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  • 1
    Online Resource
    Online Resource
    Elsevier BV ; 2009
    In:  Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics Vol. 1794, No. 11 ( 2009-11), p. 1709-1714
    In: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Elsevier BV, Vol. 1794, No. 11 ( 2009-11), p. 1709-1714
    Type of Medium: Online Resource
    ISSN: 1570-9639
    RVK:
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2009
    detail.hit.zdb_id: 2209540-8
    SSG: 12
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  • 2
    Online Resource
    Online Resource
    Japan Petroleum Institute ; 2013
    In:  Journal of the Japan Petroleum Institute Vol. 56, No. 5 ( 2013), p. 267-279
    In: Journal of the Japan Petroleum Institute, Japan Petroleum Institute, Vol. 56, No. 5 ( 2013), p. 267-279
    Type of Medium: Online Resource
    ISSN: 1346-8804 , 1349-273X
    Language: English
    Publisher: Japan Petroleum Institute
    Publication Date: 2013
    detail.hit.zdb_id: 2152070-7
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  • 3
    Online Resource
    Online Resource
    American Society for Microbiology ; 2021
    In:  Applied and Environmental Microbiology Vol. 87, No. 12 ( 2021-05-26)
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 87, No. 12 ( 2021-05-26)
    Abstract: The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes are unique features of certain marine Gammaproteobacteria inhabiting high-pressure and/or low-temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA; 20:5 n- 3) and docosahexaenoic acid (DHA; 22:6 n- 3) are produced by a hybrid polyketide/fatty acid synthase—encoded by the pfa genes—also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by severalfold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD coding sequence, which encodes malonyl coenzyme A (malonyl-CoA) acyl carrier protein transacylase. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo . The co-occurrence of independent, yet compensatory, fatty acid biosynthetic pathways in selected marine bacteria may provide genetic redundancy to optimize fitness under extreme conditions. IMPORTANCE A defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a genetic approach, new insights into the interactions between these two biosynthetic pathways have been gained. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be nonessential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2021
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 4
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 87, No. 12 ( 2021-05-26)
    Abstract: Escherichia coli contains 12 chaperone-usher operons for biosynthesis and assembly of various fimbriae. In this study, each of the 12 operons was deleted in E. coli MG1655, and the resulting 12 deletion mutants all grew better than the wild type, especially in the nutrient-deficient M9 medium. When the plasmid pBHR68 containing the key genes for polyhydroxyalkanoate production was introduced into these 12 mutants, each mutant synthesized more polyhydroxyalkanoate than the wild-type control. These results indicate that the fimbria removal in E. coli benefits cell growth and polyhydroxyalkanoate production. Therefore, all 12 chaperone-usher operons, including 64 genes, were deleted in MG1655, resulting in the fimbria-lacking strain WQM026. WQM026 grew better than MG1655, and no fimbria structures were observed on the surface of WQM026 cells. Transcriptomic analysis showed that in WQM026 cells, the genes related to glucose consumption, glycolysis, flagellar synthesis, and biosynthetic pathways of some key amino acids were upregulated, while the tricarboxylic acid cycle-related genes were downregulated. When pBHR68 was introduced into WQM026, huge amounts of poly-3-hydroxybutyrate were produced; when the plasmid pFW01 -thrA*BC-rhtC , containing the key genes for l -threonine biosynthesis and transport, was transferred into WQM026, more l -threonine was synthesized than with the control. These results suggest that this fimbria-lacking E. coli WQM026 is a good host for efficient production of polyhydroxyalkanoate and l -threonine and has the potential to be developed into a valuable chassis microorganism. IMPORTANCE In this study, we investigated the interaction between the biosynthesis and assembly of fimbriae and intracellular metabolic networks in E. coli . We found that eliminating fimbriae could effectively improve the production of polyhydroxyalkanoate and l -threonine in E. coli MG1655. These results contribute to understanding the necessity of fimbriae and the advantages of fimbria removal for industrial microorganisms. The knowledge gathered from this study may be applied to the development of superior chassis microorganisms.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2021
