Co-orientation of replication and transcription preserves genome integrity.

In many bacteria, there is a genome-wide bias towards co-orientation of replication and transcription, with essential and/or highly-expressed genes further enriched co-directionally. We previously found that reversing this bias in the bacterium Bacillus subtilis slows replication elongation, and we proposed that this effect contributes to the evolutionary pressure selecting the transcription-replication co-orientation bias. This selection might have been based purely on selection for speedy replication; alternatively, the slowed replication might actually represent an average of individual replication-disruption events, each of which is counter-selected independently because genome integrity is selected. To differentiate these possibilities and define the precise forces driving this aspect of genome organization, we generated new strains with inversions either over approximately 1/4 of the chromosome or at ribosomal RNA (rRNA) operons. Applying mathematical analysis to genomic microarray snapshots, we found that replication rates vary dramatically within the inverted genome. Replication is moderately impeded throughout the inverted region, which results in a small but significant competitive disadvantage in minimal medium. Importantly, replication is strongly obstructed at inverted rRNA loci in rich medium. This obstruction results in disruption of DNA replication, activation of DNA damage responses, loss of genome integrity, and cell death. Our results strongly suggest that preservation of genome integrity drives the evolution of co-orientation of replication and transcription, a conserved feature of genome organization.

Oscillations by minimal bacterial suicide circuits reveal hidden facets of host-circuit physiology.

Synthetic biology seeks to enable programmed control of cellular behavior though engineered biological systems. These systems typically consist of synthetic circuits that function inside, and interact with, complex host cells possessing pre-existing metabolic and regulatory networks. Nevertheless, while designing systems, a simple well-defined interface between the synthetic gene circuit and the host is frequently assumed. We describe the generation of robust but unexpected oscillations in the densities of bacterium Escherichia coli populations by simple synthetic suicide circuits containing quorum components and a lysis gene. Contrary to design expectations, oscillations required neither the quorum sensing genes (luxR and luxI) nor known regulatory elements in the P(luxI) promoter. Instead, oscillations were likely due to density-dependent plasmid amplification that established a population-level negative feedback. A mathematical model based on this mechanism captures the key characteristics of oscillations, and model predictions regarding perturbations to plasmid amplification were experimentally validated. Our results underscore the importance of plasmid copy number and potential impact of "hidden interactions" on the behavior of engineered gene circuits - a major challenge for standardizing biological parts. As synthetic biology grows as a discipline, increasing value may be derived from tools that enable the assessment of parts in their final context.

Chemical and biological applications of digital-microfluidic devices

The advent of digital microfluidic lab-on-a-chip (LoC) technology offers a platform for developing diagnostic applications with the advantages of portability, reduction of the volumes of the sample and reagents, faster analysis times, increased automation, low power consumption, compatibility with mass manufacturing, and high throughput. Moreover, digital microfluidics is being applied in other areas such as airborne chemical detection, DNA sequencing by synthesis, and tissue engineering. In most diagnostic and chemical-detection applications, a key challenge is the preparation of the analyte for presentation to the on-chip detection system. Thus, in diagnostics, raw physiological samples must be introduced onto the chip and then further processed by lysing blood cells and extracting DNA. For massively parallel DNA sequencing, sample preparation can be performed off chip, but the synthesis steps must be performed in a sequential on-chip format by automated control of buffers and nucleotides to extend the read lengths of DNA fragments. In airborne particulate-sampling applications, the sample collection from an air stream must be integrated into the LoC analytical component, which requires a collection droplet to scan an exposed impacted surface after its introduction into a closed analytical section. Finally, in tissue-engineering applications, the challenge for LoC technology is to build high-resolution (less than 10 microns) 3D tissue constructs with embedded cells and growth factors by manipulating and maintaining live cells in the chip platform. This article discusses these applications and their implementation in digital-microfluidic LoC platforms. © 2007 IEEE.

Programming stress-induced altruistic death in engineered bacteria.

