifetime fitness, whereas other proposed mechanisms of sexual selection do not. Thus, EPP drives sexual selection in this, and possibly other, socially monogamous species.     

Protein interactions within and between two F-type type IV secretion systems

Bacterial type IV secretion systems (T4SSs) can mediate conjugation. The T4SS from Neisseria gonorrhoeae possesses the unique ability to mediate DNA secretion into the extracellular environment. The N. gonorrhoeae T4SS can be grouped with F-type conjugative T4SSs based on homology. We tested 17 proteins important for DNA secretion by N. gonorrhoeae for protein interactions. The BACTH-TM bacterial two-hybrid system was successfully used to study periplasmic interactions. By determining if the same interactions were observed for F-plasmid T4SS proteins and when one interaction partner was replaced by the corresponding protein from the other T4SS, we aimed to identify features associated with the unique function of the N. gonorrhoeae T4SS as well as generic features of F-type T4SSs. For both systems, we observed already described interactions shared by homologs from other T4SSs as well as new and described interactions between F-type T4SS-specific proteins. Furthermore, we demonstrate, for the first-time, interactions between proteins with homology to the conserved T4SS outer membrane core proteins and F-type-specific proteins and we confirmed two of them by co-purification. The F-type-specific protein TraH_N was found to localize to the outer membrane and the presence of significant amounts of TraH_N in the outer membrane requires TraG_N.