Lateral Transfers of Serine Hydroxymethyltransferase (glyA) and UDP-N-Acetylglucosamine Enolpyruvyl Transferase (murA) Genes from Free-living Actinobacteria to the Parasitic Chlamydiae

The chlamydiae are important human and animal pathogens which form a phylogentically distinct lineage within the Bacteria. There is evidence that some genes in these obligate intracellular parasites have undergone lateral exchange with other free-living organisms. In the present work, we describe two interesting cases of lateral gene transfer between chlamydiae and actinobacteria, which have been identified based on the shared presence of conserved inserts in two important proteins. In the enzyme serine hydroxymethyltransferase (SHMT or GlyA protein), which links amino acid and nucleotide metabolisms by generating the key intermediate for one-carbon transfer reactions, two conserved inserts of 3 and 31 amino acids (aa) are uniquely present in various chlamydiae species as well as in a subset of Actinobacteria and in the Treponema species. Similarly, in the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which is involved in the synthesis of cell wall peptidoglycan, a 16-aa conserved insert is specifically present in various sequenced chlamydiae and a subset of actinobacteria (i.e., Streptomyces, Actinomyces, Tropheryma, Bifidobacterium, Leifsonia, Arthrobacter, and Brevibacterium). To determine the phylogenetic depths of the GlyA and MurA inserts, the fragments of these genes from two chlamydiae-like species, Simkania negevensis and Waddlia chondrophila, were PCR amplified and sequenced. The presence of the corresponding inserts in both these species strongly indicates that these inserts are distinctive characteristics of the Chlamydiales order. In phylogenetic trees based on GlyA and MurA protein sequences, the chlamydiae species (and also the Treponema species in the case of GlyA) branched with a high affinity with various insert-containing actinobacteria within a clade of other actinobacteria. These results provide strong evidence that the shared presence of these indels in these bacteria is very likely a consequence of ancient lateral gene transfers from actinobacteria to chlamydiae. Pairwise sequence identity and the branching pattern of the GlyA homologues in the phylogenetic tree indicates that the glyA gene was initially transferred from an actinobacteria to an ancestor of the Treponema genus and from there it was acquired by the common ancestor of the Chlamydiales. [Reviewing Editor: Dr. Siv Andersson]     

Direct Comparisons of 2D and 3D Dental Microwear Proxies in Extant Herbivorous and Carnivorous Mammals

The analysis of dental microwear is commonly used by paleontologists and anthropologists to clarify the diets of extinct species, including herbivorous and carnivorous mammals. Currently, there are numerous methods employed to quantify dental microwear, varying in the types of microscopes used, magnifications, and the characterization of wear in both two dimensions and three dimensions. Results from dental microwear studies utilizing different methods are not directly comparable and human quantification of wear features (e.g., pits and scratches) introduces interobserver error, with higher error being produced by less experienced individuals. Dental microwear texture analysis (DMTA), which analyzes microwear features in three dimensions, alleviates some of the problems surrounding two-dimensional microwear methods by reducing observer bias. Here, we assess the accuracy and comparability within and between 2D and 3D dental microwear analyses in herbivorous and carnivorous mammals at the same magnification. Specifically, we compare observer-generated 2D microwear data from photosimulations of the identical scanned areas of DMTA in extant African bovids and carnivorans using a scanning white light confocal microscope at 100x magnification. Using this magnification, dental microwear features quantified in 2D were able to separate grazing and frugivorous bovids using scratch frequency; however, DMTA variables were better able to discriminate between disparate dietary niches in both carnivorous and herbivorous mammals. Further, results demonstrate significant interobserver differences in 2D microwear data, with the microwear index remaining the least variable between experienced observers, consistent with prior research. Overall, our results highlight the importance of reducing observer error and analyzing dental microwear in three dimensions in order to consistently interpret diets accurately.     

Helicobacter anseris sp. nov. and Helicobacter brantae sp. nov., isolated from feces of resident Canada geese in the greater Boston area

Numbers of nonmigratory Canada geese have increased substantially in the past decade, and they have become a nuisance in some urban areas. Because of their close contact with humans in parks and areas adjacent to surface waterways, contact with their feces poses a zoonotic risk. A total of 97 geese from 10 separate geographic locales in the greater Boston area had their feces sampled for detection of Helicobacter spp. Identification of Helicobacter spp. based on 16S rRNA genus-specific helicobacter primers was noted in 39 of 97 (40.2%) DNA fecal extracts. Twenty-seven (27.8%) of these geese had helicobacters isolated from their feces. A urease-positive novel species, Helicobacter anseris, based on phenotypic, biochemical, and 16S rRNA analyses, was isolated from 20 geese from seven different flocks. A second, novel, urease-negative Helicobacter sp., H. brantae, was identified in seven geese. Four geese had both novel Helicobacter spp. cultured from their feces. Whether these two novel helicobacters pose a zoonotic risk, similar to other enteric helicobacters (e.g., H. canadensis, previously isolated from diarrheic and bacteremic humans and from geese in Europe), will require further studies.     