Programmed death is often associated with a bacterial stress response. This behavior appears paradoxical, as it offers no benefit to the individual. This paradox can be explained if the death is 'altruistic': the killing of some cells can benefit the survivors through release of 'public goods'. However, the conditions where bacterial programmed death becomes advantageous have not been unambiguously demonstrated experimentally. Here, we determined such conditions by engineering tunable, stress-induced altruistic death in the bacterium Escherichia coli. Using a mathematical model, we predicted the existence of an optimal programmed death rate that maximizes population growth under stress. We further predicted that altruistic death could generate the 'Eagle effect', a counter-intuitive phenomenon where bacteria appear to grow better when treated with higher antibiotic concentrations. In support of these modeling insights, we experimentally demonstrated both the optimality in programmed death rate and the Eagle effect using our engineered system. Our findings fill a critical conceptual gap in the analysis of the evolution of bacterial programmed death, and have implications for a design of antibiotic treatment.

Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells.

The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen and a main cause of genital and ocular diseases. During its intracellular cycle, C. trachomatis replicates inside a membrane-bound vacuole termed an "inclusion". Acquisition of lipids (and other nutrients) from the host cell is a critical step in chlamydial replication. Lipid droplets (LD) are ubiquitous, ER-derived neutral lipid-rich storage organelles surrounded by a phospholipids monolayer and associated proteins. Previous studies have shown that LDs accumulate at the periphery of, and eventually translocate into, the chlamydial inclusion. These observations point out to Chlamydia-mediated manipulation of LDs in infected cells, which may impact the function and thereby the protein composition of these organelles. By means of a label-free quantitative mass spectrometry approach we found that the LD proteome is modified in the context of C. trachomatis infection. We determined that LDs isolated from C. trachomatis-infected cells were enriched in proteins related to lipid metabolism, biosynthesis and LD-specific functions. Interestingly, consistent with the observation that LDs intimately associate with the inclusion, a subset of inclusion membrane proteins co-purified with LD protein extracts. Finally, genetic ablation of LDs negatively affected generation of C. trachomatis infectious progeny, consistent with a role for LD biogenesis in optimal chlamydial growth.

Suppression of conformational heterogeneity at a protein-protein interface.

Staphylococcal protein A (SpA) is an important virulence factor from Staphylococcus aureus responsible for the bacterium's evasion of the host immune system. SpA includes five small three-helix-bundle domains that can each bind with high affinity to many host proteins such as antibodies. The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure was not properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. Here we report the 2.3-Å structure of a fully folded SpA domain in complex with Fc. Our structure indicates that there are extensive structural rearrangements necessary for binding Fc, including a general reduction in SpA conformational heterogeneity, freezing out of polyrotameric interfacial residues, and displacement of a SpA side chain by an Fc side chain in a molecular-recognition pocket. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur when SpA binds its multiple binding partners. Suppression of conformational heterogeneity may be an important structural paradigm in functionally plastic proteins.

Methylation-dependent T cell immunity to Mycobacterium tuberculosis heparin-binding hemagglutinin.

Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides. Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice. Methylated HBHA provides high levels of protection against M. tuberculosis challenge in mice, whereas nonmethylated HBHA does not. Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guérin, the only available anti-tuberculosis vaccine. Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.

Changes in the properties of Bacillus thuringiensis after prolonged culture in a rich medium

AIM: To investigate the effect of repeated culture in a rich medium on certain genetic, metabolic, pathogenic and structural characteristics of fresh isolates of Bacillus thuringiensis. METHODS AND RESULTS: Four strains of B. thuringiensis, which had been isolated in vegetative form from leaf surfaces, were grown for 500 generations in batch culture in a rich medium. One of the strains, S4g, differed from the parent in the following respects: greater cell width; changed plasmid profile; complete loss of ability to produce delta-endotoxins; loss of ability to produce beta-exotoxin and disruption of vip3 gene; radically different fatty acid composition; and altered metabolic activity. Two of the other evolved strains (S1g and S6g) showed differences in fatty acid profiles compared with the parents. Genetic finger-printing showed that there were also mutations in the cry genes of two of the evolved strains (S1g and S2g). The delta-endotoxins of strain S6g were significantly less toxic to the larvae of Pieris brassica compared with those of the parent and it also differed in the plasmid content. CONCLUSION: Radical and unpredictable changes can occur in fresh isolates of B. thuringiensis when subjected to growth in the laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first analysis of a Gram positive and biotechnologically significant bacterium after repeated laboratory culture. It is of great relevance to the biotechnological exploitation of B. thuringiensis that prolonged growth of environmental isolates on laboratory culture media can have profound effects on their structure, genome and virulence determinants.

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).

