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Methods in Enzymology 2024Structural biology research of terpene synthases (TSs) has provided a useful basis to understand their catalytic mechanisms in producing diverse terpene products with...
Structural biology research of terpene synthases (TSs) has provided a useful basis to understand their catalytic mechanisms in producing diverse terpene products with polycyclic ring systems and multiple chiral centers. However, compared to the large numbers of>95,000 terpenoids discovered to date, few structures of TSs have been solved and the understanding of their catalytic mechanisms is lagging. We here (i) introduce the basic catalytic logic, the structural architectures, and the metal-binding conserved motifs of TSs; (ii) provide detailed experimental procedures, in gene cloning and plasmid construction, protein purification, crystallization, X-ray diffraction data collection and structural elucidation, for structural biology research of TSs; and (iii) discuss the prospects of structure-based engineering and de novo design of TSs in generating valuable terpene molecules, which cannot be easily achieved by chemical synthesis.
Topics: Alkyl and Aryl Transferases; Crystallography, X-Ray; Terpenes; Cloning, Molecular; Models, Molecular; Protein Conformation
PubMed: 38942516
DOI: 10.1016/bs.mie.2024.03.012 -
Metabolic Engineering Jun 2024Vitamin B [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low...
Vitamin B [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel "PEACES" system by (1) expressing the NAD kinase gene ppnk from Clostridium glutamicum and the NADP-dependent gapC from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.
PubMed: 38942195
DOI: 10.1016/j.ymben.2024.06.006 -
Microbiological Research Jun 2024Plasmids orchestrate bacterial adaptation across diverse environments and facilitate lateral gene transfer within bacterial communities. Their presence can perturb host...
Plasmids orchestrate bacterial adaptation across diverse environments and facilitate lateral gene transfer within bacterial communities. Their presence can perturb host metabolism, creating a competitive advantage for plasmid-free cells. Plasmid stability hinges on efficient replication and partition mechanisms. While plasmids commonly encode histone-like nucleoid-structuring (H-NS) family proteins, the precise influence of plasmid-encoded H-NS proteins on stability remains elusive. In this study, we examined the conjugative plasmid pMBL6842, harboring the hns gene, and observed its positive regulation of parAB transcription, critical for plasmid segregation. Deletion of hns led to rapid plasmid loss, which was remedied by hns complementation. Further investigations unveiled adverse effects of hns overexpression on the bacterial host. Transcriptome analysis revealed hns's role in regulating numerous bacterial genes, impacting both host growth and swimming motility in the presence of the hns gene. Therefore, our study unveils the multifaceted roles of H-NS in both plasmid stability and host physiology, underscoring its biological significance and paving the way for future inquiries into the involvement of H-NS in horizontal gene transfer events.
PubMed: 38941922
DOI: 10.1016/j.micres.2024.127817 -
Water Research Jun 2024Waterborne pathogens are threatening public health globally, but profiling multiple human pathogenic bacteria (HPBs) in various polluted environments is still a...
Waterborne pathogens are threatening public health globally, but profiling multiple human pathogenic bacteria (HPBs) in various polluted environments is still a challenge due to the absence of rapid, high-throughput and accurate quantification tools. This work developed a novel chip, termed the HPB-Chip, based on high-throughput quantitative polymerase chain reactions (HT-qPCR). The HPB-Chip with 33-nL reaction volume could simultaneously complete 10,752 amplification reactions, quantifying 27 HPBs in up to 192 samples with two technical replicates (including those for generating standard curves). Specific positive bands of target genes across different species and single peak melting curves demonstrated high specificity of the HPB-Chip. The mixed plasmid serial dilution test validated its high sensitivity with the limit of quantification (LoD) of averaged 82 copies per reaction for 25 target genes. PCR amplification efficiencies and R coefficients of standard curves of the HPB-Chip averaged 101 % and 0.996, respectively. Moreover, a strong positive correlation (Pearson' r: 0.961-0.994, P < 0.001) of HPB concentrations (log copies/L) between HPB-Chip and conventional qPCR demonstrated high accuracy of the HPB-Chip. Subsequently, the HPB-Chip has been successfully applied to absolutely quantify 27 HPBs in municipal and hospital wastewater treatment plants (WWTPs) after PMA treatment. A total of 17 HPBs were detected in the 6 full-scale WWTPs, with an additional 19 in the hospital WWTP. Remarkably, Acinetobacter baumannii, Legionella pneumophila, and Arcobacter butzler were present in the final effluent of each municipal WWTP. Overall, the HPB-Chip is an efficient and accurate high-throughput quantification tool to comprehensively and rapidly quantify 27 HPBs in the environment.
