Saccharomyces pastorianus, a natural lager-brewing yeast hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus, using whole genome shotgun sequencing. Each strain of S. pastorianus has lost species-specific portions of its genome and has undergone extensive recombination, producing chimeric chromosomes. We predicted 30 breakpoints that we confirmed at the single nucleotide level by designing species-specific primers that flank each breakpoint, and then sequencing the PCR product. These rearrangements are the result of recombination between areas of homology between the two subgenomes, rather than repetitive elements such as transposons or tRNAs. Interestingly, 28/30 S. cerevisiae- S. eubayanus recombination breakpoints are located within genic regions, generating chimeric genes. Furthermore we show evidence for the reuse of two breakpoints, located in HSP82 and KEM1, in strains of proposed independent origin.Gross chromosomal rearrangements have the potential to be evolutionarily advantageous to an adapting organism. The generation of a hybrid species increases opportunity for recombination by bringing together two homologous genomes. We sought to define the location of genomic rearrangements in three strains of  Saccharomycetous yeast occurs readily in natural and industrial environments Saccharomyces pastorianus, previously classified as Saccharomyces carlsbergensis, is a natural hybrid between Saccharomyces cerevisiae and a Saccharomyces uvarum-like species S. uvarum-like species has most recently been identified as the Argentinean-isolate Saccharomyces eubayanus, which shows 99.5% identity to the non-S. cerevisiae portion of S. pastorianusS. pastorianus is thought to have arisen by spontaneous hybridisation in brewery conditions, maintained by human selection for colder brewing temperatures, a preference that is conferred by its S. uvarum-like parent Hybridisation in S. pastorianusS. cerevisiae and one haploid S. eubayanus genome with significant loss of S. cerevisiae genes, whereas Group 2 strains contain one haploid S. eubayanus genome and a diploid S. cerevisiae genome. The differences between these two groups suggest that they may have had independent evolutionary origins, a theory given weight by both the aforementioned array-CGH analysis So far, there are only two whole genome lager yeast sequences available, Weihenstephan 34/70 S. pastorianus is thought to be either Ty-mediated flor wine yeast Lager yeast chromosomes have been shown to have undergone recombination, generating chimeric chromosomes composed of genetic material from both parental species S. pastorianus strains to both characterise genomic breakpoints and shed further light on their formation and retention. We chose strains that have been used in a previous microarray study to provide a source of validation for our sequencing S. pastorianus: two of the chosen strains of S. pastorianus are of Group 1 (DBVPG 6033 and DBVPG 6261) and one is of Group 2 (DBVPG 6257) We sequenced three S. cerevisiae- S. eubayanus breakpoints at the single nucleotide level and identified both nearby repetitive elements and regions of homology. Significantly, we found that the majority of genomic breakpoints occurred within protein coding regions, generating chimeric genes. Furthermore, the presence of identical breakpoints in KEM1 and HSP82 is evidence of breakpoint reuse between strains of proposed independent origin.We have confirmed the location of many S. pastorianus, DBVPG 6033 (Saccharomyces carlsbergensis type strain), DBVPG 6261 (Saccharomyces monacensis type strain) and DBVPG 6257 were sequenced using the SOLiD 4 Next Generation Sequencing platform and mapped to S. cerevisiae (sacCer2) and S. uvarum (sacBay MIT), which are representative of the S. pastorianus subgenomes. We used sacBay MIT as the reference genome for S. eubayanus due to its fully available sequence, which is purportedly 7% diverged from S. eubayanusS. cerevisiae chromosomes in the UCSC Genome Browser (http://genome.ucsc.edu/) is reported in S. eubayanus reads mapped to contigs were viewed in the Integrative Genomics Viewer (http://www.broadinstitute.org/igv). SOLiD sequencing allowed us to ascertain the approximate chromosomal copy number in each strain using a hierarchical cluster analysis of relative median read depth across multiple regions .The genomic DNA of three strains of  regions . In tota regions . These cS. pastorianus shows a high degree of aneuploidy and the chromosomal composition between strains is highly variable. Strains DBVPG 6033, 6261 and 6257 have eight, seven and sixteen complete S. cerevisiae chromosomes, respectively , supporting the widely held notion that lager yeasts tend to inherit and/or retain only their S. eubayanus mitochondria S. cerevisiae sequence in strains DBVPG 6033 and DBVPG 6257, but not DBVPG 6261  to identify candidate breakpoints based on variations in S. cerevisiae read copy number across each chromosome  or the S. uvarum reference genome . These may represent gene conversion from a small tract in the S. eubayanus to the S. cerevisiae subgenome rather than deletion, since each breakpoint generated a chimeric gene  and QCR2)  . InteresALD2 is involved in the oxidation of ethanol and TDH2 is a component of the tetramer glyceraldehyde-3-phosphate dehydrogenase, which is required for gluconeogenesis. Two further chimeric genes also play a role in energy metabolism. GPH1 is involved in glycogen mobilisation and GAL80 is a repressor of GAL genes in the absence of galactose. With the large genetic redundancy in all three strains of S. pastorianus, especially DBVPG 6257, which contains approximately two S. cerevisiae sets of chromosomes, chimeric gene copies may or may not significantly affect the hybrid organism.Interestingly, two genes found in the chimeric form are involved in ethanol metabolism, a key biochemical pathway in lager fermentation. GPH1 in the lager strain CMBS-33 contains a disruptive base insertion within its initial S. eubayanus sequence, and the resultant gene is not expressed MEP2, an ammonium permease, in clones of lab-created hybrids of S. cerevisiae and S. uvarum that were evolved under nitrogen-limiting conditions LG-FLO1 gene, involved in flocculation, appears to have been inactivated in non-flocculent lager yeast by a non-reciprocal translocation of S. cerevisiae YIL169c into its C-terminal region in various non-flocculent strains SSU1-R was generated by recombination between the promoter regions of SSU1 and ECM34 genes and has been found to grant increased sulphite resistance compared to the wild type allele Previous studies on the functionality and fitness of chimeric genes show mixed results. The chimeric gene S. pastorianus strains KEM1) and XVI (HSP82) were common to all the S. pastorianus strains analysed in our study , we manually recorded the nearest repetitive genomic feature to each breakpoint  transposons, since this portion of the data is mapped only to contigs. Despite the lack of proximity of repetitive elements to breakpoints as a trend, one breakpoint gene, TDH2 on chromosome XII in DBVPG 6257 is situated adjacent to an ARS, a feature known to promote chromosomal translocation S. uvarum transposon present on a contig at the site of one breakpoint on chromosome XIII , DBVPG 6261 (GSY134) and DBVPG 6257 (GSY132) were obtained from DBVPG Industrial Yeasts Collection, University of Perugia, Italy. Yeast was grown at 25°C, 200 rpm for 20 hours in YPD  and genomic DNA extracted using Wizard Genomic DNA Purification Kit (Promega).Saccharomyces cerevisiae, sacCer2 was obtained via the UCSC Genome Browser (http://genome.ucsc.edu/). Genome sequencing reference strain for Saccharomyces uvarum, sacBay MIT, was obtained from the Saccharomyces Genome Database .Genome sequencing reference strain for S. pastorianus was sequenced using Next Generation Sequencing Applied Biosystems SOLiD 4 platform to generate 50 bp single-end reads. Using BFAST (http://sourceforge.net/projects/bfast/files/), the reads were mapped to the S. cerevisiae reference genome “sacCer2” obtained from UCSC (http://genome.ucsc.edu/), which includes 16 chromosomes, the