Biology subjects

Lechuga, A.

Publications and source records attributed to Lechuga, A..

4 recordsLinked to original sources

bioRxiv2021

Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative yeast two hybrid-high throughput sequencing approach

AO_SCPLOWBSTRACTC_SCPLOWBacillus virus Bam35 is the model Betatectivirus and member of the Tectiviridae family, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. The interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein-protein interactions network for a tectivirus-host system by studying the Bam35-Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and Illumina high-throughput sequencing. We generated and thoroughly analyzed a genomic library of Bam35s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait-prey couples. In total, this screen resulted in the detection of over 4,000 potential interactions, of which 183 high-confidence interactions were defined as part of the core virus-host interactome. Overall, host metabolism proteins and peptidases are particularly enriched within the detected interactions, distinguishing this host-phage system from the other reported host-phage protein-protein interaction networks (PPIs). Our approach also suggests biological roles for several Bam35 proteins of unknown function, resulting in a better understanding of the Bam35-B. thuringiensis interaction at the molecular level. AO_SCPLOWUTHORC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWSUMMARYC_SCPLOWMembers of the family Tectiviridae, composed of non-tailed icosahedral, membrane-containing bacteriophages, have been increasingly scrutinized in recent years for their possible role in the origin of dsDNA viruses. In particular, the genus Betatectivirus receives increased attention as these phages can infect clinical strains as well as industrially relevant members of the B. cereus group. However, little is known about the interactions between these temperate viruses and their hosts. Here, we present the first high-throughput study of tectivirus-host protein-protein interactions focusing on Bam35, model virus of betatectiviruses, and its host B. thuringiensis, an important entomopathogenic bacterium. We adapted the well-known technique yeast-two-hybrid and integrated high-throughput sequencing and bioinformatics for the downstream analysis of the results which enables large-scale analysis of protein-protein interactions. In total, 182 detected interactions show an enrichment in host metabolic proteins and peptidases, in contrast with the current knowledge on host-phage PPIs. Specific host-viral protein-protein interactions were also detected enabling us to propose functions for uncharacterized proteins.

microbiology↗

bioRxiv2021

Unlimited cooperativity of Betatectivirus SSB, a novel DNA binding protein related to an atypical group of SSBs from protein-primed replicating bacterial viruses

Bam35 and related betatectiviruses are tail-less bacteriophages that prey on members of the Bacillus cereus group. These temperate viruses replicate their linear genome by a protein-primed mechanism. In this work, we have identified and characterized the product of the viral ORF2 as a single-stranded DNA binding protein (hereafter B35SSB). B35SSB binds ssDNA with great preference over dsDNA or RNA in a sequence-independent, highly cooperative manner that results in a non-specific stimulation of DNA replication. We have also identified several aromatic and basic residues, involved in base-stacking and electrostatic interactions, respectively, that are required for effective protein-ssDNA interaction. Although SSBs are essential for DNA replication in all domains of life as well as many viruses, they are very diverse proteins. However, most SSBs share a common structural domain, named OB-fold. Protein-primed viruses could constitute an exception, as no OB-fold DNA binding protein has been reported. Based on databases searches as well as phylogenetic and structural analyses, we showed that B35SSB belongs to a novel and independent group of SSBs. This group contains proteins encoded by protein-primed viral genomes from unrelated viruses, spanning betatectiviruses and {Phi}29 and close podoviruses, and they share a conserved pattern of secondary structure. Sensitive searches and structural predictions indicate that B35SSB contains a conserved domain resembling a divergent OB-fold, which would constitute the first occurrence of an OB-fold-like domain in a protein-primed genome. HighlightsO_LIBam35 ORF 2 product encodes a viral single-stranded DNA binding protein (B35SSB). C_LIO_LIB35SSB binds ssDNA in a highly cooperative manner but with no sequence specificity. C_LIO_LIB35SSB-ssDNA binding is mediated by base-stacking and ionic interactions. C_LIO_LIBam35 and {Phi}29-related SSBs form a novel group of SSBs from protein-primed viruses. C_LIO_LIThe B35-{Phi}29 SSBs group shares a highly divergent OB-fold-like domain. C_LI

biochemistry↗

bioRxiv2020

The fully resolved genome of Bacillus thuringiensis HER1410 reveals a cry-containing chromosome, two megaplasmids & an integrative plasmidial prophage