PubMed: 38941866
DOI: 10.1016/j.watres.2024.121927 -
Communications Biology Jun 2024The Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system is a candidate 'virulence factor present in multiple pathogenic species of the Mollicutes, including the...
The Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system is a candidate 'virulence factor present in multiple pathogenic species of the Mollicutes, including the fast-growing species Mycoplasma feriruminatoris. The MIB-MIP system cleaves the heavy chain of host immunoglobulins, hence affecting antigen-antibody interactions and potentially facilitating immune evasion. In this work, using -omics technologies and 5'RACE, we show that the four copies of the M. feriruminatoris MIB-MIP system have different expression levels and are transcribed as operons controlled by four different promoters. Individual MIB-MIP gene pairs of M. feriruminatoris and other Mollicutes were introduced in an engineered M. feriruminatoris strain devoid of MIB-MIP genes and were tested for their functionality using newly developed oriC-based plasmids. The two proteins are functionally expressed at the surface of M. feriruminatoris, which confirms the possibility to display large membrane-associated proteins in this bacterium. However, functional expression of heterologous MIB-MIP systems introduced in this engineered strain from phylogenetically distant porcine Mollicutes like Mesomycoplasma hyorhinis or Mesomycoplasma hyopneumoniae could not be achieved. Finally, since M. feriruminatoris is a candidate for biomedical applications such as drug delivery, we confirmed its safety in vivo in domestic goats, which are the closest livestock relatives to its native host the Alpine ibex.
Topics: Bacterial Vaccines; Mycoplasma; Animals; Bacterial Proteins; Immunoglobulins; Gene Expression Regulation, Bacterial; Mycoplasma Infections; Goats
PubMed: 38942984
DOI: 10.1038/s42003-024-06497-8 -
Scientific Reports Jun 2024Microbial NAT enzymes, which employ acyl-CoA to acylate aromatic amines and hydrazines, have been well-studied for their role in xenobiotic metabolism. Some homologues...
Microbial NAT enzymes, which employ acyl-CoA to acylate aromatic amines and hydrazines, have been well-studied for their role in xenobiotic metabolism. Some homologues have also been linked to secondary metabolism, but this function of NAT enzymes is not as well-known. For this comparative study, we surveyed sequenced microbial genomes to update the list of formally annotated NAT genes, adding over 4000 new sequences (mainly bacterial, but also archaeal, fungal and protist) and portraying a broad but not universal distribution of NATs in the microbiocosmos. Localization of NAT sequences within microbial gene clusters was not a rare finding, and this association was evident across all main types of biosynthetic gene clusters (BGCs) implicated in secondary metabolism. Interrogation of the MIBiG database for experimentally characterized clusters with NAT genes further supports that secondary metabolism must be a major function for microbial NAT enzymes and should not be overlooked by researchers in the field. We also show that NAT sequences can be associated with bacterial plasmids potentially involved in horizontal gene transfer. Combined, our computational predictions and MIBiG literature findings reveal the extraordinary functional diversification of microbial NAT genes, prompting further research into their role in predicted BGCs with as yet uncharacterized function.
