Interaction of the gas vesicle proteins GvpA, GvpC, GvpN, and GvpO of Halobacterium salinarum

The interactions of the four gas vesicle proteins GvpA, C, N, and O were investigated by split-GFP and pulldown assays. GvpA forms the ribs of the gas vesicle shell, whereas GvpC is attached to the exterior surface and stabilizes the gas vesicle structure. The AAA-ATPase GvpN as well as GvpO is found in much lower amounts. GvpN and GvpO formed homodimers and also the GvpN/GvpO heterodimer; both interacted with the C-terminal domain of GvpC when tested by split-GFP. When analyzed by pulldown assays, GvpN and GvpO also selected GvpA. The N-and C-terminal fragments of GvpC dimerized as Cterm/Cterm and Cterm/Nterm, but not as Nterm/Nterm. These interactions at both termini might lead to a network of GvpC molecules at the gas vesicle surface. However, a GvpA/GvpC interaction was not detectable, suggesting that the contact of both proteins is either mediated by another Gvp, or requires different structures that might form when GvpA is aggregated in the gas vesicle shell. Interactions of GvpA, C, N, and O were also studied with the accessory proteins GvpF through GvpM by split-GFP. GvpN bound GvpL only, whereas GvpO interacted with GvpF, I, and L, and the C-terminal domain of GvpC contacted GvpF, H, I, and L. GvpA/GvpA interactions were difficult to detect by split-GFP, but GvpA selected except for GvpI, K, and L all other accessory Gvp in pulldown assays. We will discuss the implications of these findings on gas-vesicle assembly

Heavy Metal Ion Stress on Halobacterium salinarum R1 Planktonic Cells and Biofilms

Halobacterium salinarum R1 is an extremely halophilic archaeon, able to attach to the surface and to form characteristic biofilm structures under physiological conditions. However, the effect of environmental stress factors like heavy metals on biofilms was still unknown. Here, we report on the first insights into H. salinarum biofilm formation when exposed to copper, nickel and zinc and describe the effects of metal ions on the architecture of mature biofilms. We also studied the effects on gene expression in planktonic cells. Investigation of planktonic growth and cell adhesion in the presence of sub-lethal metal concentrations yielded an up to 60% reduced adhesion in case of copper and a significantly enhanced adhesion in case of zinc, whereas nickel treatment had no effect on adhesion. A PMA-qPCR assay was developed to quantify live/dead cells in planktonic cultures and mature biofilms, enabling the investigation of cell vitality after metal exposure. An increased resistance was observed in biofilms with up to 80% in case of copper- and up to 50% in case of zinc exposure compared to planktonic cells. However, nickel-treated biofilms showed no significant increase of cell survival. Microscopic investigation of the architecture of mature biofilms exposed to lethal metal concentrations demonstrated an increased detachment and the formation of large microcolonies after copper treatment, whereas the number of adherent cells increased strongly in nickel-exposed biofilms. In contrast, zinc exposed-biofilms showed no differences compared to the control. Analysis of the expression of genes encoding putative metal transporters by qRT-PCR revealed specific changes upon treatment of the cells with heavy metals. Our results demonstrate diverse effects of heavy metal ions on H. salinarum and imply a metal-specific protective response of cells in biofilms

Overlapping activator sequences determined for two oppositely oriented promoters in halophilic Archaea

Transcription of the genomic region involved in gas vesicle formation in Halobacterium salinarum (p-vac) and Haloferax mediterranei (mc-vac) is driven by two divergent promoters, PA and PD, separated by only 35 nt. Both promoters are activated by the transcription activator GvpE which in the case of PmcA requires a 20-nt sequence (UAS) consisting of two conserved 8-nt sequence portions located upstream of BRE. Here, we determined the two UAS elements in the promoter region of p-vac by scanning mutageneses using constructs containing PpD (without PpA) fused to the bgaH reporter gene encoding an enzyme with β-galactosidase activity, or the dual reporter construct pApD with PpD fused to bgaH and PpA to an altered version of gvpA. The two UAS elements found exhibited a similar extension and distance to BRE as previously determined for the UAS in PmcA. Their distal 8-nt portions almost completely overlapped in the centre of PpD–PpA, and mutations in this region negatively affected the GvpE-mediated activation of both promoters. Any alteration of the distance between BRE and UAS resulted in the loss of the GvpE activation, as did a complete substitution of the proximal 8-nt portion, underlining that a close location of UAS and BRE was very important

Effect of Mutations in GvpJ and GvpM on Gas Vesicle Formation of Halobacterium salinarum

The two haloarchaeal proteins, GvpM and GvpJ, are homologous to GvpA, the major gas vesicle structural protein. All three are hydrophobic and essential for gas vesicle formation. The effect of mutations in GvpJ and GvpM was studied in Haloferax volcanii transformants by complementing the respective mutated gene with the remaining gvp genes and inspecting the cells for the presence of gas vesicles (Vac⁺). In case of GvpJ, 56 of 66 substitutions analyzed yielded Vac⁻ ΔJ + Jmut transformants, indicating that GvpJ is very sensitive to alterations, whereas ten of the 38 GvpM variants resulted in Vac⁻ ΔM + Mmut transformants. The variants were also tested by split-GFP for their ability to interact with their partner protein GvpL. Some of the alterations leading to a Vac⁻ phenotype affected the J/L or M/L interaction. Also, the interactions J/A and J/M were studied using fragments to exclude an unspecific aggregation of these hydrophobic proteins. Both fragments of GvpJ interacted with the M1–25 and M60–84 fragments of GvpM, and fragment J1–56 of GvpJ interacted with the N-terminal fragment A1–22 of GvpA. A comparison of the results on the three homologous proteins indicates that despite their relatedness, GvpA, GvpJ, and GvpM have unique features and cannot substitute each other

The high school student in the school district spase and inner school space:one understanding through the questionnaire

textabstractA novel papain inhibitory protein (SPI) from Streptomyces mobaraensis was studied to measure its inhibitory effect on bacterial cysteine protease activity (Staphylococcus aureus SspB) and culture supernatants (Porphyromonas gingivalis, Bacillus anthracis). Further, growth of Bacillus anthracis, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio cholerae was completely inhibited by 10 μM SPI. At this concentration of SPI, no cytotoxicity was observed. We conclude that SPI inhibits bacterial virulence factors and has the potential to become a novel therapeutic treatment against a range of unrelated pathogenic bacteria. Copyrigh