De novo assembly of transcriptomes from a B73 maize line introgressed with a QTL for resistance to gray leaf spot disease reveals a candidate allele of a lectin receptor-like kinase

Gray leaf spot (GLS) disease in maize, caused by the fungus Cercospora zeina, is a threat to maize production globally. Understanding the molecular basis for quantitative resistance to GLS is therefore important for food security. We developed a de novo assembly pipeline to identify candidate maize resistance genes. Near-isogenic maize lines with and without a QTL for GLS resistance on chromosome 10 from inbred CML444 were produced in the inbred B73 background. The B73-QTL line showed a 20% reduction in GLS disease symptoms compared to B73 in the field (p = 0.01). B73-QTL leaf samples from this field experiment conducted under GLS disease pressure were RNA sequenced. The reads that did not map to the B73 or C. zeina genomes were expected to contain novel defense genes and were de novo assembled. A total of 141 protein-coding sequences with B73-like or plant annotations were identified from the B73-QTL plants exposed to C. zeina. To determine whether candidate gene expression was induced by C. zeina, the RNAseq reads from C. zeina-challenged and control leaves were mapped to a master assembly of all of the B73-QTL reads, and differential gene expression analysis was conducted. Combining results from both bioinformatics approaches led to the identification of a likely candidate gene, which was a novel allele of a lectin receptor-like kinase named L-RLK-CML that (i) was induced by C. zeina, (ii) was positioned in the QTL region, and (iii) had functional domains for pathogen perception and defense signal transduction. The 817AA L-RLK-CML protein had 53 amino acid differences from its 818AA counterpart in B73. A second "B73-like" allele of L-RLK was expressed at a low level in B73-QTL. Gene copy-specific RT-qPCR confirmed that the l-rlk-cml transcript was the major product induced four-fold by C. zeina. Several other expressed defense-related candidates were identified, including a wall-associated kinase, two glutathione s-transferases, a chitinase, a glucan beta-glucosidase, a plasmodesmata callose-binding protein, several other receptor-like kinases, and components of calcium signaling, vesicular trafficking, and ethylene biosynthesis. This work presents a bioinformatics protocol for gene discovery from de novo assembled transcriptomes and identifies candidate quantitative resistance genes

A reservoir of 'historical' antibiotic resistance genes in remote pristine Antarctic soils

Background: Soil bacteria naturally produce antibiotics as a competitive mechanism, with a concomitant evolution, and exchange by horizontal gene transfer, of a range of antibiotic resistance mechanisms. Surveys of bacterial resistance elements in edaphic systems have originated primarily from human-impacted environments, with relatively little information from remote and pristine environments, where the resistome may comprise the ancestral gene diversity. Methods: We used shotgun metagenomics to assess antibiotic resistance gene (ARG) distribution in 17 pristine and remote Antarctic surface soils within the undisturbed Mackay Glacier region. We also interrogated the phylogenetic placement of ARGs compared to environmental ARG sequences and tested for the presence of horizontal gene transfer elements flanking ARGs. Results: In total, 177 naturally occurring ARGs were identified, most of which encoded single or multi-drug efflux pumps. Resistance mechanisms for the inactivation of aminoglycosides, chloramphenicol and beta-lactam antibiotics were also common. Gram-negative bacteria harboured most ARGs (71%), with fewer genes from Gram-positive Actinobacteria and Bacilli (Firmicutes) (9%), reflecting the taxonomic composition of the soils. Strikingly, the abundance of ARGs per sample had a strong, negative correlation with species richness (r=-0.49, P < 0.05). This result, coupled with a lack of mobile genetic elements flanking ARGs, suggests that these genes are ancient acquisitions of horizontal transfer events. Conclusions: ARGs in these remote and uncontaminated soils most likely represent functional efficient historical genes that have since been vertically inherited over generations. The historical ARGs in these pristine environments carry a strong phylogenetic signal and form a monophyletic group relative to ARGs from other similar environments

Resistance sniffer : an online tool for prediction of drug resistance patterns of Mycobacterium tuberculosis isolates using next generation sequencing data