mitochondrial genome and the 2 micron plasmid. The ‘-a 3’ flag of the post-process step was used to obtain unique best scoring alignments. The S. cerevisiae ORFs were used to find S. eubayanus consensus ORFs in the S. eubayanus reference strain “sacBay MIT” obtained from SGD (http://www.yeastgenome.org/). BFAST files were filtered to retrieve sets of reads with 0, 0–1 or 0–5 mismatches to each reference genome. Generally, 0 mismatches was found to be the most suitable cut-off value for the S. cerevisiae data and 0–5 mismatches for the S. eubayanus data, having the best agreement to previous microarray data by Dunn and Sherlock (2008).The genomic DNA of three strains of S. pastorianus SOLiD sequence mapped to the pre-annotated S. cerevisiae genome sequence via the UCSC Genome Browser (http://genome.ucsc.edu/) to identify both S. cerevisiae chromosome copy number and potential chimeric chromosomes comprising both S. cerevisiae and S. eubayanus sequence. These candidate breakpoint regions were identified visually by their abrupt and sustained reduction in S. cerevisiae reads along a chromosome. Due to difficulties in mapping and analysing repetitive regions, telomeres were excluded from the analysis. Similarly, changes in read number due to the presence of a yeast transposon (Ty) or other repetitive element were excluded.We used the S. cerevisiae genome was broken down into 26 regions  S. eubayanus chromosomes. We have assumed the S. eubayanus genome to be collinear with the highly related S. uvarum genome. The ‘coverage’ tool from the Bedtools suite of programs (http://bedtools.readthedocs.org/en/latest/) was used to obtain the read depth for every position in each of the regions under consideration. The analysis used the mapped reads at a cutoff of 0 mismatches for the S. cerevisiae data and 0–5 mismatches for the S. eubayanus data as previously described. For each region the median depth of coverage values were calculated using a custom Peal script excluding 0 coverage values.To estimate chromosomal copy number we first broke down each genome into regions. The iae data . Pre-exiS. cerevisiae and S. eubayanus regions the read depth data of each region were grouped into clusters using hierarchical cluster analysis software S. pastorianus using Ward’s method to generate read depth clusters which are displayed as dendrograms  to identify potential chimeric chromosomes comprising both S. cerevisiae and S. eubayanus sequence. These candidate breakpoint regions were identified by their abrupt and sustained reduction in S. cerevisiae read depth along a chromosome. Species-specific primers were designed to flank each predicted breakpoint area. The S. cerevisiae primers were designed using S. cerevisiae sequence obtained directly from the UCSC Genome Browser (http://genome.ucsc.edu/). The S. eubayanus primers were designed by finding the S. uvarum orthologue of the nearest S. cerevisiae gene using the SGD Synteny Viewer (http://www.yeastgenome.org). This orthologue was then mapped to S. pastorianus to find consensus sequences for the S. eubayanus portion of the S. pastorianus genome. The S. eubayanus-specific primer was then designed within this consensus sequence. Candidate primers were generated for both S. cerevisiae and S. eubayanus sequences using Primer 3 (http://frodo.wi.mit.edu). To circumvent the potential for non-specific binding between the two closely related subgenomes, these primers were then carefully selected for species-specificity using the Fungal BLAST tool in SGD (http://www.yeastgenome.org). For ease of amplification, primers were designed to anneal no more than a few thousand base pairs apart but with sufficient sequence either side of the breakpoint for clear identification of each subgenome. Primer sequences are available in We used the PCR conditions were optimised for each breakpoint to obtain pure homogeneous chimeric sequence. The PCR product was separated by electrophoresis on 1% (w/v) agarose gel. PCR products were purified prior to sequencing using QIAquick PCR Purification Kit . The purified PCR products were Sanger sequenced at GATC Biotech (Germany).S. pastorianus strain was aligned to S. cerevisiae  and S. eubayanus , where available, or S. uvarum  using Clustal Omega . Low quality ends of breakpoint sequences were trimmed before alignment. The breakpoint region in each sequence was determined as the area of identical nucleotides between the parental species flanked by S. cerevisiae-like sequence on one side and S. eubayanus-like sequence on the other side of the region. The start of S. cerevisiae-like sequence was determined by nucleotide match to S. cerevisiae but mismatch to either S. eubayanus or S. uvarum. The start of S. eubayanus-like sequence was determined by nucleotide match to S. eubayanus or S. uvarum but mismatch to S. cerevisiae.Each sequence covering each breakpoint in each S. cerevisiae sequence and S. eubayanus or S. uvarum sequence at the junction of the pre-determined breakpoint region. Reading frame was checked using Expasy Translate (http://web.expasy.org/translate/).The proposed length of each chimeric gene was determined by merging S. pastorianus was calculated using S. cerevisiae and either S. eubayanus  or S. uvarum sequences obtained from SGD (http://www.yeastgenome.org/) and Clustal Omega  Amino acid identity was calculated similarly. Chimeric nucleotide sequences were first converted to protein sequences using Expasy Translate Tool (http://web.expasy.org/translate/).Percentage nucleotide identity between each subgenome and http://www.ebi.ac.uk/ena/data/view/PRJEB4654; Sanger sequenced reads covering the breakpoint regions have been submitted to the European Nucleotide Archive under the accession numbers HG803141–HG803169 at http://www.ebi.ac.uk/ena/data/view/HG803141-HG803169.Raw reads from this study have been deposited at the European Nucleotide Archive under the accession number PRJEB4654 at Figure S1Mapping of the 2-micron plasmid to S. cerevisiae sequence. The 2-micron plasmid DNA from each strain of S. pastorianus is mapped to S. cerevisiae sequence using the UCSC Genome Browser. The scale on the Y axis is capped at a read depth of 4500.(TIF)Click here for additional data file.Figure S2Multiple alignment of each S. pastorianus breakpoint sequence to the parental species. The region sequenced over each breakpoint in each S. pastorianus strain was aligned with S. cerevisiae  and either S. eubayanus  or S. uvarum  ORF sequences, using Clustal Omega. The two intergenic breakpoints were aligned using nucleotide sequence upstream or downstream from the nearest ORF. The ORF sequences obtained from the Saccharomyces Genome Database are taken from Cliften et al.et al. S. cerevisiae nucleotide (shown in blue) and the first unique S. eubayanus or S. uvarum nucleotide (shown in red). All gene sequences are 5′-3′.(PDF)Click here for additional data file.Figure S3Hierarchical cluster analysis of read depth of S. pastorianus chromosomes. The median read depth for 26 regions covering the S. cerevisiae-like chromosomes (shown in S. eubayanus-like chromosomes (sample contigs across each chromosome) were clustered independently and for each strain of S. pastorianus. Results from each hierarchical cluster analysis using Ward’s method are shown as a dendrogram. The blue boxes indicate the copy number assigned to that cluster.shown in  and 47 r(PDF)Click here for additional data file.Table S1Breakpoints which were not successfully amplified by PCR.(DOC)Click here for additional data file.Table S2Analysis of the breakpoint region in each strain of S. pastorianus.(DOC)Click here for additional data file.Table S3Analysis of the copy number of S. cerevisiae-derived chromosomes in each strain of S. pastorianus using median read depth across multiple regions.(XLSX)Click here for additional data file.Table S4Analysis of the copy number of S. eubayanus-derived chromosomes in each strain of S. pastorianus using median read depth across multiple regions.(XLSX)Click here for additional data file.Table S5Primer sequences for amplification of breakpoint regions.(DOC)Click here for additional data file.


