Homologues of the protein constituents of the Klebsiella pneumoniae (Klebsiella oxytoca) type II secreton (T2S), the Pseudomonas aeruginosa type IV pilus/fimbrium biogenesis machinery (T4P) and the Methanococcus voltae flagellum biogenesis machinery (Fla) have been identified. Known constituents of these systems include (1) a major prepilin (preflagellin), (2) several minor prepilins (preflagellins), (3) a prepilin (preflagellin) peptidase/methylase, (4) an ATPase, (5) a multispanning transmembrane (TM) protein, (6) an outer-membrane secretin (lacking in Fla) and (7) several functionally uncharacterized envelope proteins. Sequence and phylogenetic analyses led to the conclusion that, although many of the protein constituents are probably homologous, extensive sequence divergence during evolution clouds this homology so that a common ancestry can be established for all three types of systems for only two constituents, the ATPase and the TM protein. Sequence divergence of the individual T2S constituents has occurred at characteristic rates, apparently without shuffling of constituents between systems. The same is probably also true for the T4P and Fla systems. The family of ATPases is much larger than the family of TM proteins, and many ATPase homologues function in capacities unrelated to those considered here. Many phylogenetic clusters of the ATPases probably exhibit uniform function. Some of these have a corresponding TM protein homologue although others probably function without one. It is further shown that proteins that compose the different phylogenetic clusters in both the ATPase and the TM protein families exhibit unique structural characteristics that are of probable functional significance. The TM proteins are shown to have arisen by at least two dissimilar intragenic duplication events, one in the bacterial kingdom and one in the archaeal kingdom. The archaeal TM proteins are twice as large as the bacterial TM proteins, suggesting an oligomeric structure for the latter. OverviewThree related types of prokaryotic envelope protein complexes include (putative) prepilin proteins with highly similar hydrophobic N-terminal segments of approximately 20 amino acyl residues. These putative prepilins can assemble into filamentous structures which compose parts of (1) the type II secretion system (T2S), (2) the type IV piliation/fimbriation system (T4P) (both of Gramnegative bacteria) and (3) the flagellar system (Fla) of archaea. T2S, also called the type II secreton or the main terminal branch (MTB; TC #3.A.15) of the general secretory pathway (TC #3.A.5;Cao & Saier, 2003), represents the major pathway for exoprotein transport from the periplasm across the outer membrane in a wide variety of Gramnegative bacteria (Pugsley, 1993a). The type II secreton is composed of a core of around 12 proteins, some of which are not present in all type II secretons and others of which appear to be dispensable for secreton function Pugsley, 1993a;Sandkvist, 2001). In this review, secreton components will be referred to accordi...

Mechanosensitive (MS) channels were first demonstrated in bacterial cells by using patch clamp analysis of giant bacterial protoplasts and by fusion of membranes with liposomes. Both approaches indicated the presence of high-conductance channels in the membranes of gram-positive and gram-negative bacteria (15,29,53,60). Initially the data were greeted with scepticism, based on the similarity of the conductances of MS channels to those of porins and the recognized need of the cytoplasmic membrane to exhibit tight control over H ϩ permeability in order to effect energy transduction. Activation of MS channels by membrane-intercalating amphipathic compounds suggested that these channels are sensitive to mechanical perturbations in the lipid bilayer (22,28). Support for the presence of channels was provided by the discovery and reconstitution of two distinct channel activities from Escherichia coli, each with unique properties (52). Further support came from the discovery that the efflux of solutes from E. coli cells in response to a lowering of the external osmolarity could be prevented by gadolinium ions, which are classical inhibitors of MS channels in higher organisms (6).A landmark event was the purification and cloning of the first MS channel protein, MscL, from E. coli. This heroic piece of biochemistry required that each fraction derived from the solubilized and fractionated membrane be reconstituted into liposomes and the MS channel activity be measured (51). Availability of the amino-terminal sequence of the protein led to identification of the gene. Following this breakthrough, a new age of MS channel protein structure-function analysis dawned (7,(9)(10)(11)42), culminating in the crystal structure of a mycobacterial MscL channel (13) (Fig. 1). Extensive genetic and biophysical analyses of MscL protein movement in real time, coupled with model building, electron paramagnetic resonance spectroscopy, and site-directed spin labeling studies, provided an explanation of how the protein can exist in at least two states-one tightly closed and the other creating a large pore in the membrane (23,42,48,49) (Fig. 2). MS channels are now thought to be important to many bacteria (8) and archaea (20,21,24).The genetic advances with MscL posed a further problemwhy does an mscL null mutant lack an apparent physiological phenotype? Patch clamp analysis had revealed the presence of at least two MS channels in E. coli membranes, and subsequent studies led to the possibility that five or more genetically distinct channels exist (5). Such apparent biochemical redundancy implied that observation of a phenotype might require the construction of a mutant lacking more than one channel protein. Preliminary support for the protective role of MscL was discovered by expressing the channel in Vibrio and observing protection from hypoosmotic shock (38). The discovery of the structural gene for MscS, the second major MS channel in E. coli, allowed this functional hypothesis to be tested (25). Through the genetic analysis of a missense mu...

Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes L-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for L-isoleucine export, we have used transposon mutagenesis to isolate mutants of C. glutamicum sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices. brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports L-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of L-leucine and L-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in ␣-proteobacteria but not in other prokaryotes analyzed.

SignificanceThe phenomenon of genetic balance is characterized by the finding that addition or subtraction of a single chromosome to the whole set is more detrimental than altering the dosage of the complete complement. The molecular basis of this principle has not been studied previously in a comprehensive manner. In this study, the set of all five trisomic chromosomes and a ploidy series of diploid, triploid, and tetraploid in Arabidopsis were examined for global gene expression modulations. The results indicate an impact of genomic stoichiometry on the landscape of gene expression, which has implications for how gene expression operates, the evolution of duplicate genes, and the underlying basis of quantitative traits.

Summary Remodeling DNA methylation in mammalian genomes can be global as seen in pre-implantation embryos and primordial germ cells (PGCs), or locus-specific, which can regulate neighboring gene expression. In PGCs global and locus-specific DNA demethylation occur in sequential stages, with an initial global decrease in methylated cytosines (stage I) followed by a Tet methylcytosine dioxygenase (Tet)-dependent decrease in methylated cytosines that act at imprinting control regions (ICRs) and meiotic genes (stage II). The purpose of the two-stage mechanism is unclear. Here we show that Dnmt1 preserves DNA methylation through stage I at ICRs and meiotic gene promoters, and is required for the pericentromeric enrichment of 5hmC. We discovered that the functional consequence of abrogating two-stage DNA demethylation in PGCs was precocious germline differentiation leading to hypogonadism and infertility. Therefore, bypassing stage-specific DNA demethylation has significant consequences for progenitor germ cell differentiation and the ability to transmit DNA from parent to offspring.

BackgroundGram-negative bacteria have developed a limited repertoire of solutions for secreting proteins from the cytoplasmic compartment to the exterior of the cell. Amongst the spectrum of secreted proteins are the intimins and invasins (the Int/Inv family; TC# 1.B.54) which are characterized by an N-terminal β-barrel domain and a C-terminal surface localized passenger domain. Despite the important role played by members of this family in diseases mediated by several species of the Enterobacteriaceae, there has been little appreciation for the distribution and diversity of these proteins amongst Gram-negative bacteria. Furthermore, there is little understanding of the molecular events governing secretion of these proteins to the extracellular milieu.Principal Findings In silico approaches were used to analyze the domain organization and diversity of members of this secretion family. Proteins belonging to this family are predominantly associated with organisms from the γ-proteobacteria. Whilst proteins from the Chlamydia, γ-, β- and ε-proteobacteria possess β-barrel domains and passenger domains of various sizes, Int/Inv proteins from the α-proteobacteria, cyanobacteria and chlorobi possess only the predicted β-barrel domains. Phylogenetic analyses revealed that with few exceptions these proteins cluster according to organismal type, indicating that divergence occurred contemporaneously with speciation, and that horizontal transfer was limited. Clustering patterns of the β-barrel domains correlate well with those of the full-length proteins although the passenger domains do so with much less consistency. The modular subdomain design of the passenger domains suggests that subdomain duplication and deletion have occurred with high frequency over evolutionary time. However, all repeated subdomains are found in tandem, suggesting that subdomain shuffling occurred rarely if at all. Topological predictions for the β-barrel domains are presented.ConclusionBased on our in silico analyses we present a model for the biogenesis of these proteins. This study is the first of its kind to describe this unusual family of bacterial adhesins.

Secondary metabolites (SM) produced by fungi and bacteria have long been of exceptional interest owing to their unique biomedical ramifications. The traditional discovery of new natural products that was mainly driven by bioactivity screening has now experienced a fresh new approach in the form of genome mining. Several bioinformatics tools have been continuously developed to detect potential biosynthetic gene clusters (BGCs) that are responsible for the production of SM. Although the principles underlying the computation of these tools have been discussed, the biological background is left underrated and ambiguous. In this review, we emphasize the biological hypotheses in BGC formation driven from the observations across genomes in bacteria and fungi, and provide a comprehensive list of updated algorithms/tools exclusively for BGC detection. Our review points to a direction that the biological hypotheses should be systematically incorporated into the BGC prediction and assist the prioritization of candidate BGC.

Twin-arginine targeting (Tat) protein secretion systems consist of two protein types, members of the TatA and TatC families. Homologues of these proteins are found in many archaea, bacteria, chloroplasts and mitochondria. Every prokaryotic organism with a fully sequenced genome exhibits either neither family member, or between one and three paralogues of these two family members. The Arabidopsis thaliana genome encodes three of each. Although many mitochondrially encoded TatC homologues have been identified, corresponding TatA homologues have not been found in this organelle. Phylogenetic analyses reveal that most prokaryotic Tat systems consist of one TatC homologue and two sequence-divergent TatA homologues (TatA and TatB). When only one TatA homologue is present, TatB is missing, and when three TatA homologues are present, the third one arose by duplication of TatA, not TatB. Further, homologues most resembling TatB are more sequence-divergent than those more closely resembling TatA. In contrast to the TatA family, the TatC family shows phylogenetic clustering in strict accordance with organismal type. These results are discussed in terms of their probable structural, functional and evolutionary significance.