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International Journal of Molecular... Aug 2023Numerous observations have supported the idea that various types of noncoding RNAs, including tRNA fragments (tRFs), are involved in communications between the host and...
Numerous observations have supported the idea that various types of noncoding RNAs, including tRNA fragments (tRFs), are involved in communications between the host and its microbial community. The possibility of using their signaling function has stimulated the study of secreted RNAs, potentially involved in the interspecies interaction of bacteria. This work aimed at identifying such RNAs and characterizing their maturation during transport. We applied an approach that allowed us to detect oligoribonucleotides secreted by () or inside cells. Four tRFs imported by cells co-cultured with these bacteria were obtained via chemical synthesis, and all of them affected the growth of . Their successive modifications in the culture medium and recipient cells were studied by high-throughput cDNA sequencing. Instead of the expected accidental exonucleolysis, in the milieu, we observed nonrandom cleavage by endonucleases continued in recipient cells. We also found intramolecular rearrangements of synthetic oligonucleotides, which may be considered traces of intermediate RNA circular isomerization. Using custom software, we estimated the frequency of such events in transcriptomes and secretomes of and observed surprising reproducibility in positions of such rare events, assuming the functionality of ring isoforms or their permuted derivatives in bacteria.
Topics: Escherichia coli; Introduced Species; Reproducibility of Results; RNA, Transfer; Culture Media; RNA
PubMed: 37629141
DOI: 10.3390/ijms241612960 -
Bioactive Materials Jul 2024Tumor metabolite regulation is intricately linked to cancer progression. Because lactate is a characteristic metabolite of the tumor microenvironment (TME), it supports...
Tumor metabolite regulation is intricately linked to cancer progression. Because lactate is a characteristic metabolite of the tumor microenvironment (TME), it supports tumor progression and drives immunosuppression. In this study, we presented a strategy for antitumor therapy by developing a nanogold-engineered (R.r-Au) that consumed lactate and produced hydrogen for optical biotherapy. We leveraged a cryogenic micromolding approach to construct a transdermal therapeutic cryomicroneedles (CryoMNs) patch integrated with R.r-Au to efficiently deliver living bacterial drugs. Our long-term storage studies revealed that the viability of R.r-Au in CryoMNs remained above 90%. We found that the CryoMNs patch was mechanically strong and could be inserted into mouse skin. In addition, it rapidly dissolved after administering bacterial drugs and did not produce by-products. Under laser irradiation, R.r-Au effectively enhanced electron transfer through Au NPs actuation into the photosynthetic system of and enlarged lactate consumption and hydrogen production, thus leading to an improved tumor immune activation. Our study demonstrated the potential of CryoMNs-R.r-Au patch as a minimally invasive delivery approach for living bacterial drugs. This research opens up new avenues for nanoengineering bacteria to transform tumor metabolites into effective substances for tumor optical biotherapy.
PubMed: 38698917
DOI: 10.1016/j.bioactmat.2024.03.032 -
Applied Microbiology and Biotechnology Mar 2024Environmental concerns about residues and the traditional disposal methods are driving the search for more environmentally conscious processes, such as pyrolysis and...
Environmental concerns about residues and the traditional disposal methods are driving the search for more environmentally conscious processes, such as pyrolysis and gasification. Their main final product is synthesis gas (syngas) composed of CO, CO, H, and methane. Syngas can be converted into various products using CO-tolerant microorganisms. Among them, Rhodospirillum rubrum is highlighted for its biotechnological potential. However, the extent to which high doses of CO affect its physiology is still opaque. For this reason, we have studied R. rubrum behavior under high levels of this gas (up to 2.5 bar), revealing a profound dependence on the presence or absence of light. In darkness, the key variable affected was the lag phase, where the highest levels of CO retarded growth to more than 20 days. Under light, R. rubrum ability to convert CO into CO and H depended on the presence of an additional carbon source, such as acetate. In those conditions where CO was completely exhausted, CO fixation was unblocked, leading to a diauxic growth. To enhance R. rubrum tolerance to CO in darkness, a UV-accelerated adaptive laboratory evolution (UVa-ALE) trial was conducted to isolate clones with shorter lag phases, resulting in the isolation of clones 1.4-2B and 1.7-2A. The adaptation of 1.4-2B was mainly based on mutated enzymes with a metabolic function, while 1.7-3A was mostly affected at regulatory genes, including the anti-repressor PpaA/AerR. Despite these mutations having slight effects on biomass and pigment levels, they successfully provoked a significant reduction in the lag phase (-50%). KEYPOINTS: • CO affects principally R. rubrum lag phase (darkness) and growth rate (light) • CO is converted to CO/H during acetate uptake and inhibits CO fixation (light) • UVa-ALE clones showed a 50% reduction in the lag phase (darkness).
