Towards the selection of a produced water enrichment for biological gas hydrate inhibitors.

Economic concerns associated with the recovery of non-conventional hydrocarbon reserves include unexpected ice as well as ice-like gas hydrate formation. Antifreeze proteins (AFPs) inhibit ice growth, and experiments with fish, plant, and insect AFPs have shown promise of effective gas hydrate inhibition in lab-scale experiments. If produced on an industrial scale, AFPs could provide a more environmentally friendly alternative to kinetic inhibitors, but a large-scale production of these AFPs is not currently feasible. We believe that these difficulties could be surmounted by the production of microbial AFPs, but to date, only a few such proteins have been identified and purified, and none of these are associated with hydrocarbon reserves. Here, we have used ice-affinity and freeze-thaw stress to select microbes derived from oil and gas formation water, or produced water, as a source of anaerobic microbial communities. Ice-affinity successfully incorporated anaerobic bacteria under aerobic conditions, and the mixed culture had ice-associating properties. Under these conditions, ice-affinity selection does not result in cultivatable isolates, but similar, cultivable microbes were obtained following freeze-thaw selection under anaerobic conditions. Since these mixed cultures inhibited the growth of ice crystals, they also have the potential to inhibit hydrate growth. Overall, freeze-thaw selection provides a promising first step towards the isolation of microbes capable of the inhibition of ice and gas hydrate growth, for possible application for energy exploration and recovery at high-latitudes and in-deep, cold waters.

Cross-tolerance between osmotic and freeze-thaw stress in microbial assemblages from temperate lakes.

Osmotic stress can accompany increases in solute concentrations because of freezing or high-salt environments. Consequently, microorganisms from environments with a high-osmotic potential may exhibit cross-tolerance to freeze stress. To test this hypothesis, enrichments derived from the sediment and water of temperate lakes with a range of salt concentrations were subjected to multiple freeze-thaw cycles. Surviving isolates were identified and metagenomes were sampled prior to and following selection. Enrichments from alkali lakes were typically the most freeze-thaw resistant with only 100-fold losses in cell viability, and those from freshwater lakes were most susceptible, with cell numbers reduced at least 100,000-fold. Metagenomic analysis suggested that selection reduced assemblage diversity more in freshwater samples than in those from saline lakes. Survivors included known psychro-, halo- and alkali-tolerant bacteria. Characterization of freeze-thaw-resistant isolates from brine and alkali lakes showed that few isolates had ice-associating activities such as antifreeze or ice nucleation properties. However, all brine- and alkali-derived isolates had high intracellular levels of osmolytes and/or appeared more likely to form biofilms. Conversely, these phenotypes were infrequent amongst the freshwater-derived isolates. These observations are consistent with microbial cross-tolerance between osmotic and freeze-thaw stresses.

The engrailed homeobox genes are required in multiple cell lineages to coordinate sequential formation of fissures and growth of the cerebellum.

The layered cortex of the cerebellum is folded along the anterior-posterior axis into lobules separated by fissures, allowing the large number of cells needed for advanced cerebellar functions to be packed into a small volume. During development, the cerebellum begins as a smooth ovoid structure with two progenitor zones, the ventricular zone and upper rhombic lip, which give rise to distinct cell types in the mature cerebellum. Initially, the cerebellar primordium is divided into five cardinal lobes, which are subsequently further subdivided by fissures. The cellular processes and genes that regulate the formation of a normal pattern of fissures are poorly understood. The engrailed genes (En1 and En2) are expressed in all cerebellar cell types and are critical for regulating formation of specific fissures. However, the cerebellar cell types that En1 and En2 act in to control growth and/or patterning of fissures has not been determined. We conditionally eliminated En2 or En1 and En2 either in both progenitor zones and their descendents or in the two complementary sets of cells derived from each progenitor zone. En2 was found to be required only transiently in the progenitor zones and their immediate descendents to regulate formation of three fissures and for general growth of the cerebellum. In contrast, En1 and En2 have overlapping functions in the cells derived from each progenitor zone in regulating formation of additional fissures and for extensive cerebellar growth. Furthermore, En1/2 function in ventricular zone-derived cells plays a more significant role in determining the timing of initiation and positioning of fissures, whereas in upper rhombic lip-derived cells the genes are more important in regulating cerebellar growth. Our studies reveal the complex manner in which the En genes control cerebellar growth and foliation in distinct cell types.

Prospecting for ice association: characterization of freeze-thaw selected enrichment cultures from latitudinally distant soils.

