First synthetic yeast chromosome revealed

As a eukaryote, a category that includes humans and other animals, S. cerevisiae has a more complex genome than Venter's parasite. The synthetic yeast chromosome — which has been stripped of some DNA sequences and other elements — is 272,871 base pairs long, representing about 2.5% of the 12-million-base-pair S. cerevisiae genome.The researchers, who report their accomplishment in Science¹, have formed an international consortium to create a synthetic version of the full S. cerevisiae genome within 5 years.





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Total Synthesis of a Functional Designer Eukaryotic Chromosome

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871–base pair designer eukaryotic chromosome, synIII, which is based on the 316,617–base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.

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Bacterial solutions to multicellularity: a tale of biofilms, filaments and fruiting bodies

Although bacteria frequently live as unicellular organisms, many spend at least part of their lives in complex communities, and some have adopted truly multicellular lifestyles and have abandoned unicellular growth. These transitions to multicellularity have occurred independently several times for various ecological reasons, resulting in a broad range of phenotypes. In this Review, we discuss the strategies that are used by bacteria to form and grow in multicellular structures that have hallmark features of multicellularity, including morphological differentiation, programmed cell death and patterning. In addition, we examine the evolutionary and ecological factors that lead to the wide range of coordinated multicellular behaviours that are observed in bacteria.

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El corazón púrpura de la fotosíntesis

The structure of a photosynthetic complex from a purple bacterium reveals a new class of light-harvesting protein and the channels that might allow electron-transporting molecules to escape this otherwise closed system.

Light-harvesting complex 2 (LH2) contains two groups of bacteriochlorophyll (BChL) pigments, B800 and B850. Light energy absorbed by B800 is transferred to B850 (blue arrows). It is then passed to another group of BChLs (B880) in light-harvesting complex 1 (LH1), and finally into the reaction centre (RC). The excited RC fully reduces ubiquinone molecules (Q), which leave the LH1–RC complex and pass into the membrane, transferring electrons (e⁻) to the cytochrome bc₁ complex; this transfer forms part of a cyclic electron-transport pathway (red arrows) that drives photosynthesis. This pathway is completed by a soluble protein, cytochrome c₂ (Cyt c₂). The periplasm is the region between the cell membrane and the cell wall of the bacterium.

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Structural Studies of Planctomycete Gemmata obscuriglobus Support Cell Compartmentalisation in a Bacterium.

Members of phylum Planctomycetes have been proposed to possess atypical cell organisation for the Bacteria, having a structure of sectioned cells consistent with internal compartments surrounded by membranes. Here via electron tomography we confirm the presence of compartments in the planctomycete Gemmata obscuriglobus cells. Resulting 3-D models for the most prominent structures, nuclear body and riboplasm, demonstrate their entirely membrane - enclosed nature. Immunogold localization of the FtsK protein also supports the internal organisation of G.obscuriglobus cells and their unique mechanism of cell division. We discuss how these new data expand our knowledge on bacterial cell biology and suggest evolutionary consequences of the findings.

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Taking aim at wall teichoic acid synthesis: new biology and new leads for antibiotics.

Wall teichoic acids are a major and integral component of the Gram-positive cell wall. These structures are present across all species of Gram-positive bacteria and constitute roughly half of the cell wall. Despite decades of careful investigation, a definitive physiological function for wall teichoic acids remains elusive. Advances in the genetics and biochemistry of wall teichoic acid synthesis have led to a new understanding of the complexity of cell wall synthesis in Gram-positive bacteria. Indeed, these innovations have provided new molecular tools available to probe the synthesis and function of these cell wall structures. Among recent discoveries are unexpected roles for wall teichoic acid in cell division, coordination of peptidoglycan synthesis and β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). Notably, wall teichoic acid biogenesis has emerged as a bona fide drug target in S. aureus, where remarkable synthetic-viable interactions among biosynthetic genes have been leveraged for the discovery and characterization of novel inhibitors of the pathway.