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 9 ( 2022-05-10)
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 9 ( 2022-05-10)
    Abstract: Isopropanol dehydrogenase (IPADH) is one of the most attractive options for nicotinamide cofactor regeneration due to its low cost and simple downstream processing. However, poor thermostability and strict cofactor dependency hinder its practical application for bioconversions. In this study, we simultaneously improved the thermostability (433-fold) and catalytic activity (3.3-fold) of IPADH from Brucella suis via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H) by 1.23 × 10 6 -fold. When these variants were employed in three typical bioredox reactions to drive the synthesis of important chiral pharmaceutical building blocks, they outperformed the commonly used cofactor regeneration systems (glucose dehydrogenase [GDH], formate dehydrogenase [FDH] , and lactate dehydrogenase [LDH]) with respect to efficiency of cofactor regeneration. Overall, our study provides two promising IPADH variants with complementary cofactor specificities that have great potential for wide applications. IMPORTANCE Oxidoreductases represent one group of the most important biocatalysts for synthesis of various chiral synthons. However, their practical application was hindered by the expensive nicotinamide cofactors used. Isopropanol dehydrogenase (IPADH) is one of the most attractive biocatalysts for nicotinamide cofactor regeneration. However, poor thermostability and strict cofactor dependency hinder its practical application. In this work, the thermostability and catalytic activity of an IPADH were simultaneously improved via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H). The resultant variants show great potential for regeneration of nicotinamide cofactors, and the engineering strategy might serve as a useful approach for future engineering of other oxidoreductases.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 6
    Online Resource
    Online Resource
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 13 ( 2022-07-12)
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 13 ( 2022-07-12)
    Abstract: Lipoic acid is an organosulfur cofactor essential for several key enzyme complexes in oxidative and one-carbon metabolism. It is covalently bound to the lipoyl domain of the E2 subunit in some 2-oxoacid dehydrogenase complexes and the H-protein in the glycine cleavage system. Lipoate-protein ligase (Lpl) is involved in the salvage of exogenous lipoate and attaches free lipoate to the E2 subunit or the H-protein in an ATP-dependent manner. In the hyperthermophilic archaeon Thermococcus kodakarensis , TK1234 and TK1908 are predicted to encode the N- and C-terminal regions of Lpl, respectively. TK1908 and TK1234 recombinant proteins form a heterodimer and together displayed significant ligase activity toward octanoate in addition to lipoate when a chemically synthesized octapeptide was used as the acceptor. The proteins also displayed activity toward other fatty acids, indicating broad fatty acid specificity. On the other hand, lipoyl synthase from T. kodakarensis only recognized octanoyl-peptide as a substrate. Examination of individual proteins indicated that the TK1908 protein alone was able to catalyze the ligase reaction although with a much lower activity. Gene disruption of TK1908 led to lipoate/serine auxotrophy, whereas TK1234 gene deletion did not. Acyl carrier protein homologs are not found on the archaeal genomes, and the TK1908/TK1234 protein complex did not utilize octanoyl-CoA, raising the possibility that the substrate of the ligase reaction is octanoic acid itself. Although Lpl has been considered as an enzyme involved in lipoate salvage, the results imply that in T. kodakarensis , the TK1908 and TK1234 proteins function in de novo lipoyl-protein biosynthesis. IMPORTANCE Based on previous studies in bacteria and eukaryotes, lipoate-protein ligases (Lpls) have been considered to be involved exclusively in lipoate salvage. The genetic analyses in this study on the lipoate-protein ligase in T. kodakarensis , however, suggest otherwise and that the enzyme is additionally involved in de novo protein lipoylation. We also provide biochemical evidence that the lipoate-protein ligase displays broad substrate specificity and is capable of ligating acyl groups of various chain-lengths to the peptide substrate. We show that this apparent ambiguity in Lpl is resolved by the strict substrate specificity of the lipoyl synthase LipS in this organism, which only recognizes octanoyl-peptide. The results provide relevant physiological insight into archaeal protein lipoylation.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 7