Mycobacterium tuberculosis. Considering that the mycobacterial cell wall constitutes a significant barrier to drug penetration, the aim of this study was to conduct a comparative genomics analysis of the repertoire of enzymes involved in peptidoglycan (PG) remodelling to determine the potential of exploiting this area of bacterial metabolism for the discovery of new drug targets.Mycobacteria comprise diverse species including non-pathogenic, environmental organisms, animal disease agents and human pathogens, notably in silico analysis of 19 mycobacterial species/clinical strains for the presence of genes encoding resuscitation promoting factors (Rpfs), penicillin binding proteins, endopeptidases, L,D-transpeptidases and N-acetylmuramoyl-L-alanine amidases. Our analysis reveals extensive genetic multiplicity, allowing for classification of mycobacterial species into three main categories, primarily based on their rpf gene complement. These include the M. tuberculosis Complex (MTBC), other pathogenic mycobacteria and environmental species. The complement of these genes within the MTBC and other mycobacterial pathogens is highly conserved. In contrast, environmental strains display significant genetic expansion in most of these gene families. Mycobacterium leprae retains more than one functional gene from each enzyme family, underscoring the importance of genetic multiplicity for PG remodelling. Notably, the highest degree of conservation is observed for N-acetylmuramoyl-L-alanine amidases suggesting that these enzymes are essential for growth and survival.We conducted an PG remodelling enzymes in a range of mycobacterial species are associated with extensive genetic multiplicity, suggesting functional diversification within these families of enzymes to allow organisms to adapt. N-acetyl muramic acid (NAM) moiety of the PG,11,12. Pde chain-9,17, FiMycobacterium tuberculosis, the causative agent of tuberculosis, there is a dire need for new drugs with novel modes of action. The increased prevalence of drug resistant strains has raised concerns regarding the sustainability of the current treatment regimen. To address this, several aspects of mycobacterial metabolism are being assessed for potential new drug targets and six environmental species including Mycobacterium smegmatis. Mycobacterium leprae is listed separately due to its substantially reduced genome which emerges as an outlier in the analysis.The comparative genomics analysis for PG remodelling enzymes in mycobacterial species obtained from this study is summarised in Table Micrococcus luteus, Table rpfC (Rv1884c in M. tuberculosis) homologues have been annotated as present in all mycobacteria[M. tuberculosis rpfC homologue is absent from environmental species. Artemis Comparison Tool (ACT) whole genome alignment reveals that the region encoding rpfC in M. tuberculosis is absent in M. smegmatis and all other environmental mycobacteria (data not shown). Thus, based on gene synteny, there is no direct rpfC homologue in these strains. However, there is a local duplication of rpfE in all the environmental strains (annotated as MSMEG_4643 in M. smegmatis), Table rpfE2, as a homologue of MSMEG_4643, rather than a homologue of Rv1884c. As RpfE interacts with the Rpf Interacting Protein A (RipA)[M. smegmatis and other environmental bacteria.The environmental species retain three bacteria, our anarpfC and rpfD homologues to pathogenic and MTBC strains, along with the duplication of rpfB in some environmental species, raises interesting questions regarding the nature of growth stimulation in these organisms. These differences suggest that the latter require fewer secreted Rpfs and are more reliant on the membrane bound RpfB homologue. This could be related to the fact that environmental organisms are required to grow in diverse niches of varying size and complexity making them more dependent on localised growth stimulatory activity through a membrane bound Rpf rather than paracrine signalling from diffusible Rpfs produced by neighbouring organisms. It is noteworthy that of all five homologues in M. tuberculosis, deletion of rpfB individually or in combination with rpfA results in colony forming defects and prolonged time to reactivation from chronic infection in mice[The restriction of  in mice,34,35.The role of Rpfs in TB disease in humans remains enigmatic. It has been demonstrated that sputum from patients with active TB disease, before the initiation of treatment, is characterised by a population of dormant bacteria that require Rpfs for growth. These dponA1  are a large family of evolutionarily related cell wall associated enzymes, that bind β-lactam antibiotics,49. PBPs, Rv3682), Figure as ponA3, Table pbpA; Rv0016c,[pbpB; Rv2163c,[M. smegmatis where gene deletion or depletion manifests in altered cell morphology and antibiotic resistance profiles[ponA2 - there is a distal duplication of PBP-lipo in the environmental strains, Table Class B PBP proteins PbpA Rv0016c,), PbpB Rv2163c,) and PBPRv2163c,) are pre163c, anprofiles. In this6c,, PbpdacB2-encoded DD-CPase is not affected by penicillin – though it does bind the antibiotic[M. tuberculosis[M. tuberculosis H37Rv encodes three distinct DD-CPase homologues: dacB1 (Rv3330), dacB2 (Rv2911) and Rv3627c, Table dacB2 (Rv2911) homologue, leading to consecutive numbering of the resulting duplicated genes for example, MSMEG_2432 and MSMEG_2433 in M. smegmatis. In addition, a distant DD-CPase homologue (annotated as MSMEG_1900 in M. smegmatis) was identified in the environmental strains, as well as in M. abscessus but not in the other pathogenic mycobacteria and MTBC, Table M. tuberculosis by in silico analysis through their predicted ß-lactamase domains and are grouped among Class C PBPs[D,D-carboxypeptidases (DD-CPases) are designated as Class C PBPs and are generally present in high abundance. DD-CPastibiotic. Inhibiterculosis. In thiserculosis. Our anas C PBPs. AnalysiM. tuberculosis that display strong homology to ripA, Table Mycobacterium abscessus and M. leprae, pathogenic mycobacteria retain all five of these homologues. Environmental strains display enhanced expansion of endopeptidases, with the exception of the ripD homologue (Rv1566c). The functional consequence of this remains unknown but it is noteworthy that these strains have also expanded their rpfE and rpfB gene repertoire, suggesting that the multiplicity in this case allows for a greater number of RipA-RpfB/E protein complexes, as well as for protein complexes with different subunit composition. Dysregulated expression of RipA leads to dramatic alterations in cellular morphology and growth[Endopeptidases are enzymes that cleave within the stem peptides in PG. In this study, we focus on the Nlp/P60 class of endopeptidases, which cleave within the stem peptides between positions 2 and 3 as exemplified by RipA, Figure d growth suggestiM. tuberculosis (100% amino acid identity over 58% coverage) and similar domain organization[M. leprae, Table Mycobacterium marinum homologues of Rv1477 and Rv1478, iipA and iipB (MMAR_2284 and MMAR_2285 respectively), Table RipB displays strong sequence homology RipA in nization, but lacnization. More renization. Both Rinization. This suStructural analysis of RipD reveals alterations in the catalytic domain, consistent with the inability of this protein to hydrolyse PG. NeverthM. tuberculosis results in altered colony morphology, attenuated growth in vitro, defective PDIM production and reduced colonisation of mouse lungs in the murine model of TB infection[Rv2190c encodes another NlpC/P60-type PG hydrolase in mycobacteria. Deletion of this gene in nfection. ConsistM. tuberculosis[M. abscessus[M. tuberculosis[Mt1 and LdtMt2 (Rv0116c and Rv2518c respectively) were experimentally shown to affect M. tuberculosis H37Rv morphology, growth characteristics and antibiotic susceptibility in vivo[Mt2 places the extramembrane domain 80–100 Å from the membrane surface and indicates that this enzyme is able to remodel PG within this spatial region of the PG sacculus[Mt1 and LdtMt2 in M. tuberculosis resulted in morphological defects and altered virulence in the murine model of TB infection[ldtMt3 (Rv1433), which is absent from the pathogen Mycobacterium ulcerans and from the environmental species Mycobacterium vanbaalenii, M sp. MCS and M. sp. KMS, yet its presence in M. leprae suggests functional importance. As with RipA, M. gilvum shows the greatest expansion of the ldt genes. Biochemical