Mutations Affecting HVO_1357 or HVO_2248 Cause Hypermotility in Haloferax volcanii, Suggesting Roles in Motility Regulation

Abstract

Motility regulation plays a key role in prokaryotic responses to environmental stimuli. Here, we used a motility screen and selection to isolate hypermotile Haloferax volcanii mutants from a transposon insertion library. Whole genome sequencing revealed that hypermotile mutants were predominantly affected in two genes that encode HVO_1357 and HVO_2248. Alterations of these genes comprised not only transposon insertions but also secondary genome alterations. HVO_1357 contains a domain that was previously identified in the regulation of bacteriorhodopsin transcription, as well as other domains frequently found in two-component regulatory systems. The genes adjacent to hvo_1357 encode a sensor box histidine kinase and a response regulator, key players of a two-component regulatory system. None of the homologues of HVO_2248 have been characterized, nor does it contain any of the assigned InterPro domains. However, in a significant number of Haloferax species, the adjacent gene codes for a chemotaxis receptor/transducer. Our results provide a foundation for characterizing the root causes underlying Hfx. volcanii hypermotility.

Identification and characterization of structural and regulatory cell-shape determinants inHaloferax volcanii

“…Transposon mutants JK3, JK5, and SAH1 were identified through individual motility stabs of the transposon library and subsequent screening for hypermotility. These screens were performed as previously described (34). Whole genome sequencing was performed on isolated transposon mutants via Illumina sequencing performed by SeqCenter (Pittsburgh, PA, USA).…”

“…Out of five hypermotile strains identified through stab-inoculation screens (34), four formed predominantly rods, with some disks in later growth phases, but only one exclusively formed rods (Fig. 1): SAH1, which contains a transposon insertion upstream of hvo_2176 (position 2,049,208) (34). We repeated the screen and assessed the motility of an additional 1500 transposon insertion mutants, five of which were hypermotile.…”

“…The main goal for this initial proteomics experiment was to determine whether the experimental design would facilitate identification of proteins involved in shape determination, subsequently allowing us to generate additional shape-defective gene deletion strains. These strains would be derived from a single parent strain rather than using transposon mutants that often have secondary mutations (34).…”

The Best of Both Worlds: Discovery-Driven Learning through a Lab-Seminar Approach

“…In their proposals, students incorporated concepts learned through reading and class discussions of primary literature and experimental techniques experienced during their lab work (see Appendix S4). Since our students did not have enough time to identify genes of interest, we asked them to write their proposals on genes identified in published literature ( 12 ). We encouraged students to include rigor and reproducibility in their proposals and consider factors such as feasibility, available resources, and allotted time.…”

Open Issues for Protein Function Assignment in Haloferax volcanii and Other Halophilic Archaea

“…Its genome has been sequenced and carefully annotated [ 1 , 10 , 11 ]. A plethora of biological aspects have been successfully tackled in this species, with examples including DNA replication [ 4 ]; cell division and cell shape [ 12 , 13 , 14 , 15 , 16 ]; metabolism [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ]; protein secretion [ 26 , 27 , 28 , 29 ]; motility and biofilms [ 30 , 31 , 32 , 33 , 34 , 35 ]; mating [ 36 ]; signaling [ 37 ]; virus defense [ 38 ]; proteolysis [ 39 , 40 , 41 , 42 , 43 , 44 ]; posttranslational modification (N-glycosylation; SAMPylation) [ 45 , 46 , 47 , 48 , 49 , 50 ]; gene regulation [ 21 , 25 , 51 , 52 , 53 , 54 , 55 ]; microproteins [ 56 , 57 , 58 ] and small noncoding RNAs (sRNAs) […”