Biology subjects

Motnenko, A.

Publications and source records attributed to Motnenko, A..

3 recordsLinked to original sources

bioRxiv2025

Burkholderia Genes Involved in Extracellular Bioplastic Degradation Revealed by Tn-seq and CRISPR-Cas

Bioplastics represent promising alternatives to petroleum-based plastics, yet their biodegradation remains insufficiently understood. Identifying bacteria capable of degrading bioplastics extracellularly could enhance end-of-life management practices. To investigate Burkholderias capacities for the degradation of medium-chain-length polyhydroxyalkanoate (mcl-PHA), we screened a panel of Burkholderia strains and identified such capacity in strains of B. gladioli, B. multivorans, and B. vietnamiensis. To elucidate the genetic basis of this activity, we performed transposon mutagenesis followed by activity-based screening and Tn-seq on B. vietnamiensis LMG 16232. Disrupted genetic elements in transposon mutants with negative phenotypes were further investigated using a CRISPR-associated transposase (CAST) system. These included a lipase production gene cluster, encoding two putative triacylglycerol lipases and a chaperone, and genes coding for a a A24 family peptidase, a TetR/AcrR family transcriptional regulator and a type II secretion system (T2SS) protein. Complete loss or reduced extracellular mcl-PHA depolymerase activity was observed in the CAST mutants, validating their involvement in mcl-PHA degradation. Notably, only one of the two lipases encoded in the lipase production gene cluster was responsible for mcl-PHA degradation, suggesting that while lipases may show substrate promiscuity, lipase functional annotation does not necessarily imply mcl-PHA depolymerization. Docking experiments using the amino acid sequences of the two lipases supported these findings. Together, we identify a gene coding for an active mcl-PHA depolymerase in B. vietnamiensis and demonstrate the power of combining activity-based screening, Tn-seq, and CAST to rapidly establish gene-to-function links. ImportanceDue to their versatile metabolism, Burkholderia strains play critical roles in degradation of multiple compounds in the environment. Here we show that several Burkholderia species can extracellularly degrade medium-chain-length polyhydroxyalkanoates (mcl-PHAs), a promising class of bioplastics. By integrating transposon mutagenesis, Tn-seq, and CRISPR-associated transposase (CAST) technologies, we identify and validate key genetic determinants involved in mcl-PHA degradation in B. vietnamiensis. These genes encode a lipase, a secretion system component, and regulatory factors, underscoring the complexity and specificity of microbial bioplastic degradation pathways. These findings not only advance our understanding of PHA biodegradation but also identifies B. vietnamiensis as as a source of enzymes capable of degrading extracellular mcl-PHA.

microbiology↗

bioRxiv2024

Cell envelope structural and functional contributions to antibiotic resistance in Burkholderia cenocepacia

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of the Burkholderia cepacia complex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants in B. cenocepacia K56-2, focusing on cell envelope structural and functional processes. We identified structures mechanistically important for resistance to singular and multiple antibiotic classes. For example, the polymeric O-antigen was important for resistance to cationic antibiotics, while defects in peptidoglycan precursor synthesis specifically increased susceptibility to cycloserine and revealed a new putative amino acid racemase. Susceptibility to novobiocin, avibactam, and the LpxC inhibitor, PF-04753299, was linked to the BpeAB-OprB efflux pump, suggesting these drugs are substrates for this pump in B. cenocepacia. Additionally, disruption of the periplasmic disulfide bond formation system caused pleiotropic defects on outer membrane integrity and {beta}-lactamase activity. Our findings highlight the layering of resistance mechanisms in the structure and function of the cell envelope. Consequently, we point out processes that can be targeted for developing antibiotic potentiators. ImportanceThe Gram-negative cell envelope is a double-layered physical barrier that protects cells from extracellular stressors, such as antibiotics. The Burkholderia cell envelope is known to contain additional modifications that reduce permeability. We investigated Burkholderia cell envelope factors contributing to antibiotic resistance from a genome-wide view by re-examining data from a transposon mutant library exposed to an antibiotic panel. We identified susceptible phenotypes for defects in structures and functions in the outer membrane, periplasm, and cytoplasm. Overall, we show that resistance linked to the cell envelope is multifaceted and provides new targets for the development of antibiotic potentiators.

microbiology↗

bioRxiv2022

Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium

The cell envelope of the Gram-negative Burkholderia cepacia complex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals, such as those living with cystic fibrosis. To systematically identify cell envelope-associated resistance and susceptibility determinants at the genome level, we constructed a high-density, randomly-barcoded transposon mutant library in the clinical isolate B. cenocepacia K56-2 and exposed it to a panel of more than twenty cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profiled over a hundred new functional associations and identified novel mediators of antibiotic susceptibility in the Bcc cell envelope. We revealed new connections between {beta}-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism, which highlight a vulnerability in sharing this lipid intermediate. We then show that the clinically relevant synergy of the {beta}-lactam/{beta}-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. Importantly, we found that avibactam more strongly potentiates the activity of aztreonam and meropenem than ceftazidime in a panel of Bcc clinical isolates. Finally, we characterize for first time in the Bcc the iron and receptor-dependent activity of the novel siderophore-cephalosporin antibiotic, cefiderocol. Overall, our work has implications for antibiotic target prioritization, and for using additional combinations of {beta}-lactam/{beta}-lactamase inhibitors that can extend the utility of our current clinical arsenal of antibacterial therapies. Author SummaryWhile the Gram-negative cell envelope is a major barrier to antibiotic action, we have an incomplete picture of how each component contributes to antibiotic resistance. To answer this question from a genome-wide perspective in a model of antibiotic-resistant bacteria, we constructed a library of randomly-barcoded transposon mutants in Burkholderia cenocepacia and exposed it to a panel of diverse cell envelope-targeting antibiotics. We identified individual genes and whole pathways associated with antibiotic resistance, including several novel players. Focusing on clinically relevant antibiotics, our strategy dissected how the synergy of the important {beta}-lactam/{beta}-lactamase inhibitor combination ceftazidime/avibactam primarily depends upon inhibition of a single {beta}-lactamase. Additionally, we are the first to characterise the molecular basis of the antibiotic mechanism of cefiderocol in Burkholderia, a new and very potent siderophore-cephalosporin conjugate drug. Encouragingly, we found that cefiderocol susceptibility was greatest at physiological iron concentrations. Overall, we highlight several avenues that can be taken to enhance or develop new therapeutic strategies against Burkholderia infection.

microbiology↗