    Online Resource
    Online Resource
    American Society for Microbiology ; 2020
    In:  Applied and Environmental Microbiology Vol. 86, No. 23 ( 2020-11-10)
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 86, No. 23 ( 2020-11-10)
    Abstract: The compositions of Octopus Spring and Mushroom Spring (Yellowstone National Park, Wyoming, USA) microbial mats have been thoroughly studied, but the compositions of the effluent waters that flow above the mats have not. In this study, cells in the mats and overflowing waters of both springs were investigated at multiple sites where Synechococcus spp. are the dominant cyanobacteria (ca. 72°C to ca. 50°C), and on several dates. In addition to microscopic analyses of stained and autofluorescent cells, 16S rRNA gene sequencing was used to characterize the major taxa present and a protein-encoding gene ( psaA ) was sequenced and analyzed by ecotype simulation to predict species of Synechococcus . The mats of both springs were similar in terms of the downstream distribution of predominant taxa detected previously. However, waters above these mats were predominated by taxa that reside in upstream mats or communities above the upper-temperature limit of the mat. A disturbance/recolonization study was performed at a site normally predominated by Synechococcus species adapted to low temperatures. After removing indigenous Synechococcus cells, Synechococcus species adapted to higher temperatures, which were predominant in the water overflowing this site, colonized the newly forming mat. Differences in recolonization under reduced and UV-screened irradiance suggested that, in addition to physical transport, environmental conditions likely select for species that are better adapted to these different conditions and can influence mat recovery. A transport model was developed and used to predict that, in Mushroom Spring, erosion predominates in the narrower and deeper upstream effluents and deposition predominates over erosion in wider and shallower downstream effluents. IMPORTANCE In flowing aquatic systems, cell erosion and deposition are important to the dispersal of cells from one location to another. Very little is known about microbial dispersal and the physical processes that underlie it. This study demonstrates its importance to colonization of downstream surfaces and especially to the recolonization and functioning of disturbed sites. Ecological systems in flowing environments are often, roughly speaking, pseudosteady, in that nutrients enter the system and by-products leave at relatively steady rates. Over time, material inputs and outputs must balance. Measurements of input fluxes (e.g., growth rates and proxies, such as photosynthesis rates) are frequent. However, erosion and deposition of cells are seldom measured and ecological significance is sometimes neglected. The importance of these parameters is immediately evident in any attempt to construct a model of long-time community behavior, as spatial ecological structure is significantly impacted and can be dominated by migration of organisms, even in small numbers.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2020
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 8
    Online Resource
    Online Resource
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 3 ( 2022-02-08)
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 3 ( 2022-02-08)
    Abstract: Single-cell isolation and cultivation play an important role in studying physiology, gene expression, and functions of microorganisms. A series of single-cell isolation technologies have been developed, among which single-cell ejection technology is one of the most promising. Single-cell ejection technology has applied laser-induced forward transfer (LIFT) techniques to isolate bacteria, but the viability (or recovery rate) of cells after sorting has not been clarified in current research. In this work, to keep the cells alive as long as possible, we propose a three-layer LIFT system (top layer, 25-nm aluminum film; second layer, 3 μm agar media; third layer, liquid containing bacteria) for the isolation and cultivation of single Gram-negative ( Escherichia coli ), Gram-positive ( Lactobacillus rhamnosus GG [LGG]), and eukaryotic ( Saccharomyces cerevisiae ) microorganisms. The experiment results showed that the average survival rates for ejected pure single cells were 63% for Saccharomyces cerevisiae , 22% for E. coli DH5α, and 74% for LGG. In addition, we successfully isolated and cultured the green fluorescent protein (GFP)-expressing E. coli JM109 from a mixture containing complex communities of soil bacteria by fluorescence signal. The average survival rate of E. coli JM109 was