Biology subjects

Griffitts, J. S.

Publications and source records attributed to Griffitts, J. S..

7 recordsLinked to original sources

bioRxiv2025

The evolutionary genomics of incipient endosymbiosis in wild rhizobia bacteria

The advent of endosymbiosis underlies evolutionary innovation and ecosystem function. However, whether free-living partners tend to benefit or exploit each other during incipient endosymbiosis remains a dilemma. Rhizobia bacteria are plant endosymbionts capable of initiating root nodules and fixing nitrogen due to genes carried on mobile genetic elements (MGEs) such as the symbiosis island (SI). We conjugated marked SIs into the genomes of nonnodulating strains, which was sufficient to generate de novo root nodule-forming endosymbionts. Most novel endosymbionts originated as commensals that incurred no detectable costs to host plants, in contrast to predictions of exploitation. In fact, a third of endosymbionts originated as nitrogen fixing mutualists. Consistent with phylogenetic limits to transfer of MGE function, novel endosymbionts derived from more closely related SI donor and recipient strains showed greater nitrogen fixation. However, we did not detect phylogenetic limits to SI transmission, which could reflect selfish selection for generalized horizontal transfer of this MGE. In fact, the SI was able to displace other genomic elements residing at its characteristic tRNA gene insertion site. We thus provide genetic, genomic, and functional evidence of how MGEs can potentiate and constrain major evolutionary transitions to expand bacterial niches, with cascading effects on host organisms.

evolutionary biology↗

bioRxiv2025

Mobile genetic elements shape the evolution of wild bacterial pangenomes in the face of biotic and abiotic selective pressures in the soil

Mobile genetic elements (MGEs) provide a critical reservoir of diversity that potentiates bacterial local adaptation. However, the degree to which natural populations of MGEs and the chromosomes that host them share congruent or divergent evolutionary histories remains an open question. MGE and host chromosome phylogenies could show congruence or independence, depending on rates of MGE gain and loss, costs and benefit of MGE carriage, compatibility, and gene flow. We investigated phylogenetic congruence for two critical niche-expanding MGEs and host chromosomes using 302 genomes of wild symbiotic nitrogen-fixing Mesorhizobium bacteria. The MGEs comprise: 1) a nickel resistance island (NRI), which confers adaptation to nickel-enriched serpentine soils across replicated natural populations, and 2) a more extensive symbiosis island (SI), which enables symbiotic nitrogen fixation with a host legume. We demonstrate that the NRI, like the SI, is transmissible and its acquisition confers nickel adaptation. NRI acquisition often incurs no detectable cost, consistent with conditional neutrality during MGE-mediated adaptation. Phylogenies for the NRI, SI, and chromosome are congruent across wild strains, consistent with a history of vertical co-inheritance and/or MGE transmission primarily among close relatives. This congruence does not appear to result from barriers to gene flow, as diverse MGE and host chromosome haplotypes are broadly distributed and co-occur. Thus, despite ample ecological opportunity for admixture, distant relatives rarely transmitted MGEs, consistent with selection to maintain co-adapted genomic compartments. The SI has stronger phylogenetic congruence with the chromosome than does the smaller NRI, supporting the hypothesis that MGE complexity limits horizontal transfer among more divergent lineages. However, rare horizontal NRI transmission events among distant relatives and NRI loss appear to occur when haplotypes migrate across environments. Thus, environmentally dependent selection on migrants may periodically disrupt co-adapted MGE and chromosome haplotypes and serve as an engine of diversity.

evolutionary biology↗

bioRxiv2025

Regulation of a nickel tolerance operon conserved in Mesorhizobium strains from serpentine soils

The Mesorhizobium nre (nickel resistance) operon has previously been shown to mediate Ni tolerance in serpentine soils with naturally high concentrations of this transition metal. In most serpentine-derived strains evaluated, the putative efflux genes nreX and nreY are conserved, along with a small gene (nreA) encoding a CsoR/RcnR-family transcriptional regulator. CsoR/RcnR-family regulators are small (around 100 amino acids in length), they bind transition metals, and they use an unconventional and poorly understood DNA-binding mechanism. NreA is 93 amino acids in length and belongs to a poorly characterized clade within the CsoR/RcnR family. This investigation is focused on regulatory DNA elements that functionally interact with Mesorhizobium NreA, as well as amino acid residues in NreA that influence its regulatory activity. We show that NreA is a transcriptional repressor that is responsive to exogenous Ni. The Ni-responsive promoter is immediately upstream of the nreAXY operon, resulting in a leaderless transcript. A partially palindromic operator sequence occupies the spacer region between the -35 and -10 promoter elements. Mutational analysis of the operator highlights functionally crucial base pairs occupying opposite ends of the palindromic operator. Changes to conserved residues in the NreA polypeptide result in varying effects, with changes predicted to prevent Ni binding leading to a super-repressor phenotype. Structural modeling of the NreA-operator complex provides a plausible mechanism for DNA binding by a tetrameric form of NreA, with DNA contact sites along a positively charged surface, and the modeled contact sites agree well with the mutational analysis. IMPORTANCEBacteria employ diverse mechanisms for maintaining optimal intracellular levels of bioactive metals. Isolates of Mesorhizobium bacteria from nickel-rich serpentine soils possess a small genetic region controlling Ni efflux. This region is subject to transcriptional regulation via the small repressor protein NreA. In this work, the essential components of the NreA protein and the DNA operator with which it interacts are defined, enabling potential adaptation of this system for metal sensing or other technologies requiring a compact inducible gene expression module.

