Deletion of the Sm1 encoding motif in the lsm gene results in distinct changes in the transcriptome and enhanced swarming activity of Haloferax cells.

Members of the Sm protein family are important for the cellular RNA metabolism in all three domains of life. The family includes archaeal and eukaryotic Lsm proteins, eukaryotic Sm proteins and archaeal and bacterial Hfq proteins. While several studies concerning the bacterial and eukaryotic family members have been published, little is known about the archaeal Lsm proteins. Although structures for several archaeal Lsm proteins have been solved already more than ten years ago, we still do not know much about their biological function, however one can confidently propose that the archaeal Lsm proteins will also be involved in RNA metabolism. Therefore, we investigated this protein in the halophilic archaeon Haloferax volcanii. The Haloferax genome encodes a single Lsm protein, the lsm gene overlaps and is co-transcribed with the gene for the ribosomal L37.eR protein. Here, we show that the reading frame of the lsm gene contains a promoter which regulates expression of the overlapping rpl37R gene. This rpl37R specific promoter ensures high expression of the rpl37R gene in exponential growth phase. To investigate the biological function of the Lsm protein we generated a lsm deletion mutant that had the coding sequence for the Sm1 motif removed but still contained the internal promoter for the downstream rpl37R gene. The transcriptome of this deletion mutant was compared to the wild type transcriptome, revealing that several genes are down-regulated and many genes are up-regulated in the deletion strain. Northern blot analyses confirmed down-regulation of two genes. In addition, the deletion strain showed a gain of function in swarming, in congruence with the up-regulation of transcripts encoding proteins required for motility.

Generation and phenotyping of a collection of sRNA gene deletion mutants of the haloarchaeon Haloferax volcanii.

The haloarchaeon Haloferax volcanii was shown to contain 145 intergenic and 45 antisense sRNAs. In a comprehensive approach to unravel various biological roles of haloarchaeal sRNAs in vivo, 27 sRNA genes were selected and deletion mutants were generated. The phenotypes of these mutants were compared to that of the parent strain under ten different conditions, i.e. growth on four different carbon sources, growth at three different salt concentrations, and application of four different stress conditions. In addition, cell morphologies in exponential and stationary phase were observed. Furthermore, swarming of 17 mutants was analyzed. 24 of the 27 mutants exhibited a difference from the parent strain under at least one condition, revealing that haloarchaeal sRNAs are involved in metabolic regulation, growth under extreme conditions, regulation of morphology and behavior, and stress adaptation. Notably, 7 deletion mutants showed a gain of function phenotype, which has not yet been described for any other prokaryotic sRNA gene deletion mutant. Comparison of the transcriptomes of one sRNA gene deletion mutant and the parent strain led to the identification of differentially expressed genes. Genes for flagellins and chemotaxis were up-regulated in the mutant, in accordance with its gain of function swarming phenotype. While the deletion mutant analysis underscored that haloarchaeal sRNAs are involved in many biological functions, the degree of conservation is extremely low. Only 3 of the 27 genes are conserved in more than 10 haloarchaeal species. 22 of the 27 genes are confined to H. volcanii, indicating a fast evolution of haloarchaeal sRNA genes.

Antibacterial effector/immunity systems: it's just the tip of the iceberg.

Bacteria do not live anchoretic; rather they are constantly in touch with their eukaryotic hosts and with other bacteria sharing their habitat. Therefore, bacteria have evolved sophisticated proteinaceous weapons. To harm other bacteria, they produce antibacterial effector proteins, which they either release into the environment or export via direct intercellular contact. Contact-dependent killing is mediated by two specialized secretion systems, the type V and VI secretion system, whereas contact-independent processes hijack other transport mechanisms. Regardless of the transport system, cells co-express immunity proteins to protect themselves from suicide and fratricide. In general, effector protein activities and secretion mechanisms differ between Gram-positive and Gram-negative bacteria and evidence is emerging that different effector/immunity systems act synergistically and thus extend the bacterial armory.

Structural Insights into the Effector - Immunity System Tae4/Tai4 from Salmonella typhimurium.

Type-6-secretion systems of Gram-negative bacteria are widely distributed needle-like multi-protein complexes that are involved in microbial defense mechanisms. During bacterial competition these injection needles dispense effector proteins into the periplasm of competing bacteria where they induce degradation of the peptidoglycan scaffold and lead to cell lysis. Donor cells co-produce immunity proteins and shuttle them into their own periplasm to prevent accidental toxication by siblings. Recently, a plethora of previously unidentified hydrolases have been suggested to be peptidoglycan degrading amidases. These hydrolases are part of effector/immunity pairs that have been associated with bacterial warfare by type-6-secretion systems. The Tae4 and Tai4 operon encoded by Salmonella typhimurium is one of these newly discovered effector/immunity pairs. The Tae4 effector proteins induce cell lysis by cleaving the γ-D-glutamyl-L-meso-diaminopimelic acid amide bond of acceptor stem muropeptides of the Gram-negative peptidoglycan. Although homologues of the Tae4/Tai4 system have been identified in various different pathogens, little is known about the functional mechanism of effector protein activity and their inhibition by the cognate immunity proteins. Here, we present the high-resolution crystal structure of the effector Tae4 of S. typhimurium in complex with its immunity protein Tai4. We show that Tae4 contains a classical NlpC/P60-peptidase core which is common to other effector proteins of the type-6-secretion system. However, Tae4 has unique structural features that are exclusively conserved within the family of Tae4 effectors and which are important for the substrate specificity. Most importantly, we show that although the overall structure of Tai4 is different to previously described immunity proteins, the essential mode of enzyme inhibition is conserved. Additionally, we provide evidence that inhibition in the Tae4/Tai4 heterotetramer relies on a central Tai4 dimer in order to acquire functionality.