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SecA is required for membrane targeting of the cell division protein DivIVA in vivo

The conserved protein DivIVA is involved in different morphogenetic processes in Gram-positive bacteria. In Bacillus subtilis, the protein localizes to the cell division site and cell poles, and functions as a scaffold for proteins that regulate division site selection, and for proteins that are required for sporulation. To identify other proteins that bind to DivIVA, we performed an in vivo cross-linking experiment. A possible candidate that emerged was the secretion motor ATPase SecA. SecA mutants have been described that inhibit sporulation, and since DivIVA is necessary for sporulation, we examined the localization of DivIVA in these mutants. Surprisingly, DivIVA was delocalized, suggesting that SecA is required for DivIVA targeting. To further corroborate this, we performed SecA depletion and inhibition experiments, which provided further indications that DivIVA localization depends on SecA. Cell fractionation experiments showed that SecA is important for binding of DivIVA to the cell membrane. This was unexpected since DivIVA does not contain a signal sequence, and is able to bind to artificial lipid membranes in vitro without support of other proteins. SecA is required for protein secretion and membrane insertion, and therefore its role in DivIVA localization is likely indirect. Possible alternative roles of SecA in DivIVA folding and/or targeting are discussed.

 Control experiments to confirm the cross-linking of SecA with DivIVA. (A) Pull-down experiment with His-DivIVA, DivIVA-GFP-His and GFP-His as bait proteins (strains BSN50, 3308 and BSN73, respectively). Pull-down fractions were analyzed by Western blotting for the presence of SecA (upper panel). To demonstrate that the bait proteins were pulled down efficiently, fractions were analyzed by Western blotting using DivIVA (middle panel) and GFP antisera (lower panel). (B) Pull-down experiment using SecA-His and GFP-His as bait proteins (strains BSN131, and 4097, respectively). Protein fractions were separated by SDS-PAGE (upper panel), and the presence of DivIVA was analyzed by Western blotting (lower panel). SecY, a known interaction partner of SecA, was pulled down with SecA-His. The protein band at around 55 kDa is elongation factor Tu (TufA), which appears to be an unspecific by-catch. The theoretical position of DivIVA on the SDS PAGE gel is indicated in brackets.

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Ancient Life in the Information Age

All known organisms share a number of fundamental features that, taken together, point to a common evolutionary history: DNA as the chief molecule of genetic inheritance, proteins as the primary functional molecules, and RNA as an informational intermediate between the two. The simplest explanation for why organisms share these common features is that they are inherited from a last universal common ancestor (LUCA), which sits at the root of the tree of life. Most studies of gene duplications that occurred prior to the first branch on the tree place LUCA in between the Bacteria and the common ancestor of the Archaea and Eukarya, the three taxonomic domains of cellular life.

The availability of the genome sequences from so many species across the tree of life has made it possible to look for common genomic traits that were most likely inherited from LUCA.

Some studies have estimated there to be fewer than 100 LUCA-derived gene families, while others count more than 1,000, depending on how conservatively the methods rule out genes on suspicion of horizontal gene transfer or how liberally they include genes that appear to have been present in LUCA, but subsequently lost. Despite the conflicting results, the new data are yielding insight into ancient life on Earth.

The majority of ancient gene families identified in almost all of these studies are involved in the translation of genetic information into proteins. These ancient gene families represent a range of translation functions, from regulation to ribosomal components. The genetic code at the core of translation is also highly conserved across life. In all likelihood, the enzymes responsible for establishing the genetic code by attaching amino acids to particular tRNAs evolved prior to the time of LUCA, although their evolutionary histories are obscured by subsequent horizontal gene transfers between bacteria and archaea. These results depict a translation system in LUCA that was probably similar to and as sophisticated as those of organisms alive today.

In contrast, few genes involved in the synthesis of DNA are conserved across the tree of life. The enzymes responsible for making deoxyribonucleotides from ribonucleotides exist in three distinct families that only show a weak signature of common descent in their active sites. The only DNA polymerase enzymes that are common across the evolutionary tree are those involved in repair, not the polymerases presently responsible for copying complete chromosomes. RNA polymerases from bacteria, archaea, and eukaryotes, on the other hand, do appear to have been inherited from LUCA, and may have previously functioned as DNA polymerases as well. Taken together, these observations suggest that DNA genomes replaced a genome composed of RNA just prior to or perhaps just after the time of LUCA.