microbiology↗

bioRxiv2023

Micrococcin cysteine-to-thiazole conversion through transient interactions between a scaffolding protein and two modification enzymes

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, we demonstrate complex formation between TclI, TclJ and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core. IMPORTANCEThiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications (PTMs) to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin.

biochemistry↗

bioRxiv2023

Three genes controlling streptomycin susceptibility in Agrobacterium fabrum

Streptomycin is a commonly used antibiotic for its efficacy against diverse bacteria. The plant pathogen Agrobacterium fabrum is a model for studying pathogenesis and interkingdom gene transfer. Streptomycin-resistant variants of A. fabrum are commonly employed in genetic analyses, yet mechanisms of resistance and susceptibility to streptomycin in this organism have not previously been investigated. We observe that resistance to a high concentration of streptomycin arises at high frequency in A. fabrum and we link this trait to the presence of a chromosomal gene (strB) encoding a putative aminoglycoside phosphotransferase. We show how strB, along with rpsL (encoding ribosomal protein S12) and rsmG (encoding a 16S rRNA methyltransferase) modulate streptomycin sensitivity in A. fabrum.

microbiology↗

bioRxiv2021

Engineering efficient termination of bacteriophage T7 RNA polymerase transcription

The bacteriophage T7 expression system is one of the most prominent transcription systems used in biotechnology and molecular-level research. However, T7 RNA polymerase is prone to read-through transcription due to its high processivity. As a consequence, enforcing efficient transcriptional termination is difficult. The termination hairpin found natively in the T7 genome is adapted to be inefficient, exhibiting 62% termination efficiency in vivo and even lower efficiency in vitro. In this study, we engineered a series of sequences that outperform the efficiency of the native terminator hairpin. By embedding a previously discovered 8-nucleotide T7 polymerase pause sequence within a synthetic hairpin sequence, we observed in vivo termination efficiency of 91%; by joining two short sequences into a tandem 2-hairpin structure, termination efficiency was increased to 98% in vivo and 91% in vitro. This study also tests the ability of these engineered sequences to terminate transcription of the Escherichia coli RNA polymerase. Two out of three of the most successful T7 polymerase terminators also facilitated termination of the bacterial polymerase with around 99% efficiency.

molecular biology↗

bioRxiv2017

Robustness encoded across essential and accessory replicons in an ecologically versatile bacterium

Bacterial genome evolution is characterized by gains, losses, and rearrangements of functional genetic segments. The extent to which genotype-phenotype relationships are influenced by large-scale genomic alterations has not been investigated in a high-throughput manner. In the symbiotic soil bacterium Sinorhizobium meliloti, the genome is composed of a chromosome and two large extrachromosomal replicons (pSymA and pSymB, which together constitute 45% of the genome). Massively parallel transposon insertion sequencing (Tn-seq) was employed to evaluate contributions of chromosomal genes to fitness in both the presence and absence of these extrachromosomal replicons. Ten percent of chromosomal genes from diverse functional categories are shown to genetically interact with pSymA and pSymB. These results demonstrate the pervasive robustness provided by the extrachromosomal replicons, which is further supported by constraint-based metabolic modelling. A comprehensive picture of core S. meliloti metabolism was generated through a Tn-seq-guided in silico metabolic network reconstruction, producing a core network encompassing 726 genes. This integrated approach facilitated functional assignments for previously uncharacterized genes, while also revealing that Tn-seq alone misses over a quarter of wild type metabolism. This work highlights the strong functional dependencies and epistatic relationships that may arise between bacterial replicons and across a genome, while also demonstrating how Tn-seq and metabolic modelling can be used together to yield insights not obtainable by either method alone.

systems biology↗