demonstrated to be 25.3%. In this study, the isolated and cultured single colonies were further confirmed by colony PCR and sequencing. Such precise sorting and cultivation techniques of live single microbial cells could be coupled with other microscopic approaches to isolate single microorganisms with specific functions, revealing their roles in the natural community. IMPORTANCE We developed a laser-induced forward transfer (LIFT) technology to accurately isolate single live microbial cells. The cultivation recovery rates of the ejected single cells were 63% for Saccharomyces cerevisiae , 22% for E. coli DH5α, and 74% for Lactobacillus rhamnosus GG (LGG). With coupled LIFT with a fluorescence microscope, we demonstrated that single cells of GFP-expressing E. coli JM109 were sorted according to fluorescence signal from a complex community of soil bacteria and subsequently cultured with 25% cultivation recovery rate. This single-cell live sorting technology could isolate single microbes with specific functions, revealing their roles in the natural community.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 9
    Online Resource
    Online Resource
    American Society for Microbiology ; 2023
    In:  Applied and Environmental Microbiology Vol. 89, No. 3 ( 2023-03-29)
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 89, No. 3 ( 2023-03-29)
    Abstract: Microorganisms have successfully predominated deep-sea ecosystems, while we know little about their adaptation strategy to multiple environmental stresses therein, including high hydrostatic pressure (HHP). Here, we focused on the genus Halomonas , one of the most widely distributed halophilic bacterial genera in marine ecosystems and isolated a piezophilic strain Halomonas titanicae ANRCS81 from Antarctic deep-sea sediment. The strain grew under a broad range of temperatures (2 to 45°C), pressures (0.1 to 55 MPa), salinities (NaCl, 0.5 to 17.5%, wt/vol), and chaotropic agent (Mg 2+ , 0 to 0.9 M) with either oxygen or nitrate as an electron acceptor. Genome annotation revealed that strain ANRCS81 expressed potential antioxidant genes/proteins and possessed versatile energy generation pathways. Based on the transcriptomic analysis, when the strain was incubated at 40 MPa, genes related to antioxidant defenses, anaerobic respiration, and fermentation were upregulated, indicating that HHP induced intracellular oxidative stress. Under HHP, superoxide dismutase (SOD) activity increased, glucose consumption increased with less CO 2 generation, and nitrate/nitrite consumption increased with more ammonium generation. The cellular response to HHP represents the common adaptation developed by Halomonas to inhabit and drive geochemical cycling in deep-sea environments. IMPORTANCE Microbial growth and metabolic responses to environmental changes are core aspects of adaptation strategies developed during evolution. In particular, high hydrostatic pressure (HHP) is the most common but least examined environmental factor driving microbial adaptation in the deep sea. According to recent studies, microorganisms developed a common adaptation strategy to multiple stresses, including HHP, with antioxidant defenses and energy regulation as key components, but experimental data are lacking. Meanwhile, cellular SOD activity is elevated under HHP. The significance of this research lies in identifying the HHP adaptation strategy of a Halomonas strain at the genomic, transcriptomic, and metabolic activity levels, which will allow researchers to bridge environmental factors with the ecological function of marine microorganisms.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 10
    Online Resource
    Online Resource
    American Society for Microbiology ; 2020
    In:  Applied and Environmental Microbiology Vol. 86, No. 21 ( 2020-10-15)
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 86, No. 21 ( 2020-10-15)
    Abstract: CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N 2 ) fixation were targeted for dCas9-mediated repression. First, the nif operon ( nifHI 1 I 2 DKEN ) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD . Remarkably, growth of M. acetivorans with N 2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was 〉 90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance 〉 85% and impaired but did not abolish growth of M. acetivorans with N 2 . Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1 , encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N 2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans . IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    RVK:
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2020
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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