The effective control of multidrug resistant tuberculosis (MDR-TB) relies upon the timely diagnosis and correct treatment of all tuberculosis cases. Whole genome sequencing (WGS) has great potential as a method for the rapid diagnosis of drug resistant Mycobacterium tuberculosis (Mtb) isolates. This method overcomes most of the problems that are associated with current phenotypic drug susceptibility testing. However, the application of WGS in the clinical setting has been deterred by data complexities and skill requirements for implementing the technologies as well as clinical interpretation of the next generation sequencing (NGS) data. The proposed diagnostic application was drawn upon recent discoveries of patterns of Mtb clade-specific genetic polymorphisms associated with antibiotic resistance. A catalogue of genetic determinants of resistance to thirteen anti-TB drugs for each phylogenetic clade was created. A computational algorithm for the identification of states of diagnostic polymorphisms was implemented as an online software tool, Resistance Sniffer (http://resistance-sniffer.bi.up. ac.za/), and as a stand-alone software tool to predict drug resistance in Mtb isolates using complete or partial genome datasets in different file formats including raw Illumina fastq read files. The program was validated on sequenced Mtb isolates with data on antibiotic resistance trials available from GMTV database and from the TB Platform of South African Medical Research Council (SAMRC), Pretoria. The program proved to be suitable for probabilistic prediction of drug resistance profiles of individual strains and large sequence data sets.The South African National Research Foundation (NRF)https://www.elsevier.com/locate/ijmmam2020BiochemistryGeneticsMicrobiology and Plant Patholog

Differences in precipitation regime shape microbial community composition and functional potential in Namib Desert soils

Precipitation is one of the major constraints influencing the diversity, structure, and activity of soil microbial communities in desert ecosystems. However, the effect of changes in precipitation on soil microbial communities in arid soil microbiomes remains unresolved. In this study, using 16S rRNA gene high-throughput sequencing and shotgun metagenome sequencing, we explored changes in taxonomic composition and functional potential across two zones in the Namib Desert with contrasting precipitation regime. We found that precipitation regime had no effect on taxonomic and functional alpha-diversity, but that microbial community composition and functional potential (beta-diversity) changed with increased precipitation. For instance, Acidobacteriota and ‘resistance to antibiotics and toxic compounds’ related genes were relatively more abundant in the high-rainfall zone. These changes were largely due to a small set of microbial taxa, some of which were present in low abundance (i.e. members of the rare biosphere). Overall, these results indicate that key climatic factors (i.e. precipitation) shape the taxonomic and functional attributes of the arid soil microbiome. This research provides insight into how changes in precipitation patterns associated with global climate change may impact microbial community structure and function in desert soils.A Free standing and Research and Development Programme Grant funded by the National Research Foundation (NRF) of South Africa.https://link.springer.com/journal/248hj2023BiochemistryGeneticsMicrobiology and Plant Patholog

Phenolic compound degradation by Pseudomonas syringae phylogroup 2 strains

It has recently been shown that Pseudomonas syringae strains pathogenic to woody hosts belonging to phylogroup (PG) 2 lack phenolic compound degradation pathways such as the beta-ketoadipate and protocatechuate pathways. The aim of this study was to analyse a selection of P. syringae PG 2 genomes, including those used previously to determine if they had other phenolic compound degradation pathways and to determine whether or not they were functional. Twenty-one publicly available genomes of PG 2 strains were analyzed. These strains had previously been isolated from both woody and herbaceous hosts. Phenolic degradation enzymes were present in 5 (23%) of the strains analysed, originating from both woody and herbaceous hosts. Hypothetical pathways were proposed to determine if catechol, anthranilate and benzoic acid were degraded by these strains. Both spectrophotometric and HPLC were used to determine phenolic compound degradation. The five strains with phenolic degradation enzymes were able to metabolize catechol, and HRI-W 7924 and MAFF 301072 could also metabolize anthranilate and benzoate, respectively. The study showed that even though some PG 2 strains lack the beta-ketoadipate and protocatechuate pathways, they still have phenolic compound degrading enzymes that may play a role in virulence.The Horticultural Knowledge Group (HORTGRO) and National Research Foundation (NRF).https://link.springer.com/journal/421612019-07-01am2019BiochemistryForestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant PathologyZoology and Entomolog

Clade-Specific Distribution of Antibiotic Resistance Mutations in the Population of Mycobacterium tuberculosis - Prospects for Drug Resistance Reversion

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a leading cause of death in humans worldwide. The emergence of antibiotic-resistant strains of Mtb is a threat to tuberculosis control. A general belief is that drug resistance is acquired by Mtb during antibiotic treatment by accumulation of spontaneous mutations. Also, it is known that the drug resistance mutations (DRM) have an associated fitness cost, reducing the transmissibility and virulence of resistant strains. In this work we show that many canonical DRM are clade specific; i.e. they occur only in specific genetic lineages of Mtb and depend on a specific genetic context necessary for the reduction of the fitness cost and sustainability of the drug resistance phenotype. Dependence of the drug resistance on occurrence of genetic variants of multiple genes and specific activities of the encoded proteins allows combating the drug resistance by impairing the global genetic context. A new drug, FS-1, reverses antibiotic resistance by compromising this genetic context and aggravating the fitness cost of DRM