characterisation of all five M. tuberculosis H37Rv homologues, LdtMt1 - LdtMt5, confirms PG cross-linking and/or ß-lactam acylating enzyme activities in all of these enzymes[M. tuberculosis raises interesting questions with respect to the functional consequences of the expansion of this protein family in environmental strains, which may require greater flexibility in Ldt function.L,D-transpeptidases (Ldt) are a group of carbapenem sensitive enzymes in erculosis that conabscessus and M. terculosis exhibit y in vivo. The crysacculus. More renfection. A notab enzymes. This ac enzymes,71. MoreEscherichia coli and other organisms, and can be organised into 2 main families containing either an amidase_2 or amidase_3 – type domain[E. coli  have specific substrate requirements governed by the structural confirmation of the NAM carbohydrate moiety. Knockout of these amidases results in chaining phenotypes, abnormal cell morphologies and/or increased susceptibility to certain antibiotics[Bacillus subtilis[M. tuberculosis, two containing the amidase_2 domain  and two the amidase_3 domain , Table M. tuberculosis confirms that this enzyme is able to bind and cleave muramyl dipeptide[ami4) from non-MTBC pathogens and its presence in the MTBC and environmental strains. M. leprae retains only the ami1 and ami2 genes – both containing the amidase_3 domain. This suggests that amidase_2 domain amidase activity is dispensable specifically in this species, but required for peptidoglycan remodelling in the other pathogenic mycobacteria.While endopeptidases and transpeptidases are responsible for cleavage within or between peptide stems, amidases act to remove the entire peptide stem from the glycan strands, cleaving between the NA/GM moiety and the L-Ala in the first position of the stem peptide, Figure e domain,9,72. Thibiotics. Amidase subtilis,76. The ipeptide. The amiin vitro growth and division of M. leprae, as it can only be grown in animal models. From our analysis, it is apparent that M. leprae habours notable genetic redundancy for PG remodelling enzymes [http://tree.bio.ed.ac.uk/software/figtree). Functional annotation of each of the M. tuberculosis proteins was identified at InterScanPro[The 19 mycobacterial strain sequences used in this study were all complete and either published,78,81-90ercuList, GenoLisercuList or TBDBercuList. Where nercuList, utilisibrowser). Where ibrowser) was utilbrowser). Phylogebrowser) alignmenbrowser) analysisrScanPro, for PFArScanPro, signal rScanPro and membrScanPro.The authors declare that they have no competing interests.BDK conceived and designed the study. EEM conducted all the bioinformatics analyses and compiled the manuscript. SS and CE provided intellectual input on certain aspects of the study. All authors approve of the final content in the manuscript.Figure S2. Phylogenetic relationship between Class A penicillin binding proteins (PonA family) from various mycobacteria. Figure S3. Phylogenetic relationship between Class B penicillin binding proteins (Pbp family) from various mycobacteria. Figure S4. Phylogenetic relationship between Class C penicillin binding proteins (DD-carboxypeptidases) from various mycobacteria. Figure S5. Phylogenetic relationship between endopeptidases (Nlp/P60 – domain containing proteins) from various mycobacteria. Figure S6. Phylogenetic relationship between L,D-transpeptidases from various mycobacteria. Figure S7. Phylogenetic relationship between N-acetylmuramoyl-L-alanine from various mycobacteria.Phylogenetic relationship between Resuscitation Promoting Factors from various mycobacteria. Click here for fileTable S2. Bioinformatics sites used for analysis.Mycobacterial strains included in this study. Click here for file


Plasmodium infection.The effects of helminth co-infection on malaria in humans remain uncertain. This study aimed to evaluate the nature of association of intestinal helminths with prevalence and clinical outcomes of Plasmodium and intestinal helminth infections were diagnosed using Giemsa-stained blood films and Kato-Katz technique, respectively. Haemoglobin level was determined using a haemocue machine.A cross-sectional study involving 1,065 malaria suspected febrile patients was conducted at Dore Bafeno Health Center, Southern Ethiopia, from December 2010 to February 2011. Plasmodium parasites . Among 702 patients who provided stool samples, 53.8%, 31.6% and 19.4% were infected with intestinal helminths, Plasmodium alone and with both Plasmodium and intestinal helminths, respectively. The prevalence of infections with Ascaris lumbricoides (A. lumbricoides), Trichuris trichiura (T. trichiura), Schistosoma mansoni (S. mansoni) and hookworm (9.8%) were 35.9%, 15.8%, 11.7% and 9.8%, respectively. Out of the 222 (31.6%) Plasmodium infected cases, 9 (4.1%) had severe malaria. P. falciparum infection was more common in febrile patients infected with A. lumbricoides alone (21.3%), T. trichiura alone (23.1%) and S. mansoni alone (23.1%) compared to those without intestinal helminth infections (9.3%) . Prevalence of non-severe malaria was significantly higher in individuals infected with intestinal helminths than in those who were not infected with intestinal helminths . The chance of developing non-severe P. falciparum malaria were 2.6, 2.8 and 3.3 times higher in individuals infected with A. lumbricoides alone, T. trichiura alone and S. mansoni alone, respectively, compared to intestinal helminth-free individuals . The odds ratio for being infected with non-severe P. falciparum increased with the number of intestinal helminth species (p<0.001). Mean Plasmodium density among intestinal helminth infected individuals was significantly increased with the number of intestinal helminths species (p=0.027). Individuals who were co-infected with different species of intestinal helminths and Plasmodium showed lower mean haemoglobin concentration than individuals who were infected only with Plasmodium.Among 1,065 malaria suspected febrile patients, 28.8% were positive for A. lumbricoides, T. trichiura and S. mansoni were positively associated with P. falciparum infection. However, further studies are required to investigate how these helminths could contribute to increased prevalence of P. falciparum infection.Infections with  Plasmodium and helminth co-infections are prevalent in tropical countries, the effect of their interactions remains unclear [Plasmodium infection [P. falciparum malaria and intestinal helminths infection [Plasmodium density and intensity of hookworm infection [Although  unclear . Some stnfection -4, increnfection , decreasnfection  and incrnfection -9. Crossnfection ,11. Posinfection .On the other hand, evidence from other studies showed that helminth infections may protect from malaria or related clinical outcomes by suppressing acute clinical manifestations ,14, paraPlasmodia stages and species or altered immune response because of helminth co-infections [Plasmodium, epidemiological setting of the study area and other confounding factors could contribute for the contradictions.Still other studies reported the absence of significant association between helminth infection and malaria ,20. Thesfections . In addiP. falciparum was less common (only 4.0%), and infection with S. mansoni was absent [Plasmodium infected cases, and factors such as age, sex, nutrition and socioeconomic status were not considered in the analysis. Thus, the current study was conducted to assess the nature of association of intestinal helminths with prevalence and clinical outcomes of Plasmodium infection among malaria suspected febrile individuals attending Dore Bafeno Health Center, Southern Ethiopia.In Ethiopia, previous similar study was conducted in an area where infection with s absent . In addiA cross-sectional study involving 1,065 febrile patients (age range= 1 to 82 years) was conducted at Dore Bafeno Health Center, Sidama Zone, southern Ethiopia, December 2010 to February 2011. The health center is located in Dore Bafeno District, about 283 km south of Addis Ababa, at about 1,708 m above sea level and 7° 5' N and 38° 29 E. It has a mean annual rainfall of 80.4 mm (it varies from 15 mm to 130 mm) and mean annual temperature of 23.2°C (it varies from 21.5°C to 25°C). The study participants were malaria suspected febrile patients who did not take anti-malarial drug within the last two weeks. Almost all the study participants belonged to the Sidama ethnic group, residing in rural area with