Topics: Carbon Monoxide; Rhodospirillum rubrum; Carbon Dioxide; Acetates
PubMed: 38466440
DOI: 10.1007/s00253-024-13079-5 -
International Journal of Molecular... Mar 2024Circadian rhythms, characterized by approximately 24 h cycles, play a pivotal role in enabling various organisms to synchronize their biological activities with daily...
Circadian rhythms, characterized by approximately 24 h cycles, play a pivotal role in enabling various organisms to synchronize their biological activities with daily variations. While ubiquitous in Eukaryotes, circadian clocks remain exclusively characterized in among Prokaryotes. These rhythms are regulated by a core oscillator, which is controlled by a cluster of three genes: , , and . Interestingly, recent studies revealed rhythmic activities, potentially tied to a circadian clock, in other Prokaryotes, including purple bacteria such as , known for its applications in fuel and plastic bioproduction. However, the pivotal question of how light and dark cycles influence protein dynamics and the expression of putative circadian clock genes remains unexplored in purple non-sulfur bacteria. Unraveling the regulation of these molecular clocks holds the key to unlocking optimal conditions for harnessing the biotechnological potential of . Understanding how its proteome responds to different light regimes-whether under continuous light or alternating light and dark cycles-could pave the way for precisely fine-tuning bioproduction processes. Here, we report for the first time the expressed proteome of grown under continuous light versus light and dark cycle conditions using a shotgun proteomic analysis. In addition, we measured the impact of light regimes on the expression of four putative circadian clock genes (, , , ) at the transcriptional and translational levels using RT-qPCR and targeted proteomic (MRM-MS), respectively. The data revealed significant effects of light conditions on the overall differential regulation of the proteome, particularly during the early growth stages. Notably, several proteins were found to be differentially regulated during the light or dark period, thus impacting crucial biological processes such as energy conversion pathways and the general stress response. Furthermore, our study unveiled distinct regulation of the four genes at both the mRNA and protein levels in response to varying light conditions. Deciphering the impact of the diel cycle on purple bacteria not only enhances our understanding of their ecology but also holds promise for optimizing their applications in biotechnology, providing valuable insights into the origin and evolution of prokaryotic clock mechanisms.
Topics: Proteomics; Molecular Dynamics Simulation; Proteobacteria; Proteome; Circadian Rhythm; Circadian Clocks; Biotechnology; Bacterial Proteins
PubMed: 38474181
DOI: 10.3390/ijms25052934 -
MSystems Dec 2023vast metabolic versatility places it as a remarkable model bacterium and an excellent biotechnological chassis. The key component of photosynthesis (PS) studied in this...
vast metabolic versatility places it as a remarkable model bacterium and an excellent biotechnological chassis. The key component of photosynthesis (PS) studied in this work (HP1) stands out among the other members of PpaA/AerR anti-repressor family since it lacks the motif they all share: the cobalamin B-12 binding motif. Despite being reduced and poorly conserved, HP1 stills controls PS as the other members of the family, allowing a fast response to changes in the redox state of the cell. This work also shows that HP1 absence affects genes from relevant biological processes other than PS, including nitrogen fixation and stress response. From a biotechnological perspective, HP1 could be manipulated in approaches where PS is not necessary, such as hydrogen or polyhydroxyalkanoates production, to save energy.
Topics: Rhodospirillum rubrum; Photosynthesis; Oxidation-Reduction; Bacteria; Chromosomal Proteins, Non-Histone
PubMed: 38054698
DOI: 10.1128/msystems.00702-23 -
Protein Science : a Publication of the... Apr 2024L-Asparaginases (ASNases) catalyze the hydrolysis of L-Asn to L-Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as...
