No abstract

CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.

Summary The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex co-purifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5’ ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a ‘sea worm’ and is composed of a Cmr2-3 heterodimer ‘tail’, a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled ‘head’ containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.

Anaerobic oxidation of methane (AOM) is catalyzed by anaerobic methane-oxidizing archaea (ANME) via a reverse and modified methanogenesis pathway. Methanogens can also reverse the methanogenesis pathway to oxidize methane, but only during net methane production (i.e., “trace methane oxidation”). In turn, ANME can produce methane, but only during net methane oxidation (i.e., enzymatic back flux). Net AOM is exergonic when coupled to an external electron acceptor such as sulfate (ANME-1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis pathway and essential differences between ANME and methanogens are described by combining published information with domain based (meta)genome comparison of archaeal methanotrophs and selected archaea. These differences include abundances and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively. Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different mechanism for disposal of electrons and possibly are less versatile in electron acceptors use than ANME-2. Future research will shed light on the molecular basis of reversal of the methanogenic pathway and electron transfer in different ANME types.

In sulfate-reducing and methanogenic environments complex biopolymers are hydrolyzed and degraded by fermentative micro-organisms that produce hydrogen, carbon dioxide and short chain fatty acids. Degradation of short chain fatty acids can be coupled to methanogenesis or to sulfate-reduction. Here we study from a genome perspective why some of these micro-organisms are able to grow in syntrophy with methanogens and others are not. Bacterial strains were selected based on genome availability and upon their ability to grow on short chain fatty acids alone or in syntrophic association with methanogens. Systematic functional domain profiling allowed us to shed light on this fundamental and ecologically important question. Extra-cytoplasmic formate dehydrogenases (InterPro domain number; IPR006443), including their maturation protein FdhE (IPR024064 and IPR006452) is a typical difference between syntrophic and non-syntrophic butyrate and propionate degraders. Furthermore, two domains with a currently unknown function seem to be associated with the ability of syntrophic growth. One is putatively involved in capsule or biofilm production (IPR019079) and a second in cell division, shape-determination or sporulation (IPR018365). The sulfate-reducing bacteria Desulfobacterium autotrophicum HRM2, Desulfomonile tiedjei and Desulfosporosinus meridiei were never tested for syntrophic growth, but all crucial domains were found in their genomes, which suggests their possible ability to grow in syntrophic association with methanogens. In addition, profiling domains involved in electron transfer mechanisms revealed the important role of the Rnf-complex and the formate transporter in syntrophy, and indicate that DUF224 may have a role in electron transfer in bacteria other than Syntrophomonas wolfei as well. This article is a part of a Special Issue entitled: 18th European Bioenergetics Conference (Biochim. Biophys. Acta, Volume 1837, Issue 7, July 2014).