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]     

Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy

The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.     

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.     

Severe defect in thymic development in an insertional mutant mouse model

Transgenic mice were generated expressing NK1.1, an NK cell-associated receptor, under control of the human CD2 promoter. Unexpectedly, one of the founder lines, Tg66, showed a marked defect in thymic development characterized by disorganized architecture and small size. Mapping of the transgene insertion by fluorescence in situ hybridization revealed integration in chromosome 2, band G. Already from postnatal day 3, the thymic architecture was disturbed with a preferential loss of cortical thymic epithelial cells, a feature that became more pronounced over time. Compared with wild-type mice, total thymic cell numbers decreased dramatically between 10 and 20 days of age. Thymocytes isolated from adult Tg66 mice were predominantly immature double-negative cells, indicating a block in thymic development at an early stage of differentiation. Consequently, Tg66 mice had reduced numbers of peripheral CD4(+) and CD8(+) T cells. Bone marrow from Tg66 mice readily reconstituted thymi of irradiated wild-type as well as RAG-deficient mice. This indicates that the primary defect in Tg66 mice resided in nonhemopoietic stromal cells of the thymus. The phenotype is observed in mice heterozygous for the insertion and does not resemble any known mutations affecting thymic development. Preliminary studies in mice homozygous for transgene insertion reveal a more accelerated and pronounced phenotype suggesting a semidominant effect. The Tg66 mice may serve as a useful model to identify genes regulating thymic epithelial cell differentiation, thymic development, and function.