similar life style and earning their living as mixed-farming. Malaria is unstable in the area and occurs mainly from October to December following the heavy rainy season and from April to May following the light rain season .Clinical data including auxiliary body temperature, blood pressure, coma status, breathing status and prostration were collected by physicians. Clinical data together with laboratory results were used to group malaria cases as severe and non-severe . Socio-dApproximately, 20μl finger prick blood was collected from each febrile patient using a plastic capillary tube and 10 μl of the blood was used to prepare thick and thin blood films on a single slide for the diagnoses and count of malaria parasitaemia . The sliT. trichiura, S. mansoni and A. lumbricoides infections were quantified at the Aklilu Lemma Institute of Pathobiology (ALIPB), Addis Ababa University. Average egg counts of the two slides was multiplied by 24 to obtain egg counts per gram of stool for each of the study participants and classes of intensity was determined for different helminth species as per the WHO recommendation [Fresh stool specimens were collected from each study participant who also gave blood sample for malaria diagnosis. The stool specimens were prepared for microscopic examination using double Kato slides . Quantitendation .P. falciparum and P. vivax infection were treated with coarteum and chloroquine, respectively. Individuals who were found positive for the soil-transmitted helminths (STHs) were treated with 400mg of albendazole while praziquantel was used to treat those who were positive for S. mansoni, Taenia saginata (T. saginata), and Hymenolepis nana (H. nana). The study participants were treated free of charge. Since the study participants were selected from patients coming to the health center seeking medical treatment for malaria, they were not compensated for extra incentive to replace their transport cost or time lost from work was not given.This study was conducted after obtaining ethical clearance from the Institutional Review Board (IRB) of Aklilu Lemma Institute of Pathobiology, Addis Ababa University. As the majority of the population in the study area was illiterate the IRB endorsed oral consenting of the study participants. Permission to conduct the study was also obtained from Awassa Zuria District Health Office and Dore Bafeno Health Center. Detailed explanations about the aims, procedures, potential risks and benefit of the study were given to physicians of the health center. Individual oral consent was obtained in local language by the physicians of the health center during clinical examination and only voluntarily consented individuals were included in the study. For children younger than 18 years, assent was obtained from parents or guardians. Treatment was made based on the national protocol for treating malaria and helminth infections. Individuals who were found positive for 2-test and one-way ANOVA were used to compare malaria prevalence and mean parasitaemia or egg intensity values, respectively across categories of categorical variables. Univariable and multivariable logistic regression analyses were used to test for an association between intestinal helminths infection and prevalence of non-severe malaria or malaria related anaemia. Multiple linear regression analyses was used to evaluate differences in the means of Plasmodium density and haemoglobin level between Plasmodium-helminth co infected and those who were infected with Plasmodium alone. The analysis was adjusted for age, sex and nutritional status [Plasmodium infections, or related outcomes was evaluated. Since the study population was homogenous in their socio-economic status differences in terms of socioeconomic status were not considered during the analysis. The distribution of parasite density was skewed and square root transformation was used to make it symmetrical before using linear regression to evaluate the effect of pre-specified risk factors on this outcome. Results were considered significant whenever p-value was less than 5%.Data was computerized using Epi-data version 3.1 software  and analyzed using STATA version 11 . Pearson χl status ,30, whilPlasmodium parasites . P. falciparum infection alone or mixed infection with P. vivax was also higher in the 5–15 years old children compared to in individuals older than 15 years (p<0.01). Infection with P. vivax alone was higher (p<0.001) in the under five children and in the 5–15 years age group compared to those individuals older than 15 years. There was no significant difference in the percentage of P. falciparum and/or P. vivax infection between males and females.Among 1,065 malaria suspected febrile patients (Mean age=18.6 years), 306 (28.8%) were positive for A. lumbricoides (35.9%), T. trichiura (15.8%), S. mansoni (11.7%), hookworm (9.8%), T. saginata (3.9%), H. nana (1.4%) and Enterobius vermicularis (E. vermicularis) (0.9%). Of the 378 intestinal helminth infected individuals, 35.8%, 13.3%, 4.3% and 0.6% had single, double, triple and quadruple infections, respectively. Intestinal helminth infection was most prevalent among individuals of age 5–15 years (63.2%). A. lumbricoides and T. trichiura infections were higher among individuals in 5–15 years age group than among individuals of age >15 years.Among 1,065 febrile patients examined for malaria, 702 were volunteered to provide stool samples for the diagnosis of infection with intestinal helminths. There was no significant difference in baseline demographics between those who provided stool sample and those who did not (data not shown). Out of 702 patients examined, 53.8% were positive for at least one intestinal helminth species  for S. mansoni, 107 epg  for hookworm and 101 epg  for T. trichiura. Intensity of A. lumbricoides infection showed age related pattern with the highest  mean intensity being observed among individuals of age group 5–15 years (mean epg=3113.7) followed by individuals of age group <5 years (mean epg=2410.6). Mean intensity of hookworm infection was higher  among individuals of age <5 years (mean epg=648) followed by those individuals whose age ranges from 5–15 years (mean epg=69.6).Mean intensity of infections were 2345 egg per gram {epg}  for Plasmodium alone, and 136 (19.4%) with both Plasmodium and intestinal helminths  . Prevalence of P. falciparum infection was significantly higher in individuals infected with any intestinal helminth (s), A. lumbricoides alone, T. trichiura alone, S. mansoni alone compared to those who were not infected with intestinal helminths (9.3%)   were infected with intestinal helminths alone, 86(12.3%) with 4.5% werePlasmodium infected patients, 14 showed one or more sign and symptoms of severe malaria . Nine severe malaria cases were volunteered to provide stool samples for the diagnosis of helminth infection and 7 of them were co-infected with intestinal helminths like A. lumbricoides, T. trichiura and hook worm.Out of 306 P. falciparum mono-infection was higher among individuals infected with intestinal helminth than non-infected with intestinal helminth . The ORs changed in parallel with the number of intestinal helminth species infection (p<0.001). Individuals infected with A. lumbricoides alone (AOR=2.6), T. trichiura alone (AOR=2.8), S. mansoni alone (AOR=3.3), any intestinal helminth (AOR=2.9), T. trichiura and A. lumbricoides (AOR=3.1), and T. trichiura, A. lumbricoides, S. mansoni and hookworm (AOR=4.0) had increased odds of non-severe P. falciparum mono-infection compared to those who were not infected with any intestinal helminths  . Similarly, infection with hookworm alone was not significantly associated with any of non-severe P. falciparum and/or P. vivax infection.Of the 292 non-severe malaria cases, 213 patients were examined for intestinal helminth infection and 61.0% were positive. The odds of non-severe malaria infection was higher in individuals infected with intestinal helminth compared to intestinal helminth-free individuals {adjusted Odds Ratio (AOR) =1.57, 95% CI= 1.12, 2.21}. Similarly, the odds of non-severe Plasmodium density was significantly higher in individuals co-infected with four different intestinal helminth species  compared to those who were free from intestinal helminth infection . Similarly, there was an increasing trend in mean Plasmodium parasitaemia among individuals who were infected with T. trichiura alone, S. mansoni alone, A. lumbricoides alone and any two