Multiple-locus variable-number tandem repeat analysis genotypes of Listeria monocytogenes isolated from farms, abattoirs, and retail in Gauteng Province, South Africa

The use of multiple-locus variable-number analysis (MLVA) of tandem repeats (TRs) for subtyping Listeria monocytogenes has proven to be reliable and fast. This study determined the MLVA genotypes of 60 isolates of L. monocytogenes recovered from cattle farms, abattoirs, and retail outlets in Gauteng province, South Africa. The distribution of the 60 L. monocytogenes isolates analyzed by type of sample was as follows: raw beef (28, 46.7%), ready-to-eat beef products (9, 15.0%), beef carcass swabs (9, 15.0%), cattle environment (6, 10.0%), and cattle feces (8, 13.3%). The serogroups of the isolates were determined using PCR and the MLVA genotypes based on six selected loci. The frequency of the 60 serogroups detected was as follows: 1/2a-3a (IIa) (27, 45.0%); 4b-4d-4e (1Vb) (24, 40.0%); 1/2c-3c (IIc) (8, 13.3%); and 1/2b-3b (IIb) (1, 1.7%). MLVA successfully clustered genetically related isolates and differentiated nonrelated isolates, irrespective of their sources, sample types, and serogroups, as demonstrated by 16 MLVA pattern types detected. For serogroup 4b-4d-4e (IVb), there was no variation in TRs LM-TR2, LM-TR4, and LM-TR6, which each contained only one allele (02, 00, and 93, respectively). However, across the sources and sample types of isolates, there was variation in serogroup 4b-4d-4e (IVb): LM-TR1 contained 00, 03, and 05; LM-TR3 contained 14, 20, and 22; and LM-TR5 contained 14, 21, and 25. Similar patterns of variation in the TRs were detected in the other serogroups (1/2a-3a, 1/2b-3b, and 1/2c-3c). BioNumeric data analysis identified at least five types in Gauteng province. MLVA epidemiologically clustered the related isolates and differentiated unrelated isolates.Red Meat Research and Development, South Africa (RMRD-SA).https://www.sciencedirect.com/journal/journal-of-food-protectionProduction Animal Studie

Pre_GI : a global map of ontological links between horizontally transferred genomic islands in bacterial and archaeal genomes

The Predicted Genomic Islands database (Pre_GI) is a comprehensive repository of prokaryotic genomic islands (islands, GIs) freely accessible at http://pregi.bi.up.ac.za/index. php. Pre_GI, Version 2015, catalogues 26 744 islands identified in 2407 bacterial/archaeal chromosomes and plasmids. It provides an easy-to-use interface which allows users the ability to query against the database with a variety of fields, parameters and associations. Pre_GI is constructed to be a web-resource for the analysis of ontological roads between islands and cartographic analysis of the global fluxes of mobile genetic elements through bacterial and archaeal taxonomic borders. Comparison of newly identified islands against Pre_GI presents an alternative avenue to identify their ontology, origin and relative time of acquisition. Pre_GI aims to aid research on horizontal transfer events and materials through providing data and tools for holistic investigation of migration of genes through ecological niches and taxonomic boundaries.A research grant 86941 provided by the National Research Foundation (NRF) of South Africa. Funding for open access charge: A contribution received from the University of Pretoria APC Fund.http://database.oxfordjournals.orghttp://pregi.bi.up.ac.za/index.phpam201

Long non-coding RNAs in the human genome acquired by horizontal gene transfer

BACKGROUND : Horizontal gene transfer of mobile genetic elements is an essential component of prokaryotic evolution. These insertion events in eukaryotes and particularly in the human genome have been investigated by various methodologies with varying results. OBJECTIVE : In this paper, we implement a sequence composition approach to investigate insertions of genomic islands in the human genome. METHODS : A modified version of a prokaryotic GI identifier, SeqWord Gene Island Sniffer v.2.0, was used to predict genomic islands in the hg38 version of the human genome. RESULTS : Predicted genomic islands were enriched with long non-coding RNAs and also contributed to the acquisition and modification of proteins associated with the immune system and gonad development, albeit to a lesser extent. The estimated rate of acquisition of these genomic islands in vertebrate genomes was non-linear with regards to species divergence times with an acceleration at the time of vertebrate land invasion and during the transition of prosimians to monkeys soon after the Cretaceous-Paleogene extinction. CONCLUSION : The rapid acquisition of non-conserved long non-coding RNAs in the human genome and probably in vertebrata genomes was facilitated by horizontal gene transfer. All predicted human genomic islands and supporting information are freely accessible from http://hislands.bi.up.ac.za.The National Research Foundation (NRF) grant 105996am2019BiochemistryGeneticsMicrobiology and Plant Patholog