Production and characterization of a trehalolipid biosurfactant produced by the novel marine bacteriumRhodococcussp., strain PML026

AIMS: The aim of this study was to evaluate biosurfactant production by a novel marine Rhodococcus sp., strain PML026 and characterize the chemical nature and properties of the biosurfactant. METHODS AND RESULTS: A novel marine bacterium (Rhodococcus species; strain PML026) was shown to produce biosurfactant in the presence of hydrophobic substrate (sunflower oil). Biosurfactant production (identified as a trehalolipid) was monitored in whole-batch cultures (oil layer and aqueous phase), aqueous phase (no oil layer) and filtered (0·2mum) aqueous phase (no oil or cells; extracellular) and was shown to be closely associated with growth/biomass production. Extracellular trehalolipid levels increased postonset of stationary growth phase. Purified trehalolipid was able to reduce the surface tension of water to 29mN m(-1) at Critical Micellar Concentration (CMC) of c. 250mgl(-1) and produced emulsions that were stable to a wide range of conditions (pH 2-10, temperatures of 20-100°C and NaCl concentrations of 5-25% w/v). Separate chemical analyses of the intact trehalolipid and its constituents demonstrated the compound was in fact a mixture of homologues (>1180MW) consisting of a trehalose moiety esterified to a series of straight chain and hydroxylated fatty acids. CONCLUSIONS: The trehalolipid biosurfactant produced by the novel marine strain Rhodococcus sp. PML026 was characterized and exhibited high surfactant activity under a wide range of conditions. SIGNIFICANCE AND IMPACT OF STUDY: Strain PML026 of Rhodococcus sp. is a potential candidate for bioremediation or biosurfactant production for various applications.

The effect of pressure on leucine and thymidine incorporation by free-living bacteria and by bacteria attached to sinking oceanic particles

The effect of pressure on upper ocean free-living bacteria and bacteria attached to rapidly sinking particles was investigated through studying their ability to synthesize DNA and protein by measuring their rate of 3H-thymidine and 3H-leucine incorporation. Studies were carried out on samples from the NE Atlantic under the range of pressures (1–430 atm) encountered by sinking aggregates during their journey to the deep-sea bed. Thymidine and leucine incorporation rates per bacterium attached to sinking particles from 200 m were about six and ten times higher, respectively, than the free-living bacterial assemblage. The ratio of leucine incorporation rate per cell to thymidine incorporation rate per cell was significantly different between the larger attached (18.9:1) and smaller free-living (10.4:1) assemblages. The rates of leucine and thymidine incorporation decreased exponentially with increasing pressure for the free-living and linearly for attached bacteria, while there was no significant influence of pressure on cell numbers. At 100 atm leucine and thymidine incorporation rate per free-living bacterium was reduced to 73 and 20%, respectively, relative to that measured at 1 atm. Pressure of 100 atm reduced leucine and thymidine incorporation per attached bacterium to 94 and 70%, and at 200 atm these rates were reduced to 34 and 51%, respectively, relative to those measured at 1 atm. There was no significant uncoupling of thymidine and leucine incorporation for either the free-living or attached bacterial assemblages with increasing pressure, indicating that the processess of DNA and protein synthesis may be equally affected by increasing pressure. It is therefore unlikely that bacteria, originating from surface waters, attached to rapidly sinking particles play a role in particle remineralization below approximately 1000–2000 m. These results may help to explain the occurrence of relatively fresh aggregates on the deep-sea bed that still contain sufficient organic carbon to fuel the rapid growth of benthic micro-organisms; they also indicate that the effect of pressure on microbial processes may be important in oceanic biogeochemical cycles.

Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph

A variety of culture-independent techniques have been developed that can be used in conjunction with culture-dependent physiological and metabolic studies of key microbial organisms, in order to better understand how the activity of natural populations influences and regulates all major biogeochemical cycles. In this study, we combined DNA-stable isotope probing with metagenomics and metaproteomics to characterize an as yet uncultivated marine methylotroph that actively incorporated carbon from 13C-labeled methanol into biomass. By metagenomic sequencing of the heavy DNA, we retrieved virtually the whole genome of this bacterium and determined its metabolic potential. Through protein-stable isotope probing, the RuMP cycle was established as the main carbon assimilation pathway, and the classical methanol dehydrogenase-encoding gene mxaF, as well as three out of four identified xoxF homologues were found to be expressed. This proof-of-concept study is the first in which theculture-independent techniques of DNA- and protein-stable isotope probing have been used to characterize the metabolism of a naturally-ocurring Methylophaga-like bacterium in the marine environment (i.e. M. thiooxydans L4) and thus provides a powerful approach to access the genome and proteome of uncultivated microbes involved in key processes in the environment

