Consumidores dentro del mundo narco

A literature search was performed to find RNases and inhibitors that interact with barstar and barnase. The presence of the genes on plasmids and viruses was de- termined using blastp on the NCBI genomic database of all bacterial sequences. Searches were performed using the B. amyloliquefaciens barnase and barstar amino acid sequences as the comparators in their active form (omitting the export signals on native barnase), and repeated with amino acid sequences from the interacting RNAses and inhibtors (Table B.1). The resulting matches were compiled using the program Geneious, which filtered the entries by the terms ‘virus’ and ‘plasmid’. Re- sults were validated as occurring on viruses or plasmids manually. Blastn was used to find matches to loci of linked barnase and barstar.

2.7.4 Programs and statistics

This thesis was typeset in LaTex. Figures and graphs were generated in Excel and LaTex, with variation measured as Standard Error (SE). P-values were generated using two-tailed t-tests in Excel. Though most conditions had equal variance between them (as established with an F-test, also done in Excel), all t-tests were performed assuming unequal variance to account for those exceptions and to keep consistency across tests. Unless otherwise stated, t-tests were performed on data from the final time point of an assay, comparing each condition back to the control.

Chapter 3

Distribution of type I TA systems

across replicons, comparing between

families and other TA types

Bioinformatic techniques have been instrumental in expanding the number of known families and putative homologues of TA systems. TAs have been found on a multitude of MGEs, and are abundant on microbial chromosomes, with some species of bacteria containing more than 90 copies of type II systems (Pandey and Gerdes 2005; Ramage

et al. 2009; Leplae et al. 2011). Interesting patterns in distribution have emerged

between types (defined by antitoxin) and between families within types (categorized by relatedness of the toxin). Thus far, type II systems appear to be more broadly dispersed than type I systems, occuring across many phyla of bacteria as well as in archae (Leplae et al. 2011; Makarova et al. 2009). In addition, type II families are more commonly localized on mobile elements and more lineage independent: that is, a given family of type II TAs appears across unrelated species of bacteria (Fozo

et al. 2010; Leplae et al. 2011; Makarova et al. 2009; Weaver et al. 2009).

This differential distribution has caused some to suggests that type I systems are less horizontally mobile than type II systems (Fozo et al. 2010; Mruk and Kobayashi 2014). This hypothesis is investigated here. Historically, type I systems have been more difficult to detect in silica than type II systems, though recent computational analyses have begun to change this (Fozo et al. 2010; Findeiß et al. 2010; Sayed

et al. 2012; Kawano 2012; Fozo et al. 2008). And as our ability to detect type I TAs

advances, differences in distribution in comparison to type II systems may become less apparent.

Computational methods are used to explore the phylogenetic range of known families of type I TA systems and find new families, which may display different distributions than known families. These results are compared to findings in the existing literature on TA families. Aspects of TA system biology, and limitations of current methods are discussed which may account for why type I systems consistently differ in their phylogenetic range from other known TA systems. Most of the raw data in this chapter was generated through close collaboration with Dr. Paul Gardner.

3.1 Results and Discussion

Most type I toxins are small and membrane associated, (Kawano 2012; Fozo et al. 2008; Wang et al. 2012; Unoson and Wagner 2008; Mok et al. 2010). SymE, a nuclease that targets mRNA, is an exception (Kawano et al. 2007). Nine families of type I TA systems where chosen for analysis, all with known or predicted membrane-associated toxins: Hok, FlmA, LdrD, TxpA, Ibs, TisB, ShoB, Fst, and plasmid_Toxin (PT). TisB, Ibs, and Hok are believed to insert into the inner membrane, causing loss of membrane potential and the ghost cell phenotype (Unoson and Wagner 2008; Gerdes

et al.1986; Gerdes et al. 1986; Fozo et al. 2008; Mok et al. 2010). The Ldr toxin

causes nucleoid condensation upon overexpression (Kawano et al. 2002) and the Fst toxin causes chromosomal mis-segregation and interferes with cell division at low levels as well as disrupting cellular membranes at higher concentrations (Patel and Weaver 2006). These families were to analyzed to identify patterns between families that would facilitate finding new families of type I TAs, and their distributions were used as a source of comparison to other TA types.

A database derived from six frame translations of bacterial chromosomes and plasmids was created. The database was scanned with hidden Markov models (HMMs) for each toxin family based on amino acid sequence (Figures C.1 to C.9). All regions that were considered ’matches’ in the database (loci) were assembled into a baseline compilation of putative homologues from known families. We then attempted to group families by different methods to generate new HMMs, aggregated from the sequences of multiple type I families. This was intended to broaden the signal, decreasing the specificity of the HMM and increasing the ability to detect previously unknown families. Loci found with the aggregated HMMs were compared to the compilation of loci from known families.

3.1 Results and Discussion 55 Table 3.1: Scoring type I TA system toxins by membrane topology using TMHMM

Total1 _{Predicted transmembrane}2 _In-Out3 _In-Out4

Hok 44 39 22 17 FlmA 35 33 19 14 TxpA 1 1 0 1 ShoB 1 1 1 0 Ibs 20 1 0 1 Plasmid_toxin 11 0 0 0 Ldr 9 9 1 8 Fst 15 3 3 0 TisB 8 4 4 0

1 Number of non-redundant sequences from each family analyzed with TMHMM 2 Number of sequences that scored as transmembrane proteins

3 N terminus is inside the membrane, C terminus is out 4 N terminus is outside the membrane, C terminus is in