Structural insights into the effector-immunity system Tse1/Tsi1 from Pseudomonas aeruginosa.

During an interbacterial battle, the type-6-secretion-system (T6SS) of the human pathogen Pseudomonas aeruginosa injects the peptidoglycan(PG)-hydrolase Tse1 into the periplasm of gram-negative enemy cells and induces their lysis. However, for its own benefit, P. aeruginosa produces and transports the immunity-protein Tsi1 into its own periplasm where in prevents accidental exo- and endogenous intoxication. Here we present the high-resolution X-ray crystal structure of the lytic enzyme Tse1 and describe the mechanism by which Tse1 cleaves the γ-D-glutamyl-l-meso-diaminopimelic acid amide bond of crosslinked PG. Tse1 belongs to the superfamily of N1pC/P60 peptidases but is unique among described members of this family of which the structure was described, since it is a single domain protein without any putative localization domain. Most importantly, we present the crystal structure of Tse1 bound to its immunity-protein Tsi1 as well and describe the mechanism of enzyme inhibition. Tsi1 occludes the active site of Tse1 and abolishes its enzyme activity by forming a hydrogen bond to a catalytically important histidine residue in Tse1. Based on our structural findings in combination with a bioinfomatic approach we also identified a related system in Burkholderia phytofirmans. Not only do our findings point to a common catalytic mechanism of the Tse1 PG-hydrolases, but we can also show that it is distinct from other members of this superfamily. Furthermore, we provide strong evidence that the mechanism of enzyme inhibition between Tsi1 orthologues is conserved. This work is the first structural description of an entire effector/immunity pair injected by the T6SS system. Moreover, it is also the first example of a member of the N1pC/P60 superfamily which becomes inhibited upon binding to its cognate immunity protein.

Regulatory RNAs in Haloferax volcanii.

In organisms of all three domains of life, a plethora of sRNAs (small regulatory RNAs) exists in addition to the well-known RNAs such as rRNAs, tRNAs and mRNAs. Although sRNAs have been well studied in eukaryotes and in bacteria, the sRNA population in archaea has just recently been identified and only in a few archaeal species. In the present paper, we summarize our current knowledge about sRNAs and their function in the halophilic archaeon Haloferax volcanii. Using two different experimental approaches, 111 intergenic and 38 antisense sRNAs were identified, as well as 42 tRFs (tRNA-derived fragments). Observation of differential expression under various conditions suggests that these sRNAs might be active as regulators in gene expression like their bacterial and eukaryotic counterparts. The severe phenotypes observed upon deletion and overexpression of sRNA genes revealed that sRNAs are involved in, and important for, a variety of biological functions in H. volcanii and possibly other archaea. Investigation of the Haloferax Lsm protein suggests that this protein is involved in the archaeal sRNA pathway.

The archaeal Lsm protein binds to small RNAs.

Proteins of the Lsm family, including eukaryotic Sm proteins and bacterial Hfq, are key players in RNA metabolism. Little is known about the archaeal homologues of these proteins. Therefore, we characterized the Lsm protein from the haloarchaeon Haloferax volcanii using in vitro and in vivo approaches. H. volcanii encodes a single Lsm protein, which belongs to the Lsm1 subfamily. The lsm gene is co-transcribed and overlaps with the gene for the ribosomal protein L37e. Northern blot analysis shows that the lsm gene is differentially transcribed. The Lsm protein forms homoheptameric complexes and has a copy number of 4000 molecules/cell. In vitro analyses using electrophoretic mobility shift assays and ultrasoft mass spectrometry (laser-induced liquid bead ion desorption) showed a complex formation of the recombinant Lsm protein with oligo(U)-RNA, tRNAs, and an small RNA. Co-immunoprecipitation with a FLAG-tagged Lsm protein produced in vivo confirmed that the protein binds to small RNAs. Furthermore, the co-immunoprecipitation revealed several protein interaction partners, suggesting its involvement in different cellular pathways. The deletion of the lsm gene is viable, resulting in a pleiotropic phenotype, indicating that the haloarchaeal Lsm is involved in many cellular processes, which is in congruence with the number of protein interaction partners.