Identification of the bacteriochlorophyll homologues of Chlorobium phaeobacteroides strain UdG6053 grown at low light intensity Identification of the bacteriochlorophyll homologues of Chlorobium phaeobacteroides strain UdG6053 grown at low light intensity

Detailed APCI LC-MS/MS analysis using an improved HPLC separation reveals the green sulphur bacterium Chlorobium phaeobacteroides strain UdG6053 to contain a wider range of distinct bacteriochlorophyll homologues than has been previously recognised in Chlorobiaceae. The diversity in the homologue distribution is confirmed as arising from differences in the extent of alkylation of the macrocycle and variation in the nature of the esterifying alcohol and a novel series of bacteriochlorophyll structures has been recognised. Homologues containing esterifying alcohols other than farnesol, a number of which have not previously been reported in Chlorobiaceae, are present in high relative abundance. Confirmation of the structures of the esterifying alcohols has been obtained by hydrolysis and analysis by GC-MS.

Ice-binding proteins adsorb to their ligand via anchored clathrate waters

The main success of my thesis has been to establish the mechanism by which antifreeze proteins (AFPs) bind irreversibly to ice crystals, and hence prevent their growth. AFPs organize ice-like water on their ice-binding site, which then merges and freezes with the quasi-liquid layer of ice. This was revealed from studying the exceptionally large (ca. 1.5-MDa) Ca 2+-dependent AFP from the Antarctic bacterium Marinomonas primoryensis (MpAFP). The 34-kDa antifreeze- active region of MpAFP was predicted to fold as a novel Ca 2+-binding β-helix. Site-directed mutagenesis confirmed the model and demonstrated that its ice-binding site (IBS) consisted of solvent-exposed Thr and Asx parallel arrays on the Ca 2+-binding turns. The X-ray crystal structure of the antifreeze region was solved to a resolution of 1.7 Å. Two of the four molecules within the unit cell of the crystal had portions of their IBSs freely exposed to solvent. Identical clathrate-like cages of water molecules were present on each IBS. These waters were organized by the hydrophobic effect and anchored to the protein via hydrogen bonds. They matched the spacing of water molecules in an ice lattice, demonstrating that anchored clathrate waters bind AFPs to ice. This mechanism was extended to other AFPs including the globular type III AFP from fishes. Site-directed mutagenesis and a modified ice-etching technique demonstrated this protein uses a compound ice-binding site, comprised of two flat and relatively hydrophobic surfaces, to bind at least two planes of ice. Reinvestigation of several crystal structures of type III AFP identified anchored clathrate waters on the solvent-exposed portion of its compound IBS that matched the spacing of waters on the primary prism plane of ice. Ice nucleation proteins (INPs), which can raise the temperature at which ice forms in solution to just slightly below 0oC, have the opposite effect to AFPs. A novel dimeric β-helical model was proposed for the INP produced by the bacterium Pseudomonas borealis. Molecular dynamics simulations showed that INPs are also capable of ordering water molecules into an ice- like lattice. However, their multimerization brings together sufficient ordered waters to form an ice nucleus and initiate freezing.

Enantiocomplementary preparation of (S)- and (R)-1-pyridylalkanols via ketone reduction and alkane hydroxylation using whole cells of Pseudomonas putida UV4

A previously unreported alcohol dehydrogenase enzyme in the mutant soil bacterium Pseudomonas putida UV4 catalyses the reduction of 2-, 3- and 4-acylpyridines to afford the corresponding (S)-1-pyridyl alkanols, with moderate to high e.e., whilst under the same conditions 2,6-diacetylpyridine is readily converted to the corresponding enantiopure C2-symmetric (S,S)-diol in one step. In contrast, the toluene dioxygenase enzyme in the same organism catalyses the hydroxylation of 2- and 3-alkylpyridines to (R)-1-(2-pyridyl) and (R)-1-(3-pyridyl)alkanols. This combination of oxidative and reductive biotransformations thus provides a method for preparing both enantiomers of chiral 1-pyridyl alkanols using one biocatalyst.