Topics: Multigene Family; Secondary Metabolism; Bacteria; Archaea; Phylogeny; Arylamine N-Acetyltransferase; Fungi; Genomics; Gene Transfer, Horizontal
PubMed: 38942826
DOI: 10.1038/s41598-024-65342-4 -
STAR Protocols Jun 2024A gene-rescue experiment under a mutant background is essential to clarify gene function and the resulting biological potential in vivo. Here, we present a protocol for...
A gene-rescue experiment under a mutant background is essential to clarify gene function and the resulting biological potential in vivo. Here, we present a protocol for determining the change in interferon response by microinjecting plasmids into one-cell-stage zebrafish embryos. We describe steps for comparing the resistance potential to virus infection in wild-type and knockout zebrafish larvae following plasmid microinjection. We then detail how to link the enhanced interferon immunity to the improved resistance in knockout zebrafish larvae by gene-rescue experiments. For complete details on the use and execution of this protocol, please refer to Qu et al..
PubMed: 38941183
DOI: 10.1016/j.xpro.2024.103156 -
The Journal of Experimental Biology Jun 2024The analysis of how neural circuits function in individuals and change during evolution is simplified by the existence of neurons identified as homologous within and...
The analysis of how neural circuits function in individuals and change during evolution is simplified by the existence of neurons identified as homologous within and across species. Invertebrates, including leeches, have been used for these purposes in part because their nervous systems comprise a high proportion of identified neurons, but technical limitations make it challenging to assess the full extent to which assumptions of stereotypy hold true. Here, we introduce Minos plasmid-mediated transgenesis as a tool for introducing transgenes into the embryos of the leech Helobdella austinensis (Spiralia; Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae). We identified an enhancer driving pan-neuronal expression of markers, including histone2B:mCherry, which allowed us to enumerate neurons in segmental ganglia. Unexpectedly, we find that the segmental ganglia of adult transgenic Helobdella contain fewer and more variable numbers of neurons than in previously examined leech species.
PubMed: 38940760
DOI: 10.1242/jeb.247419 -
Journal of Radiation Research Jun 2024The ionizing radiation with high linear energy transfer (LET), such as a heavy ion beam, induces more serious biological effects than low LET ones, such as gamma- and...
The ionizing radiation with high linear energy transfer (LET), such as a heavy ion beam, induces more serious biological effects than low LET ones, such as gamma- and X-rays. This indicates a difference in the DNA damage produced by low and high LET radiations and their biological effects. We have been studying the differences in DNA damage produced by gamma-rays and carbon ion beams. Therefore, we analyze mutations induced by both ionizing radiations to discuss the differences in their biological effects in this study. pUC19 plasmid DNA was irradiated by carbon ion beams in the solution containing 1M dimethyl sulfoxide to mimic a cellular condition. The irradiated DNA was cloned in competent cells of Escherichia coli. The clones harboring some mutations in the region of lacZα were selected, and the sequence alterations were analyzed. A one-deletion mutation is significant in the carbon-irradiated DNA, and the C:G↔T:A transition is minor. On the other hand, the gamma-irradiated DNA shows mainly G:C↔T:A transversion. These results suggest that carbon ion beams produce complex DNA damage, and gamma-rays are prone to single oxidative base damage, such as 8-oxoguanine. Carbon ion beams can also introduce oxidative base damage, and the damage species is 5-hydroxycytosine. This was consistent with our previous results of DNA damage caused by heavy ion beams. We confirmed the causal DNA damage by mass spectrometry for these mutations.
PubMed: 38940734
DOI: 10.1093/jrr/rrae050 -
Microbiology Resource Announcements Jun 2024sp. strain R1 was isolated from the stem of a tomato plant and exhibited antagonism with . The complete genome sequence of this bacterium consists of one 3,874,532 bp...
sp. strain R1 was isolated from the stem of a tomato plant and exhibited antagonism with . The complete genome sequence of this bacterium consists of one 3,874,532 bp circular chromosome and two plasmids.
PubMed: 38940606
DOI: 10.1128/mra.00242-24