L-Asparaginases (ASNases) catalyze the hydrolysis of L-Asn to L-Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300-400 amino acids (typical class 1 ASNases). In contrast, the chain of ASNase from Rhodospirillum rubrum, reported here and referred to as RrA, consists of only 172 amino acid residues. RrA is homologous to the N-terminal domain of typical bacterial class 1 ASNases and exhibits millimolar affinity for L-Asn. In this study, we demonstrate that RrA belongs to a unique family of cytoplasmic, short-chain ASNases (scASNases). These proteins occupy a distinct region in the sequence space, separate from the regions typically assigned to class 1 ASNases. The scASNases are present in approximately 7% of eubacterial species, spanning diverse bacterial lineages. They seem to be significantly enriched in species that encode for more than one class 1 ASNase. Here, we report biochemical, biophysical, and structural properties of RrA, a member of scASNases family. Crystal structures of the wild-type RrA, both with and without bound L-Asp, as well as structures of several RrA mutants, reveal topologically unique tetramers. Moreover, the active site of one protomer is complemented by two residues (Tyr21 and Asn26) from another protomer. Upon closer inspection, these findings clearly outline scASNases as a stand-alone subfamily of ASNases that can catalyze the hydrolysis of L-Asn to L-Asp despite the lack of the C-terminal domain that is present in all ASNases described structurally to date.
Topics: Asparaginase; Rhodospirillum rubrum; Protein Subunits; Aspartic Acid; Catalytic Domain
PubMed: 38501449
DOI: 10.1002/pro.4920 -
ELife Jan 2024Bactofilins have emerged as a widespread family of cytoskeletal proteins with important roles in bacterial morphogenesis, but their precise mode of action is still...
Bactofilins have emerged as a widespread family of cytoskeletal proteins with important roles in bacterial morphogenesis, but their precise mode of action is still incompletely understood. In this study, we identify the bactofilin cytoskeleton as a key regulator of cell growth in the stalked budding alphaproteobacterium . We show that, in this species, bactofilin polymers localize dynamically to the stalk base and the bud neck, with their absence leading to unconstrained growth of the stalk and bud compartments, indicating a central role in the spatial regulation of cell wall biosynthesis. Database searches reveal that bactofilin genes are often clustered with genes for cell wall hydrolases of the M23 peptidase family, suggesting a functional connection between these two types of proteins. In support of this notion, we find that the M23 peptidase homolog LmdC interacts directly with bactofilin in vitro and is required for proper cell shape in vivo. Complementary studies in the spiral-shaped alphaproteobacterium again reveal a close association of its bactofilin and LmdC homologs, which co-localize at the inner curve of the cell, modulating the degree of cell curvature. Collectively, these findings demonstrate that bactofilins and M23 peptidases form a conserved functional module that promotes local changes in the mode of cell wall biosynthesis, thereby driving cell shape determination in morphologically complex bacteria.
Topics: Bacterial Proteins; Endopeptidases; Cytoskeleton; Bacteria; Cytoskeletal Proteins
PubMed: 38294932
DOI: 10.7554/eLife.86577 -
Frontiers in Bioengineering and... 2023Designing cell factories for the production of novel polyhydroxyalkanoates (PHAs) via smart metabolic engineering is key to obtain materials with tailored...
Designing cell factories for the production of novel polyhydroxyalkanoates (PHAs) via smart metabolic engineering is key to obtain materials with tailored physicochemical properties. To this end, we used the model medium-chain-length-PHA producing bacterium, KT2440 as a chassis, which is characterized by its metabolic versatility and stress tolerance. Different PHA biosynthetic modules were assembled in expression plasmids using the Golden gate/MoClo modular assembly technique to implement an orthogonal short-chain-lengh-PHA (scl-PHA) switch in a "deaf" PHA mutant. This was specifically constructed to override endogenous multilevel regulation of PHA synthesis in the native strain. We generated a panel of engineered approaches carrying the genes from and demonstrating that diverse scl-PHAs can be constitutively produced in the chassis strain to varying yields from 23% to 84% PHA/CDW. Co-feeding assays of the most promising engineered strain harboring the PHA machinery from resulted to a panel of PHBV from 0.6% to 19% C5 monomeric incorporation. Chromosomally integrated PHA machineries with high PhaC synthase dosage successfully resulted in 68% PHA/CDW production. Interestingly, an inverse relationship between PhaC synthase dosage and granule size distribution was demonstrated in the heterologous host. In this vein, it is proposed the key involvement of inclusion body protein IbpA to the heterologous production of tailored PHA in KT2440.
PubMed: 38026847
DOI: 10.3389/fbioe.2023.1275036