Bacillus thuringiensis is the most used biopesticide in agriculture. Its entomopathogenic capacity stems from the possession of plasmid-borne insecticidal crystal genes (cry), traditionally used as discriminant taxonomic feature for that species. As such, crystal and plasmid identification are key to the characterization of this species. To date, about 600 B. thuringiensis genomes have been reported, but less than 5% have been resolved, while the other draft genomes are incomplete, precluding plasmid delineation. Here we present the complete genome of Bacillus thuringiensis HER1410, a strain closely related to B. thuringiensis entomocidus and a known host for a variety of Bacillus phages. The combination of short and long-reads techniques allowed fully resolving the genome and delineation of three plasmids. This enabled the accurate detection of an unusual location of a unique cry gene, cry1Ba4, located in a genomic island near the chromosome replication origin. Two megaplasmids, pLUSID1 and pLUSID2 could be delineated: pLUSID1 (368kb), a likely conjugative plasmid involved in virulence, and pLUSID2 potentially related to the sporulation process. A smaller plasmidial prophage pLUSID3, with a dual lifestyle whose integration within the chromosome, causes the disruption of a flagellar key component. Finally, phylogenetic analysis located this strain within a clade comprising members from the B. thuringiensis serovar thuringiensis and other serovars and with B.cereus s. s. This highlights the intermingled taxonomy of B. cereus sensu lato group, where genomics alone does not support the present taxonomy between B. cereus s. s. and B. thuringiensis as species designation currently relies solely on the presence of entomocidal genes. ImportanceBacillus cereus group species have been extensively studied due to their economical and clinical relevance. This importance originally set the basis for B. cereus group members classification which are commonly based on phenotypical criteria. Sequencing era has shed light about genomic characterization of these species, showing their chromosomal genomic similarity and highlighting the role of mobile genetic elements, especially megaplasmids, in the classification and characterization of this group. However, only the 5% of the sequenced B. thuringiensis genomes have been fully resolved. Thus, here we addressed efficiently the study B. thuringiensis HER1410 genomic features by the use of a combination of short and long-reads sequencing. This methodology resulted in the high-quality assembly, which led to the identification of an uncommon location of a cry gene close to the chromosomal origin, as well as three fully resolved extrachromosomal elements, two megaplasmids and an integrative plasmidial prophage.

microbiology↗

bioRxiv2019

Rhizosphere metagenomics of mine tailings colonizing plants: assembling and selecting synthetic bacterial communities to enhance in situ bioremediation

Mine tailings phytostabilization has been proposed as a bioremediation strategy to constrain the contaminants dispersion using plants to limit the effects of erosion. Rhizospheric bacteria impact plant health and facilitate plant establishment through their metabolic functions, which could be relevant in bioremediation strategies. We studied both culturable and metagenomic diversity or rhizospheric bacteria of mine tailings colonizing plants of an abandoned mine operation in Nacozari de Garcia, Sonora, Mexico. Diversity was described through amplification of the 16S rRNA gene and whole metagenome shotgun sequencing of both environmental and cultured rhizosphere associated microbes. The culturable bacteria were assembled in a synthetic community (SC; 235 bacteria genera). Subsequently, we performed an experimental evolution setup with the SC, selecting for heavy metal resistance, microbial competition, and the ability for growing in plant-derived nutrient sources. The selection experiment show that bacteria diversity decreases from the environmental culture-free microbiomes to the mine tailings rhizospheres and the experimental evolution outcome: the synthetic community (FSC; 43 bacteria genera). The rhizosphere communities shifted from the dominance of Actinobacteria in their environment to Proteobacteria in the cultivated consortia and the synthetic communities. Both environmental and cultured metagenomes contained plant-growth promotion, heavy-metal homeostasis, and antibiotic resistance predicted genes. The FSC included predicted proteins related to plant-growth promotion such as siderophore production and plant hormone regulation proteins. We reconstructed a metagenome assembled genomic sequence named Enterobacter sp., Nacozari. The recovered Enterobacter sp. Nacozari, have predicted coding genes for direct and indirect plant growth promotion along with adhesion and oxidative stress-related proteins. The metabolic potential of the FSC presents promising features that might make it useful for plant-growth promotion in tailored phytostabilization strategies for the abandoned mine-tailings of Nacozari.

genomics↗