Improving the genetic system for Halorubrum lacusprofundi to allow in-frame deletions

Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, Antarctica. Hrr. lacusprofundi is commonly used to study adaptation to cold environments and thereby a potential source for biotechnological products. Additionally, in contrast to other haloarchaeal model organisms, Hrr. lacusprofundi is also susceptible to a range of different viruses and virus-like elements, making it a great model to study virus-host interactions in a cold-adapted organism. A genetic system has previously been reported for Hrr. lacusprofundi; however, it does not allow in-frame deletions and multiple gene knockouts. Here, we report the successful generation of uracil auxotrophic (pyrE2) mutants of two strains of Hrr. lacusprofundi. Subsequently, we attempted to generate knockout mutants using the auxotrophic marker for selection. However, surprisingly, only the combination of the auxotrophic marker and antibiotic selection allowed the timely and clean in-frame deletion of a target gene. Finally, we show that vectors established for the model organism Haloferax volcanii are deployable for genetic manipulation of Hrr. lacusprofundi, allowing the use of the portfolio of genetic tools available for H. volcanii in Hrr. lacusprofundi

The sub-cellular localization of Sulfolobus DNA replication

Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated

Replication termination and chromosome dimer resolution in the archaeon Sulfolobus solfataricus

Eubacteria and archaea possess single-circular chromosomes, yet some archaea resemble eukaryotes in using multiple origins and replication forks. Replication termination in Sulfolobus is found to occur by stochastic collision of these forks, and—unlike the situation in eubacteria—it is not linked to chromosome segregation

“Influence of plasmids, selection markers and auxotrophic mutations on Haloferax volcanii cell shape plasticity”

Haloferax volcanii and other Haloarchaea can be pleomorphic, adopting different shapes, which vary with growth stages. Several studies have shown that H. volcanii cell shape is sensitive to various external factors including growth media and physical environment. In addition, several studies have noticed that the presence of a recombinant plasmid in the cells is also a factor impacting H. volcanii cell shape, notably by favoring the development of rods in early stages of growth. Here we investigated the reasons for this phenomenon by first studying the impact of auxotrophic mutations on cell shape in strains that are commonly used as genetic backgrounds for selection during strain engineering (namely: H26, H53, H77, H98, and H729) and secondly, by studying the effect of the presence of different plasmids containing selection markers on the cell shape of these strains. Our study showed that most of these auxotrophic strains have variation in cell shape parameters including length, aspect ratio, area and circularity and that the plasmid presence is impacting these parameters too. Our results indicated that ΔhdrB strains and hdrB selection markers have the most influence on H. volcanii cell shape, in addition to the sole presence of a plasmid. Finally, we discuss limitations in studying cell shape in H. volcanii and make recommendations based on our results for improving reproducibility of such studies

The parasitic lifestyle of an archaeal symbiont

DPANN archaea are a diverse group of microorganisms characterised by small cells and reduced genomes. To date, all cultivated DPANN archaea are ectosymbionts that require direct cell contact with an archaeal host species for growth and survival. However, these interactions and their impact on the host species are poorly understood. Here, we show that a DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. During these interactions, the nanohaloarchaeon appears to enter, or be engulfed by, the host cell. Our results provide experimental evidence for a predatory-like lifestyle of an archaeon, suggesting that at least some DPANN archaea may have roles in controlling host populations and their ecology

Diversity and Potential Multifunctionality of Archaeal CetZ Tubulin-like Cytoskeletal Proteins

Tubulin superfamily (TSF) proteins are widespread, and are known for their multifaceted roles as cytoskeletal proteins underpinning many basic cellular functions, including morphogenesis, division, and motility. In eukaryotes, tubulin assembles into microtubules, a major component of the dynamic cytoskeletal network of fibres, whereas the bacterial homolog FtsZ assembles the division ring at midcell. The functions of the lesser-known archaeal TSF proteins are beginning to be identified and show surprising diversity, including homologs of tubulin and FtsZ as well as a third archaea-specific family, CetZ, implicated in the regulation of cell shape and possibly other unknown functions. In this study, we define sequence and structural characteristics of the CetZ family and CetZ1 and CetZ2 subfamilies, identify CetZ groups and diversity amongst archaea, and identify potential functional relationships through analysis of the genomic neighbourhoods of cetZ genes. We identified at least three subfamilies of orthologous CetZ proteins in the archaeal class Halobacteria, including CetZ1 and CetZ2 as well as a novel uncharacterized subfamily. CetZ1 and CetZ2 were correlated to one another as well as to cell shape and motility phenotypes across diverse Halobacteria. Among other known CetZ clusters in orders Archaeoglobales, Methanomicrobiales, Methanosarcinales, and Thermococcales, an additional uncharacterized group from Archaeoglobales and Methanomicrobiales is affiliated strongly with Halobacteria CetZs, suggesting that they originated via horizontal transfer. Subgroups of Halobacteria CetZ2 and Thermococcales CetZ genes were found adjacent to different type IV pili regulons, suggesting potential utilization of CetZs by type IV systems. More broadly conserved cetZ gene neighbourhoods include nucleotide and cofactor biosynthesis (e.g., F420) and predicted cell surface sugar epimerase genes. These findings imply that CetZ subfamilies are involved in multiple functions linked to the cell surface, biosynthesis, and motility

Data_Sheet_1_Improving the genetic system for Halorubrum lacusprofundi to allow in-frame deletions.pdf

Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, Antarctica. Hrr. lacusprofundi is commonly used to study adaptation to cold environments and thereby a potential source for biotechnological products. Additionally, in contrast to other haloarchaeal model organisms, Hrr. lacusprofundi is also susceptible to a range of different viruses and virus-like elements, making it a great model to study virus-host interactions in a cold-adapted organism. A genetic system has previously been reported for Hrr. lacusprofundi; however, it does not allow in-frame deletions and multiple gene knockouts. Here, we report the successful generation of uracil auxotrophic (pyrE2) mutants of two strains of Hrr. lacusprofundi. Subsequently, we attempted to generate knockout mutants using the auxotrophic marker for selection. However, surprisingly, only the combination of the auxotrophic marker and antibiotic selection allowed the timely and clean in-frame deletion of a target gene. Finally, we show that vectors established for the model organism Haloferax volcanii are deployable for genetic manipulation of Hrr. lacusprofundi, allowing the use of the portfolio of genetic tools available for H. volcanii in Hrr. lacusprofundi.</p

Non-linear Min protein interactions generate harmonics that signal mid-cell division in <i>Escherichia coli</i>

<div><p>The Min protein system creates a dynamic spatial pattern in <i>Escherichia coli</i> cells where the proteins MinD and MinE oscillate from pole to pole. MinD positions MinC, an inhibitor of FtsZ ring formation, contributing to the mid-cell localization of cell division. In this paper, Fourier analysis is used to decompose experimental and model MinD spatial distributions into time-dependent harmonic components. In both experiment and model, the second harmonic component is responsible for producing a mid-cell minimum in MinD concentration. The features of this harmonic are robust in both experiment and model. Fourier analysis reveals a close correspondence between the time-dependent behaviour of the harmonic components in the experimental data and model. Given this, each molecular species in the model was analysed individually. This analysis revealed that membrane-bound MinD dimer shows the mid-cell minimum with the highest contrast when averaged over time, carrying the strongest signal for positioning the cell division ring. This concurs with previous data showing that the MinD dimer binds to MinC inhibiting FtsZ ring formation. These results show that non-linear interactions of Min proteins are essential for producing the mid-cell positioning signal via the generation of second-order harmonic components in the time-dependent spatial protein distribution.</p></div