The variety of metabolic strategies observed in modern organisms demonstrates that metabolism is generally less highly conserved, which makes it harder to identify those metabolic pathways that were present in LUCA. Still, various databases organize enzymatic data into metabolic maps, which can be used to uncover highly conserved components of modern metabolic pathways. For example, a recent study combined these data with evolutionary trees of carbon-fixation genes and found that the ancestral carbon-fixation pathway was most likely an amalgam of components currently found in two separate pathways in extant archaea and bacteria: the reductive acetyl-CoA pathway and the reductive citric acid cycle (PLOS Comput Biol, 8:e1002455, 2012). Another taxonomically broad comparison study, focused on amino acid metabolism, uncovered conserved biosynthetic pathways for 8 of the 20 canonical amino acids, and conserved enzymes from pathways for another eight (Genome Biology, 9:R95, 2008).

Finally, LUCA most likely had a phospholipid membrane that set the boundaries between organisms and offered protection from the external environment. The universal presence of genes responsible for targeting proteins to membranes suggests that LUCA’s membrane was replete with proteins. Furthermore, the ubiquity of both catalytic subunits of the membrane-bound ATPase motor also implies that this membrane was impermeable enough to ions that it could be used to generate the proton gradients used by the motor to convert ADP to ATP.

While this detailed understanding of LUCA is relatively recent, Darwin proposed the idea of an early common ancestor to all life in the first edition of Origin of Species, where he wrote, “Therefore I should infer from analogy that probably all the organic beings which have ever lived on this earth have descended from some one primordial form, into which life was first breathed.” Although Darwin’s insight is brilliant for its time, the modern view shows that LUCA is not this “primordial form,” but rather a sophisticated cellular organism that, if alive today, would probably be difficult to distinguish from other extant bacteria or archaea. This means that a great detail of evolution must have taken place between the time of the origin of life and the appearance of LUCA. Continuing advances in evolutionary biology, bioinformatics, and computational biology will give us the tools to describe LUCA and the evolutionary transitions preceding it with unprecedented accuracy and detail.

Aaron David Goldman is an assistant professor of biology at Oberlin College. His research employs bioinformatics and systems biology tools to study the genome and metabolism of LUCA and their connections to evolutionary predecessors. 

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The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular.

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Sulphoglycolysis in Escherichia coli K-12 closes a gap in the biogeochemical sulphur cycle

Sulphoquinovose (SQ, 6-deoxy-6-sulphoglucose) has been known for 50 years as the polar headgroup of the plant sulpholipid in the photosynthetic membranes of all higher plants, mosses, ferns, algae and most photosynthetic bacteria. It is also found in some non-photosynthetic bacteria, and SQ is part of the surface layer of some Archaea. The estimated annual production of SQ is 10,000,000,000 tonnes (10 petagrams), thus it comprises a major portion of the organo-sulphur in nature, where SQ is degraded by bacteria. However, despite evidence for at least three different degradative pathways in bacteria, no enzymic reaction or gene in any pathway has been defined, although a sulphoglycolytic pathway has been proposed⁷. Here we show that Escherichia coli K-12, the most widely studied prokaryotic model organism, performs sulphoglycolysis, in addition to standard glycolysis. SQ is catabolised through four newly discovered reactions that we established using purified, heterologously expressed enzymes: SQ isomerase, 6-deoxy-6-sulphofructose (SF) kinase, 6-deoxy-6-sulphofructose-1-phosphate (SFP) aldolase, and 3-sulpholactaldehyde (SLA) reductase. The enzymes are encoded in a ten-gene cluster, which probably also encodes regulation, transport and degradation of the whole sulpholipid; the gene cluster is present in almost all (>91%) available E. coli genomes, and is widespread in Enterobacteriaceae. The pathway yields dihydroxyacetone phosphate (DHAP), which powers energy conservation and growth of E. coli, and the sulphonate product 2,3-dihydroxypropane-1-sulphonate (DHPS), which is excreted. DHPS is mineralized by other bacteria, thus closing the sulphur cycle within a bacterial community.

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Identification of a conserved branched RNA structure that functions as a factor-independent terminator

All cells regulate which regions of DNA are transcribed to RNA. Controlling where transcription terminates is an essential part of this regulation. In bacterial cells, RNA structures, referred to as factor-independent terminators, can interact with RNA polymerase to direct termination. These structures are typically inverted sequence repeats that form an RNA hairpin followed by several uridine residues. We identified a branched RNA structure that functions as a factor-independent terminator. The terminated product is a functional small RNA, but termination is inefficient, allowing transcription of downstream genes. Additional branched terminators are encoded in bacterial chromosomes, demonstrating that this unusual terminator is not unique. This work reveals an unappreciated structural diversity of factor-independent terminators and will inform annotation of bacterial genomes.    

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