VapC6, a ribonucleolytic toxin regulates thermophilicity in the crenarchaeote Sulfolobus solfataricus

Abstract

The phylum Crenarchaeota includes hyperthermophilic micro-organisms subjected to dynamic thermal conditions. Previous transcriptomic studies of Sulfolobus solfataricus identified vapBC6 as a heat-shock (HS)-inducible member of the Vap toxinantitoxin gene family. In this study, the inactivation of the vapBC6 operon by targeted gene disruption produced two recessive phenotypes related to fitness, HS sensitivity and a heat-dependent reduction in the rate of growth. In-frame vapBC6 deletion mutants were analyzed to examine the respective roles of each protein. Since vapB6 transcript abundance was elevated in the vapC6 deletion, the VapC6 toxin appears to regulate abundance of its cognate antitoxin. In contrast, vapC6 transcript abundance was reduced in the vapB6 deletion. A putative intergenic terminator may underlie these observations by coordinating vapBC6 expression. As predicted by structural modeling, recombinant VapC6 produced using chaperone cosynthesis exhibited heat-dependent ribonucleolytic activity toward S. solfataricus total RNA. This activity could be blocked by addition of preheated recombinant VapB6. In vivo transcript targets were identified by assessing the relative expression of genes that naturally respond to thermal stress in VapBC6-deficient cells. Preferential increases were observed for dppB-1 and tetR, and preferential decreases were observed for rpoD and eIF2 gamma. Specific VapC6 ribonucleolytic action could also be demonstrated in vitro toward RNAs whose expression increased in the VapBC6-deficient strain during heat shock. These findings provide a biochemical mechanism and identify cellular targets underlying VapBC6-mediated control over microbial growth and survival at temperature extremes.

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

“…VapC of Shigella flexneri 2a virulence plasmid pMYSH6000 and VapC of Salmonella enterica serovar Typhimurium T2 are site-specific tRNases that cleave initiator tRNA between the anticodon stem and loop (Winther and Gerdes 2011). VapC6 from Sulfolobus solfataricus targets mRNAs involved in the heat shock response (Maezato et al 2011), and VapC from nontypeable Haemophilus influenzae degrades free RNA in vitro but not double-stranded (ds) DNA or single-stranded (ss) DNA (Daines et al 2007). VapC-5 from Mycobacterium tuberculosis displays Mg 2+ -dependent ribonuclease activity (Miallau et al 2009), although the catalytic mechanism or sequence-specificity of these enzymes has not been determined.…”

A broadly applicable gene knockout system for the thermoacidophilic archaeon Sulfolobus islandicus based on simvastatin selection

“…Unfortunately, lactose selection is limited to S. solfataricus and a few S. islandicus strains such as S. islandicus REN1H1, because many strains of Sulfolobus cannot be grown efficiently in medium using lactose as the sole carbon and energy source (Berkner & Lipps, 2008;Grogan, 1989). Maltose selection has also been established in S. solfataricus, in which the malA gene was used as a marker (Maezato et al, 2011b).…”

Identification of a VapBC toxin–antitoxin system in a thermophilic bacterium Thermus thermophilus HB27

“…3c). These results strongly indicate that TtVapC does not possess substrate-or sequence-specificity, rather it degrades free RNA, such as VapC6 of Sulfolobus solfataricus (Maezato et al 2011), and those of some pathogenic bacteria (Daines et al 2007;Ramage et al 2009). …”