Mycoplasmas regulate the expression of heat-shock protein genes through CIRCE-HrcA interactions

Mycoplasmas in general are rarely exposed to severe environmental changes except during its colonization and infection processes. Genomic analysis indicates that Mycoplasma hyopneumoniae possesses the genes of a single sigma factor and the HrcA repressor of negative regulation of the heat-shock response. A perfect inverted repeat sequence (5′-CTGGCACTT-N₉-AAGTGCCAA-3′) upstream of the DnaK gene has also been identified. In the present study, we demonstrate the functionality of HrcA-CIRCE interactions using the gel electrophoretic mobility shift assay. The presence of the unique sigma factor, HrcA repressor, and the CIRCE-like sequences reveals that mycoplasmal species may all use the negative regulatory mechanism in the heat-shock response. It is conceivable that mycoplasmas may have evolved a single HrcA repressor-based mechanism which might be the most simple and economical way of controlling HSP gene expression.     

A nested gene in Streptomyces bacteria encodes a protein involved in quaternary complex formation

The gene pgaM is involved in the biosynthesis of an angucycline-type polyketide antibiotic in Streptomyces sp. PGA64. It encodes a two-domain polypeptide consisting of an N-terminal flavoprotein oxygenase and a C-terminal short-chain alcohol dehydrogenase/reductase, which are fused together at the translational level as a result of end codon deletion. Here we show that translation also initiates at an internal start codon that enables independent expression of a separate reductase subunit, PgaMred. This confirms that the gene exhibits a rare viral-like arrangement of two overlapping reading frames that allows simultaneous expression of two alternative forms of the protein. Together, these two proteins associate to form a stable non-covalent complex, the native form of PgaM. The reductase subunit PgaMred is shown to provide enzyme stability and to affect the redox state of the oxygenase domain FAD. Finally, a model for the quaternary structure of the complex that explains the necessity for a nested gene system and the unusual behaviour of the protein subunits in vitro is presented.     

Sub-terminal sequences modulating IS30 transposition in vivo and in vitro

Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR-IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5′-GAGATAATTG-3′, is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.     

The funnel approach to the precrystallization production of membrane proteins

Challenges in the production of integral membrane proteins for structural studies include low expression levels, incorrect membrane insertion, aggregation and instability. In this report, we describe a “funnel approach” to overcoming these difficulties and demonstrate its efficacy in a case study of 36 prokaryotic P-type transporters. A diverse ensemble of modified constructs is generated and tested for expression in Escherichia coli, membrane localization, detergent extraction, and homogeneity. High-throughput methodologies are implemented throughout the process to facilitate identification of promising targets. We find that the choice of promoter, the choice of source organism providing the cloned gene, and, most importantly, the position of the affinity tag have a great effect on successful production. The latter had pronounced effects at all tested levels, from expression levels observed in whole cells to the extent of membrane insertion, and even on protein function. Following the initial streamlined screening, we were able to fine-tune and produce 9 of the 36 targets as materials suitable for crystallization or other structural studies.     

Three murine anxiety models: results from multiple inbred strain comparisons

The literature surrounding rodent models of human anxiety disorders is discrepant concerning which models reflect anxiety-like behavior distinct from general activity and whether different models are measuring the same underlying constructs. This experiment compared the responses of 15 inbred mouse strains (129S1/SvlmJ, A/J, AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, CBA/J, CE/J, DBA/2J, FVB/NJ, NZB/B1NJ, PL/J, SJL/J and SWR/J) in three anxiety-like behavioral tasks (light/dark test, elevated zero-maze and open field) to examine whether responses were phenotypically and/or genetically correlated across tasks. Significant strain differences were found for all variables examined. Principal components analyses showed that variables associated with both activity and anxiety-like behaviors loaded onto one factor, while urination and defecation loaded onto another factor. Our findings differ from previous research by suggesting that general activity and anxiety-related behaviors are linked, negatively correlated and cannot easily be dissociated in these assays. However, these findings may not necessarily generalize to other unconditioned anxiety-like behavioral tests.     

Influence of elevated CO2 and O3 on IAA, IAA oxidase and peroxidase in the leaves of ginkgo trees

This study examined the impacts of elevated CO₂ or O₃ on indole-3-acetic acid (IAA) content, activities of IAA oxidase (IAAO) and peroxidase (POD) in Ginkgo biloba leaves. Plants grown in open-top chambers were exposed to ambient atmosphere (control; C), elevated CO₂ and elevated O₃ from 1 June to 30 September. An increase in IAA content and decrease in IAAO and POD activities were observed in plants exposed to elevated CO₂ compared with C. Elevated O₃ had no significant effect on IAA content and IAAO activity, but increased POD activity during the early days. When trees pre-exposed to elevated CO₂ were transferred to elevated O₃ or C, the increase in IAAO activity resulted in the decrease in IAA content. When trees pre-exposed to elevated O₃ were transferred to elevated CO₂ or C, IAA content, IAAO and POD activities showed no significant changes. The influence of POD activity on the IAA activity was low.     

Crystal structure of a soluble decoy receptor IL-22BP bound to interleukin-22

Interleukin-22 (IL-22) plays an important role in the regulation of immune and inflammatory responses in mammals. The IL-22 binding protein (IL-22BP), a soluble receptor that specifically binds IL-22, prevents the IL-22/interleukin-22 receptor 1 (IL-22R1)/interleukin-10 receptor 2 (IL-10R2) complex assembly and blocks IL-22 biological activity. Here we present the crystal structure of the IL-22/IL-22BP complex at 2.75 Å resolution. The structure reveals IL-22BP residues critical for IL-22 binding, which were confirmed by site-directed mutagenesis and functional studies. Comparison of IL-22/IL-22BP and IL-22/IL-22R1 crystal structures shows that both receptors display an overlapping IL-22 binding surface, which is consistent with the inhibitory role played by IL-22 binding protein.

Structured summary

MINT-7010533: IL-22 BP (uniprotkb:Q969J5) and IL-22 (uniprotkb:Q9GZX6) bind (MI:0407) by X-ray crystallography (MI:0114)