Freeze-thaw stress has previously been shown to alter soil community structure and function. We sought to further investigate this stress on enriched microbial consortia with the aim of identifying microbes with ice-associating adaptations that facilitate survival. Enrichments were established to obtain culturable psychrotolerant microbes from soil samples from the latitudinal extremes of the Canadian Shield plateau. The resulting consortia were subjected to consecutive freeze-thaw cycles, and survivors were putatively identified by their 16S rRNA gene sequences. Even though the northerly site was exposed to longer, colder winters and large spring-time temperature fluctuations, the selective regime similarly affected both enriched consortia. Quantitative PCR and metagenomic sequencing were used to determine the frequency of a subset of the resistant microbes in the original enrichments. The metagenomes showed 22 initial genera, only 6 survived and these were not dominant prior to selection. When survivors were assayed for ice recrystallization inhibition and ice nucleation activities, over 60% had at least one of these properties. These phenotypes were not more prevalent in the northern enrichment, indicating that regarding these adaptations, the enrichment strategy yielded seemingly functionally similar consortia from each site.

Primary cilia and Gli3 activity regulate cerebral cortical size.

During neural development patterning, neurogenesis, and overall growth are highly regulated and coordinated between different brain regions. Here, we show that primary cilia and the regulation of Gli activity are necessary for the normal expansion of the cerebral cortex. We show that loss of Kif3a, an important functional component of primary cilia, leads to the degeneration of primary cilia, marked overgrowth of the cortex, and altered cell cycle kinetics within cortical progenitors. The G1 phase of the cell cycle is shortened through a mechanism likely involving reduced Gli3 activity and a resulting increase in expression of cyclin D1 and Fgf15. The defects in Gli3 activity alone are sufficient to accelerate cell cycle kinetics and cause the molecular changes seen in brains that lack cilia. Finally, we show that levels of full-length and repressor Gli3 proteins are tightly regulated during normal development and correlate with changes in expression of two known Shh-target genes, CyclinD1 and Fgf15, and with the normal lengthening of the cell cycle during corticogenesis. These data suggest that Gli3 activity is regulated through the primary cilium to control cell cycle length in the cortex and thus determine cortical size.

Spatially restricted and developmentally dynamic expression of engrailed genes in multiple cerebellar cell types.

The cerebellum is a highly organized structure partitioned into lobules along the anterior-posterior (A-P) axis and into striped molecular domains along the medial-lateral (M-L) axis. The Engrailed (En) homeobox genes are required for patterning the morphological and molecular domains along both axes, as well as for the establishment of the normal afferent topography required to generate a fully functional cerebellum. As a means to understand how the En genes regulate multiple levels of cerebellum construction, we characterized En1 and En2 expression around birth and at postnatal day (P) 21 during the period when the cerebellum undergoes a remarkable transformation from a smooth ovoid structure to a highly foliated structure. We show that both En1 and En2 are expressed in many neuronal cell types in the cerebellum, and expression persists until at least P21. En1 and En2 expression, however, undergoes profound changes in their cellular and spatial distributions between embryonic stages and P21, and their expression domains become largely distinct. Comparison of the distribution of En-expressing Purkinje cells relative to early- and late-onset Purkinje cell M-L stripe proteins revealed that although En1- and En2-expressing Purkinje cell domains do not strictly align with those of ZEBRINII at P21, a clear pattern exists that is most evident at E17.5 by an inverse correlation between the level of En2 expression and PLCß4 and EPHA4.

Selection of low-temperature resistance in bacteria and potential applications.

Microbial consortia may harbour an array of resistance mechanisms that facilitate survival under harsh conditions, including antifreeze and ice-nucleation proteins. Antifreeze proteins lower freezing points as well as inhibit the growth of large, potentially damaging ice crystals from small ice embryos. In contrast, ice-nucleation proteins prevent supercooling and allow ice formation at high, sub-zero temperatures. Psychrophiles and psychrotolerant microbes are typically sought in extremely cold environments. However, given that geography is unlikely to present an insurmountable barrier to microbial dispersal, we reasoned that species with low-temperature adaptations should also be present, although rare, in more temperate environments. In consequence, the challenge then becomes one of selecting for rare microbes present in a larger community. Following the introductory commentary, we demonstrate that both freeze-thaw survival and ice-affinity selection can be used to identify microbes, which demonstrate low-temperature resistance, from enrichments derived from temperate environments. Selection resulted in a drastic decrease in cell abundance and diversity, allowing the isolation of a subset of resistant microbes. Depending on the origin of the consortia, these resistant microbes demonstrated cross-tolerance to osmotic stress, or a high proportion of antifreeze and/or ice-nucleation protein activities. Both types of ice-associating proteins presumably facilitate microbial survival at low temperatures. These proteins, as well as molecules that maintain osmotic balance, are also of commercial interest, with applications in the food, energy and medical industries. In addition, the resistant phenotypes described here provide a glimpse into the breadth of strategies microbes use to survive and thrive at low temperatures.