or three of these species compared to individuals who were not infected with any intestinal helminth species. The mean Plasmodium density among intestinal helminth infected individuals increased significantly as the number of intestinal helminth species increased . However, patients infected with hookworm alone showed slightly lower mean Plasmodium parasitaemia compared to individuals who had no intestinal helminth infection.Data regarding the effect of intestinal helminths co infection on malaria-related outcomes are summarized in Table A. lumbricoides, hookworm, S. mansoni, T. trichiura alone or in combination and Plasmodium showed lower mean haemoglobin levels compared to malaria cases without intestinal helminths infections. However, the mean differences were not significant in the multivariable model. Prevalence of anaemia was higher among individuals co-infected with both intestinal helminths and Plasmodium compared to those with Plasmodium infection alone. Individuals co-infected with Plasmodium and four different intestinal helminths were almost 9 times more likely to be anaemic compared to those with Plasmodium infection alone .Individuals co-infected with Plasmodium density and intensity of intestinal helminth infections . In S. mansoni and T. trichiura infected patients, the mean parasitaemia increased as the class of intensity of the helminths increased; nonetheless the regression coefficients remain non-significant after adjusting for the effects of age, sex, nutrition, and concurrent infection by other species of helminth. Conversely, the mean parasitaemia of individuals with moderate intensity of A. lumbricoides infection was relatively low compared to individuals co infected with light intensity of A. lumbricoides and Plasmodium. For hookworm infection, all the infected cases were of light intensity class (data not shown).There was not significant correlation between the mean Plasmodium was negatively correlated with intensity of intestinal helminths. However, the coefficients for the association were low. Similarly, although the mean differences were not significant, haemoglobin levels decreased more in Plasmodium infected individuals co-infected with heavy intense S. mansoni or moderate intense T. trichiura infections compared to individuals with light intensity of infections with the respective intestinal helminths (data not shown).The reduction in mean haemoglobin level among individuals co-infected with intestinal helminths and P. falciparum malaria was significantly higher in individuals infected with intestinal helminths, particularly in those with A. lumbricoides alone, T. trichiura alone or S. mansoni alone compared to individuals without intestinal helminths. Similarly, prevalence of non-severe malaria  was significantly higher in individuals infected with intestinal helminth or A. lumbricoides alone compared to individuals without intestinal helminths. These findings are similar with previous finding in children [P. falciparum malaria was also previously reported in helminth infected individuals [In this study, the likelihood of being infected with non-severe children ,11 and achildren . Increasividuals ,7,8.Plasmodium and intestinal helminth co-infected patients compared to those with only Plasmodium infection. There was a non-significant decreasing trend in mean haemoglobin levels as the number of intestinal helminths species per an individual increased. Similarly, the association of anaemia was slightly higher in individuals co-infected with Plasmodium and intestinal helminth compared to malaria cases without having intestinal helminth infections. Intestinal helminths and malaria reduce haemoglobin level differently [In concordance with previous reports ,31, a referently ,33, implPlasmodium density was slightly higher among intestinal helminth infected individuals compared with intestinal helminth free individuals. These results together with the high odds of anaemia (or decreased mean haemoglobin levels) and non-severe P. falciparum and/or P. vivax infection give the impression of a positive association between intestinal helminth infection and non-severe malaria. This association between prevalence or outcomes of malaria and intestinal helminth infections seemed to increase with an increase in the number of intestinal helminths species. This is in agreement with previous reports from Ethiopia [Plasmodium density and P. falciparum infection, respectively increased with the number of intestinal helminth species.Mean Ethiopia  and ThaiEthiopia  where thPlasmodium[Plasmodium during concomitant infection [The present findings appear to substantiate the hypothesis that clinical malaria can be aggravated during concomitant infection of intestinal helminths and lasmodium. Worms alasmodium. Helmintlasmodium. Additionfection . Helmintnfection  promotinPlasmodium density was less associated with intensity of helminth infections and results regarding mean differences of Plasmodium density between intestinal helminth free and those infected with different intestinal helminth species were contradictory. Compared to intestinal helminth free malaria cases, the mean Plasmodium density of individuals infected with A. lumbricoides alone, S. mansoni alone or T. trichiura alone was slightly higher, but lower in the case of single hookworm infection. These controversial results on mean Plasmodium changes could have arisen due to the dominancy of light intense helminth infection among cases or complex nature of helminth and Plasmodium interaction when co-exist in a host.However, P. falciparum and/or P. vivax infection. This is in agreement with previous reports from Uganda [P. vivax mono-infection and infection with different intestinal helminth species which agrees with the observation in Thailand [Plasmodium species [Individuals with hookworm infection alone were neither protected nor associated with increased non-severe m Uganda  and Zairm Uganda , but in m Uganda . The lowThailand . This co species .Previous studies documented negative ,17-19 anPlasmodium species. In addition, the effect of confounding factors such as age, sex, nutrition status, socioeconomic factors and other infections  were not usually considered in previous studies. It is well known that, level of endemicity , local climate, traditional and modern health care practices as well as community health seeking behaviors vary from country to country and even from district to district. These variations could also bring substantial differences in helminth-malaria interactions and hence a result from one country/district might not be necessarily generalized or interpreted in another country/district where the above factors are significantly different.Variation in inferences about the nature of malaria and helminth interaction among reports could be due to differences in the nature of the study participants , study design, methodology and the way data was analyzed and interpreted. In most previous studies, data analysis did not separate the effect of different helminth or Participants of the current study were more homogenous in that they were largely (>90% of the cases) similar in their life style, education level (illiterate), income level (average) and means of making a living (agriculture), place of residence  and ethnicity (Sidama). However, slight differences in socioeconomic status might have existed among the cases which could partly affect the current data. In addition, cross-sectional nature of the study design has been a limitation to make conclusion about the risk or incidence of malaria due to previous status of helminth infection.A. lumbricoides, T. trichiura and S. mansoni were positively associated with P. falciparum infection. However, further studies are required to investigate how these helminths could contribute to increased prevalence of P. falciparum infection.In the current study, infections with The authors declare they have no competing interests.AD: conceived the project idea and designed the study, collected, analyzed and interpreted the data. GM: involved in data analysis. AD, GM, ML, AA and BE involved in the drafting of the manuscript for important intellectual content. All authors read the draft manuscript and approved the final copy for submission.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/291/prepub