Alternative mating strategies in a desert grasshopper: evidence of density-dependence

Field observations revealed that, on a given day, male mating behaviour in a population of the grasshopper Ligurotettix coquilletti ranged from little or no stridulation (inactive) to relatively persistent singing (active signalling). Inactive males were usually located in the territories of active signallers. Actively signalling male achieved more matings, and also more frequently approached and mounted females in incidents terminating in the male departing the female without copulating. Individuals switched between inactive and actively-signalling behaviour during their adult lives, and males that were usually active signallers achieved greater lifetime mating success. Thus, we obtained no evidence to support the hypothesis that variation in mating behaviour was maintained by negative frequency-dependent selection. By comparing the behaviour of males in two field plots maintained at different population densities, however, we found that high density was directly related to a higher incidence of inactive behaviour. Males perched on creosote (Larrea tridentata) bushes (their host plant), and certain bushes. Mating behaviour was independent of body size and, while young (<7 days old) males tended to be inactive, neither age nor the time of adult maturation during the season could fully account for the incidence of inactive behaviour among males. Instead, we suggest that the adoption of inactive behaviour resulted partly from aggressive encounters between males.     

Crystal structures of Trypanosoma brucei and Staphylococcus aureus mevalonate diphosphate decarboxylase inform on the determinants of specificity and reactivity

Mevalonate diphosphate decarboxylase (MDD) catalyzes the ATP-dependent decarboxylation of mevalonate 5-diphosphate (MDP) to form isopentenyl pyrophosphate, a ubiquitous precursor for isoprenoid biosynthesis. MDD is a poorly understood component of this important metabolic pathway. Complementation of a temperature-sensitive yeast mutant by the putative mdd genes of Trypanosoma brucei and Staphylococcus aureus provides proof-of-function. Crystal structures of MDD from T. brucei (TbMDD, at 1.8 A resolution) and S. aureus (SaMDD, in two distinct crystal forms, each diffracting to 2.3 A resolution) have been determined. Gel-filtration chromatography and analytical ultracentrifugation experiments indicate that TbMDD is predominantly monomeric in solution while SaMDD is dimeric. The new crystal structures and comparison with that of the yeast Saccharomyces cerevisiae enzyme (ScMDD) reveal the structural basis for this variance in quaternary structure. The presence of an ordered sulfate in the structure of TbMDD reveals for the first time details of a ligand binding in the MDD active site and, in conjunction with well-ordered water molecules, comparisons with the related enzyme mevalonate kinase, structural and biochemical data derived on ScMDD and SaMDD, allows us to model a ternary complex with MDP and ATP. This model facilitates discussion of the molecular determinants of substrate recognition and contributions made by specific residues to the enzyme mechanism.     

F-actin capping (CapZ) and other contractile saphenous vein smooth muscle proteins are altered by hemodynamic stress: a proteonomic approach

Increased force generation and smooth muscle remodeling follow the implantation of saphenous vein as an arterial bypass graft. Previously, we characterized and mapped 129 proteins in human saphenous vein medial smooth muscle using two-dimensional (2-D) PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Here, we focus on actin filament remodeling in response to simulated arterial flow. Human saphenous vein was exposed to simulated venous or arterial flow for 90 min in vitro, and the contractile medial smooth muscle was dissected out and subjected to 2-D gel electrophoresis using a non-linear immobilized pH 3-10 gradient in the first dimension. Proteins were analyzed quantitatively using PDQuest 2-D software. The actin polymerization inhibitor cytochalasin B (1 microm) prevented increases in force generation after 90 min of simulated arterial flow. At this time point, there were several consistent changes in actin filament-associated protein expression (seven paired vein samples). The heat shock protein HSP27, identified as a three-spot charge train, showed a 1.6-fold increase in abundance (p = 0.01), but with reduced representation of the phosphorylated Ser(82) and Ser(15)Ser(82) isoforms (p = 0.018). The abundance of actin-capping protein alpha2 subunit CapZ had decreased 3-fold, p = 0.04. A 19-kDa proteolytic fragment of actin increased 2-fold, p = 0.04. For the four-spot charge train of gelsolin, there was reduced representation of the more acidic isoforms, p = 0.022. The abundance of other proteins associated with actin filaments, including cofilin and destrin, remained unchanged after arterial flow. Actin filament remodeling with differential expression and/or post-translational modification of proteins involved in capping the barbed end of actin filaments, HSP27 and CapZ, is an early response of contractile saphenous vein smooth muscle cells to hemodynamic stress. The observed changes would favor the generation of contractile stress fibers.