Ice-active characteristics of soil bacteria selected by ice-affinity.

As an initial screen for microorganisms that produce ice-active macromolecules, ice-affinity was used to select microorganisms from soil consortia originating from three temperate regions. Once selected and subsequently purified to single colonies, these microbes were putatively identified by 16S ribosomal RNA sequencing and assayed for various ice-active properties. Ice-affinity selection appeared to select for bacteria with ice-associating activities: inhibition of ice recrystallization; ice nucleation; ice shaping. Although none of these activities were observed in Paenibacillus amyloliticus C8, others such as Chryseobacterium sp. GL8, demonstrated both ice recrystallization inhibition and ice-shaping activities. Pseudomonas borealis DL7 was classified as a type I ice nucleator, Flavobacterium sp. GL7, was identified as a type III ice nucleator and Acinetobacter radioresistens DL5 demonstrated ice recrystallization inhibition. In all, 19 different culturable bacteria were selected from the thousands of microbes in late-summer collected soil samples. Many of the selected microbes have been previously reported in glacial ice cores or polar sea ice, and of five isolates that were further characterized, four showed ice-associating activities. These results indicate the significant potential of ice-affinity selection even with temperate climate soils, suggesting that sampling in more extreme and remote areas is not required for the isolation of ice-active bacteria.

Differential telomere processing by Borrelia telomere resolvases in vitro but not in vivo.

Causative agents of Lyme disease and relapsing fever, including Borrelia burgdorferi and Borrelia hermsii, respectively, are unusual among bacteria in that they possess a segmented genome with linear DNA molecules terminated by hairpin ends, known as telomeres. During replication, these telomeres are processed by the essential telomere resolvase, ResT, in a unique biochemical reaction known as telomere resolution. In this study, we report the identification of the B. hermsii resT gene through cross-species hybridization. Sequence comparison of the B. hermsii protein with the B. burgdorferi orthologue revealed 67% identity, including all the regions currently known to be crucial for telomere resolution. In vitro studies, however, indicated that B. hermsii ResT was unable to process a replicated B. burgdorferi type 2 telomere substrate. In contrast, in vivo cross-species complementation in which the native resT gene of B. burgdorferi was replaced with B. hermsii resT had no discernible effect, even though B. burgdorferi strain B31 carries at least two type 2 telomere ends. The B. burgdorferi ResT protein was also able to process two telomere spacing mutants in vivo that were unresolvable in vitro. The unexpected differential telomere processing in vivo versus in vitro by the two telomere resolvases suggests the presence of one or more accessory factors in vivo that are normally involved in the reaction. Our current results are also expected to facilitate further studies into ResT structure and function, including possible interaction with other Borrelia proteins.

Catalytic residues of the telomere resolvase ResT: a pattern similar to, but distinct from, tyrosine recombinases and type IB topoisomerases.

ResT is a member of the telomere resolvases, a newly discovered class of DNA breakage and reunion enzymes. These enzymes are involved in the formation of co-valently closed hairpin DNA ends that are found in linear prokaryotic chromosomes and plasmids. The hairpins are generated by telomere resolution, where the replicated linear DNA ends are processed by DNA breakage followed by joining of DNA free ends to the complementary strand of the same molecule. Previous studies have shown that ResT catalyzes hairpin formation through a two-step transesterification similar to tyrosine recombinases and type IB topoisomerases. In the present study we have probed the reaction mechanism of ResT. The enzyme was found to efficiently utilize a substrate with a 5'-bridging phosphorothiolate at each cleavage site, similar to tyrosine recombinases/type IB topoisomerases. Using such a substrate to trap the covalent protein-DNA intermediate, coupled with affinity purification and mass spectroscopy, we report a new, non-radioactive approach to directly determine the position of the amino acid in the protein, which is linked to the DNA. We report that tyrosine 335 is the active site nucleophile in ResT, strengthening the link between ResT and tyrosine recombinases/type IB topoisomerases. However, a distinct pattern of catalytic residues with similarities, but distinct differences from the above enzymes was suggested. The differences include the apparent absence of a general acid catalyst, as well as the dispensability of the final histidine in the RKHRHY hexad. Finally, two signature motifs (GRR(2X)E(6X)F and LGH(4-6X)T(3X)Y) near the catalytic residues of aligned telomere resolvases are noted.