Mycobacterium tuberculosis, the causative agent of tuberculosis. Here, we use a computational algorithm to predict sRNA candidates in the mycobacterial species M. smegmatis and M. bovis BCG and confirmed the expression of many sRNAs using Northern blotting. Thus, we have identified 17 and 23 novel sRNAs in M. smegmatis and M. bovis BCG, respectively. We have also applied a high-throughput technique (Deep-RACE) to map the 5′ and 3′ ends of many of these sRNAs and identified potential regulators of sRNAs by analysis of existing ChIP-seq datasets. The sRNAs identified in this work likely contribute to the unique biology of mycobacteria.Bacterial small RNAs (sRNAs) are short transcripts that typically do not encode proteins and often act as regulators of gene expression through a variety of mechanisms. Regulatory sRNAs have been identified in many species, including  Mycobacterium contains many clinically relevant pathogens, including Mycobacterium tuberculosis and Mycobacterium leprae, the etiologic agents of tuberculosis (TB) and leprosy, respectively. M. tuberculosis alone was responsible for 8.7 million incident cases and 1.4 million deaths globally in 2011 The genus M. tuberculosisM. tuberculosis using other experimental approaches M. tuberculosis. sRNAs have also been identified in other mycobacterial species: 34 and 15 sRNAs have been experimentally validated in Mycobcaterium bovis BCG and Mycobacterium smegmatis, respectively Bacterial “small RNAs” (sRNAs) are small (50–400 nt), typically untranslated transcripts. Many sRNAs play important roles in gene regulation in response to environmental changes sRNA Identification Protocol using High-throughput Technologies) M. bovis BCG. We selected 34 conserved sRNA candidates which we experimentally confirmed by Northern blot M. bovis BCG as well as all SIPHT predictions for M. smegmatis (these were not explored in the previous study). By combining SIPHT predictions with large-scale Northern blot validation, we have identified 23 and 17 novel sRNAs in M. bovis BCG and M. smegmatis, respectively. Thus, we have substantially increased the number of experimentally validated sRNAs in mycobacterial species. We also analyzed existing ChIP-seq datasets to identify possible regulators of sRNA expression, and used Deep-RACE, a technique that combines high throughput RNA-seq with Rapid Amplification of cDNA Reads (RACE) In a previous study, we used computational predictions from the SIPHT , and M. tuberculosis H37Rv were grown in mycomedium .M. smegmatis were predicted using the SIPHT program with the same parameters as described previously Small RNA candidates of M. bovis BCG, M. smegmatis and M. tuberculosisRNA was isolated as previously reported M. tuberculosis transcription factors extracted from a previous study M. bovis BCG and M. tuberculosis sRNAs from the current study and two previous studies M. bovis BCG sRNAs, we first identified the equivalent region of the M. tuberculosis H37Rv genome. Possible sRNA regulators were selected if the ChIP-seq peak was located within 100 bp upstream and 20 bp downstream of an sRNA 5′ end.We analyzed existing ChIP-seq datasets for 55 Deep 5′ RACE and Deep 3′ RACE were performed as previously described M. smegmatis (refseq: NC_008596) (M. bovis BCG (refseq: NC_008769)   and 144 _008769) . Tables M. smegmatis sRNA candidates were tested by Northern blot using oligonucleotides in both orientations; expression was confirmed for 18 sRNA  in M. bovis BCG . These were confirmed directly in M. smegmatis by Northern blotting in current study and listed in the 17 novel confirmed sRNAs. A homologue of Sm76 was previously identified in M. tuberculosis by RNA-seq All 93 M. smegmatis to also probe expression of these candidates in M. bovis BCG and M. tuberculosis. Although our focus was to validate M. smegmatis predictions, we fortuitously discovered homologues of 9 candidates in M. bovis BCG and 4 candidates in M. tuberculosis  were previously identified, under the nomenclature Mpr 1–21, respectively M. tuberculosis by RNA-seq M. smegmatis and M. tuberculosis and identified 20 and 5 sRNA homologues, respectively . Although we aimed to find intergenic sRNAs, half of the candidates we identified in this study overlap partially or entirely protein-coding genes in either the sense or antisense orientation , Table 2M. smegmatis  and twelve sRNAs in M. bovis BCG  were mapped completely to intergenic regions. Four sRNAs in M. smegmatis  were mapped to the sense strand of annotated protein-coding genes, and four were mapped to the antisense strand . One sRNA in M. smegmatis (Sm11) and five in M. bovis BCG  overlap partially with adjacent genes in the antisense orientation, and four sRNAs in M. bovis BCG  overlap partially with adjacent genes in the sense orientation. One sRNA in M. smegmatis (Sm47) and two in M. bovis BCG  were not classified.Nine sRNAs in cis-acting regulatory elements such as riboswitches.The location of sRNAs relative to protein-coding genes also gives clues as to their function. Regulatory sRNAs that are completely intergenic typically function by base-pairing with distally-encoded mRNAs; however, some of the sRNAs are close to the 5′ end or 3′ end of adjacent genes, suggesting possible alternative regulatory roles. sRNAs antisense to ORFs or UTRs can regulate expression of the overlapping gene Salmonella entericacis-acting regulatory elements such as riboswitches.sRNAs can be transcribed independently or generated by processing of mRNA UTRs. Several features of the sRNAs identified in this work are consistent with the sRNAs being independently transcribed from their own promoters. First, the Northern blots showed no evidence of larger bands that could correspond to pre-processed mRNAs. Second, 13 sRNAs  are orientated away from the surrounding genes. Third, 5 sRNAs  are located >200 bp from the nearest gene start/stop. Nineteen sRNAs are close to (<200 bp) upstream or downstream coding regions  and four  overlap coding regions in the sense orientation. It is formally possible that these sRNAs are generated by mRNA processing or premature termination, although the Northern blot analysis argues against this. Regardless, sRNAs processed from mRNAs could still have important regulatory functions M. tuberculosis transcription factors have recently been determined using ChIP-seq and these data are publicly available M. bovis BCG, it is highly likely that these sRNAs are conserved in M. tuberculosis given the extremely high similarity of the M. bovis BCG and M. tuberculosis genomes M. tuberculosis for transcription factors that bind close to sRNA 5′ ends, including sRNAs identified in earlier studies The regulation of sRNAs can provide important clues as to their biological functions. However, very little is currently known about regulation of mycobacterial sRNAs. The genome-wide binding profiles of many M. smegmatis and 23 novel sRNAs in M. bovis BCG, verified 5′ and 3′ ends, and list these sRNAs according to a recently-proposed annotation nomenclature. Our analysis of sRNA position relative to protein-coding genes suggests various potential roles for these sRNAs in gene regulation. Although the specific biological function of these, and all other known mycobacterial sRNAs, is not understood, we speculate that some of these sRNAs contribute to the biology of pathogenic mycobacterial species. Future studies will focus on the functional characterization of these novel sRNAs.In summary, we have identified 17 novel sRNAs in Figure S1M. smegmatis sRNAs.Northern blotting analysis for (PDF)Click here for additional data file.Figure S2M. tuberculosis with M. smegmatis and M. bovis BCG probes.Northern blotting analysis of sRNA candidates in (PDF)Click here for additional data file.Figure S3M. bovis BCG sRNAs.Northern blotting analysis for (PDF)Click here for additional data file.Figure S4Deep-RACE mapped reads of all sRNAs and adjacent gene annotations.(PDF)Click here for additional data file.Table S1The oligonucleotide sequence of all probes used for Northern Blotting analysis in this study.(PDF)Click here for additional data file.Table S2The oligonucleotide sequence of all primers used for Deep-RACE PCR.(PDF)Click here for additional data file.Table S3M. smegmatis.All 93 sRNA sequences predicted by SIPHT in (PDF)Click here for additional data file.Table S4M. bovis BCG. This list excludes predicted tRNAs.All 144 sRNA sequences predicted by SIPHT in (PDF)Click here for additional data file.Table S5All transcription factor ChIP-seq peaks located within 100 bp upstream and 20 bp downstream of sRNA 5′ ends.(PDF)Click here for additional data file.


Plant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process.Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (http://www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin.This paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Our work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects. For mucications -11. In cArabidopsis thaliana (Arabidopsis), using a BioBrick-modified plant vector system  [Zea mays [Lactobacillus lactis [Using BioBrick compatible plant vectors, we sought to modify the taste of Arabidopsis, specifically enhancing the sweetness of a bitter plant without altering sugar content. Several naturally occurring proteins are 100–3000 times sweeter than sugar by weight . BrazzeiE. coli) , Zea mayZea mays , and Lacs lactis .Synsepalum dulcificum, does not taste sweet on its own. Rather, it acts as a flavor-inverter by binding to taste receptors on the tongue in a pH-dependent manner, causing sour foods to taste sweet [E. coli [S. dulcificum glycosylation is not required for functional expression.Miraculin, isolated from the berries of the West African plant te sweet . A 1 μM te sweet . Miraculte sweet , tomato te sweet , and eve[E. coli , indicatBeyond creative gastronomy, we imagine this system being used to enhance the nutritional content of edible plants or help allergy sufferers enjoy the benefits of fresh home-grown produce Figure . The devAgrobacterium: when a plant is injured, Agrobacterium migrates to the wound site and transfers the T-DNA region of its tumor-inducing (Ti) plasmid into the plant cell [Agrobacterium’s Ti plasmid to allow transformation of heterologous DNA into plants via Agrobacterium [pat gene, or kanamycin resistance via the nptII gene, to enable the selection of successfully transformed plants.Arabidopsis is readily transformed by ant cell . The T-Dacterium . pORE vehttp://www.partsregistry.org).We developed a new set of six BioBrick DNA assembly compatible plant transformation vectors based on the pORE series Table . VectorsE. coli and the yeast Saccharomyces cerevisiae (S. cerevisiae), since the introduction of exogenous genes is faster in these organisms. Full-length miraculin and brazzein genes were commercially synthesized and codon-optimized for expression in Arabidopsis. BioBrick compatible restriction enzyme sites bracketed each open reading frame. Constructs were tagged at either the N- or C-terminus with the Strep-II tag [E. coli. Monomeric miraculin was expressed at very low levels at approximately 24 kDa regardless of tag location, which is consistent with previous work [S. cerevisiae  is likely due to yeast-specific glycosylation of the brazzein protein [We first tested the expression of standardized miraculin and brazzein genes in p-II tag  for westous work . Brazzeious work . Brazzeie Figure . The hig protein . While eAgrobacterium-mediated transformation [We successfully introduced two different BioBrick plant vectors into Arabidopsis and selected for seeds carrying genomically-integrated miraculin and brazzein transgenes. Miraculin- or brazzein-encoding DNA under control of the pENTCUP2 promoter and NosT transcriptional terminator on either the V3 (glufosinate resistance) or V4 (kanamycin resistance) BioBrick vector was introduced into Arabidopsis via ormation . TransfoGenetic engineering of plants at the industrial and laboratory scale is well established. Technological advances have yielded crops that reduce food production costs through resistance to pests, herbicide, drought, and flood . Additiohttp://www.partsregistry.org) and are available as a resource for the synthetic biology and plant engineering communities. These include vectors modified from the pORE vector series [We have modified existing plant integration vectors to make them compatible with the BioBrick assembly standard 23 , demonstr series , plant-sIn addition to using these vectors to express exogenous proteins, we have considered integrating constructs expressing hairpin RNAs  or artifWe hope that availability of plant integration vectors compatible with a common assembly standard will facilitate the use of plants as a chassis in synthetic biology. Local-scale design of food plants, in which the grower selects traits desired in their community, can be made possible through the availability of standardized, modular genetic parts. Personalized engineering of plants to modify flavor, nutritional value, or allergenicity could create a new class of designed foods that are grown and consumed at a local scale. We encourage the iGEM community to continue to explore these concepts via plant engineering.A significant barrier to the adoption of local-scale plant engineering is the uncertain regulatory landscape for the deployment of genetically modified organisms. This landscape has been defined by large-scale commercial agriculture. As the tools of synthetic biology become more accessible, efforts by small groups such as our own will continue to challenge existing regulatory frameworks. We hope that our concept of genetic engineering tools in the hands of local growers will spur discussion and debate on how to responsibly regulate the synthetic biology scenarios of the near future.E. coli DH5α using BioBrick assembly standard 23 [Gene assembly was performed in ndard 23 , and allBrazzein and miraculin were codon-optimized for expression in Arabidopsis, commercially synthesized , and assembled with the pENTCUP2 promoter and NosT transcriptional terminator. Completed constructs were subcloned from BioBrick assembly vector V0120 to BioBrick modified pORE vectors through digestion with EcoRI and PstI.Arabidopsis thaliana seeds were sterilized by washing with 70% ethanol, 0.1% Triton X-100, followed by two 95% ethanol washes and two sterile dH2O washes. Seeds were then plated on 1X Murashige & Skoog (MS) media with 0.7% agar supplemented with 150 uM carbenicillin and placed in the dark at 4°C for three days before moving to an incubator with 16 h illumination at 20°C and 8 h dark at 15°C per day to allow seeds to germinate. Once plants produced secondary leaves, they were moved to soil and allowed to mature and produce seeds. Seeds were collected and stored at 4°C.Wild-type Col-0 Agrobacterium-mediated transformation was performed according to previously reported techniques [Agrobacterium was made electro-competent by washing in cold sterile water and resuspending in 10% glycerol. Vector DNA was dialyzed to remove excess salt, and electroporated into Agrobacterium. Kanamycin-resistant colonies were grown in YEB media, spread on YEB plates, and allowed to form a lawn. Lawns were scraped and suspended in a solution of 20% YEB, 4% sucrose (w/v), and 0.024% Silwet L-77 surfactant . Wild-type Col-0 Arabidopsis flowers were dipped in the Agrobacterium solution and allowed to grow and develop seed pods. Seeds were collected from mature plants and selected on 1x MS media with 0.7% agar supplemented with 5 mg/L glufosinate or 50 μg/ml kanamycin.chniques . BrieflyE. coli, StrepII-tagged brazzein and miraculin were inserted at multiple cloning site 1 of a BioBrick-modified pET-duet vector [In BL21(DE3) t vector . Cells wS. cerevisiae, StrepII-tagged brazzein was cloned with the constitutive TEF promoter or the copper-inducible CUP1 promoter and integrated at the LEU2 locus. Transformants were grown in YEPD media with 0.3 mM CuSO4 to induce protein expression, which was measured by western blot.In PSY580a yeast Genomic DNA was extracted from Arabidopsis using the DNEasy kit (Qiagen) and amplified by PCR . Samples were diluted into SDS-PAGE loading buffer and loaded onto a 4–20% Tris/glycine/SDS acrylamide gel. α-Strep-tag II antibody  was used to measure brazzein and miraculin protein expression in E. coli and yeast, and α-miraculin antibody [Protein samples were extracted from Arabidopsis, antibody  (provideMCS: Multiple cloning site.The authors declare they have no competing interests.Agrobacterium-mediated transformation. PMB, DRB, MCI, and CMA performed PCR, qPCR and western blots; all other cloning and experiments were performed by AD, JGdW, MAG, JYQ, MLP, AMR, MRT, LW, and JCW. KAH, AV, TJB, GMC, JVS, and PAS provided general advising throughout the project. The manuscript was drafted by PMB, DRB, MCI, and CMA. All authors read and approved the final manuscript.Cloning schemes were designed by PMB, DRB, MCI, CMA, AD, JGdW, MAG, JYQ, MLP, AMR, MRT, LW, JCW, and OM. KS provided technical assistance with Arabidopsis culture and Supplementary Data for A BioBrick Compatible Strategy for Genetic Modification of Plants. Contains tables of primer sequences describing primers used to modify pORE vectors and verify integration and RNA expression of transgenes.Click here for file