scholarly journals Biodegradation of the Organic Disulfide 4,4′-Dithiodibutyric Acid by Rhodococcus spp.

2015 ◽  
Vol 81 (24) ◽  
pp. 8294-8306 ◽  
Author(s):  
Heba Khairy ◽  
Jan Hendrik Wübbeler ◽  
Alexander Steinbüchel
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Rhodococcus Erythropolis ◽  
Model Organism ◽  
Hypothetical Protein ◽  
Gene Encoding ◽  
Content Type ◽  
Gene Coding ◽  
Initial Cleavage ◽  
Genes Encoding

ABSTRACTFourRhodococcusspp. exhibited the ability to use 4,4′-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. BecauseRhodococcus erythropolisstrain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis ofR. erythropolisMI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity ofnoxRandnox. The interruption mutant generated,R. erythropolisMI2noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently,noxwas overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB).

scholarly journals Hidden resources in the Escherichia coli genome restore PLP synthesis and robust growth after deletion of the essential gene pdxB

2019 ◽  
Vol 116 (48) ◽  
pp. 24164-24173 ◽  
Author(s):  
Juhan Kim ◽  
Jake J. Flood ◽  
Michael R. Kristofich ◽  
Cyrus Gidfar ◽  
Andrew B. Morgenthaler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Essential Gene ◽  
Alternative Pathway ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Phosphoglycerate Dehydrogenase ◽  
Functional Pathway

PdxB (erythronate 4-phosphate dehydrogenase) is expected to be required for synthesis of the essential cofactor pyridoxal 5′-phosphate (PLP) in Escherichia coli. Surprisingly, incubation of the ∆pdxB strain in medium containing glucose as a sole carbon source for 10 d resulted in visible turbidity, suggesting that PLP is being produced by some alternative pathway. Continued evolution of parallel lineages for 110 to 150 generations produced several strains that grow robustly in glucose. We identified a 4-step bypass pathway patched together from promiscuous enzymes that restores PLP synthesis in strain JK1. None of the mutations in JK1 occurs in a gene encoding an enzyme in the new pathway. Two mutations indirectly enhance the ability of SerA (3-phosphoglycerate dehydrogenase) to perform a new function in the bypass pathway. Another disrupts a gene encoding a PLP phosphatase, thus preserving PLP levels. These results demonstrate that a functional pathway can be patched together from promiscuous enzymes in the proteome, even without mutations in the genes encoding those enzymes.

scholarly journals CAU-1, a Subclass B3 Metallo-β-Lactamase of Low Substrate Affinity Encoded by an Ortholog Present in the Caulobacter crescentus Chromosome

2002 ◽  
Vol 46 (6) ◽  
pp. 1823-1830 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Fabrizio Pantanella ◽  
Francesco Giuliani ◽  
Maria Cristina Thaller ◽  
Gianfranco Amicosante ◽  
...  
Keyword(s):  
Caulobacter Crescentus ◽  
Hypothetical Protein ◽  
Exchange Chromatography ◽  
Substrate Affinity ◽  
Gene Encoding ◽  
Native Form ◽  
Significant Similarity ◽  
Expression Studies ◽  
Genes Encoding ◽  
Isoelectric Ph

ABSTRACT The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-β-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C. crescentus type strain DSM4727. Expression studies confirmed the metallo-β-lactamase activity of its product, CAU-1. The enzyme produced in E. coli was purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (>9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various β-lactams were poor overall (Km values were always >100 μM and often >400 μM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. In C. crescentus, the CAU-1 enzyme is produced independently of β-lactam exposure and, interestingly, the bla CAU determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-β-lactamase in a member of the α subdivision of the class Proteobacteria.

scholarly journals Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

2013 ◽  
Vol 79 (18) ◽  
pp. 5566-5575 ◽  
Author(s):  
Jens Buchholz ◽  
Andreas Schwentner ◽  
Britta Brunnenkan ◽  
Christina Gabris ◽  
Simon Grimm ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Fed Batch ◽  
Complex Activity ◽  
Gene Encoding ◽  
Content Type ◽  
Genes Encoding ◽  
Cell Densities ◽  
Dehydrogenase Complex

ABSTRACTExchange of the nativeCorynebacterium glutamicumpromoter of theaceEgene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutateddapApromoter variants led to a series ofC. glutamicumstrains with gradually reduced growth rates and PDHC activities. Upon overexpression of thel-valine biosynthetic genesilvBNCE, all strains producedl-valine. Among these strains,C. glutamicum aceEA16 (pJC4ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of thepqoandppcgenes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities,C. glutamicum aceEA16 Δpqo Δppc(pJC4ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter)l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression ofilvBNCDinstead ofilvBNCEtransformed thel-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with aYP/Sof 0.24 mol per mol of glucose and aQPof 6.9 mM per h [0.8 g/(liter × h)]. The replacement of theaceEpromoter by thedapA-A16 promoter in the twoC. glutamicuml-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate thatC. glutamicumstrains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

scholarly journals Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

2010 ◽  
Vol 76 (9) ◽  
pp. 2884-2894 ◽  
Author(s):  
Efraín Manilla-Pérez ◽  
Alvin Brian Lange ◽  
Stephan Hetzler ◽  
Marc Wältermann ◽  
Rainer Kalscheuer ◽  
...  
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Marine Bacterium ◽  
Neutral Lipids ◽  
Nile Red ◽  
Dry Weight ◽  
Wax Ester ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Key Enzyme

ABSTRACT In many microorganisms, the key enzyme responsible for catalyzing the last step in triacylglycerol (TAG) and wax ester (WE) biosynthesis is an unspecific acyltransferase which is also referred to as wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT; AtfA). The importance and function of two AtfA homologues (AtfA1 and AtfA2) in the biosynthesis of TAGs and WEs in the hydrocarbon-degrading marine bacterium Alcanivorax borkumensis SK2 have been described recently. However, after the disruption of both the AtfA1 and AtfA2 genes, reduced but substantial accumulation of TAGs was still observed, indicating the existence of an alternative TAG biosynthesis pathway. In this study, transposon-induced mutagenesis was applied to an atfA1 atfA2 double mutant to screen for A. borkumensis mutants totally defective in biosynthesis of neutral lipids in order to identify additional enzymes involved in the biosynthesis of these lipids. At the same time, we have searched for a totally TAG-negative mutant in order to study the function of TAGs in A. borkumensis. Thirteen fluorescence-negative mutants were identified on Nile red ONR7a agar plates and analyzed for their abilities to synthesize lipids. Among these, mutant 2 M131 was no longer able to synthesize and accumulate TAGs if pyruvate was used as the sole carbon source. The transposon insertion was localized in a gene encoding a putative cytochrome c family protein (ABO_1185). Growth and TAG accumulation experiments showed that the disruption of this gene resulted in the absence of TAGs in 2 M131 but that growth was not affected. In cells of A. borkumensis SK2 grown on pyruvate as the sole carbon source, TAGs represented about 11% of the dry weight of the cells, while in the mutant 2 M131, TAGs were not detected by thin-layer and gas chromatography analyses. Starvation and lipid mobilization experiments revealed that the lipids play an important role in the survival of the cells. The function of neutral lipids in A. borkumensis SK2 is discussed.

scholarly journals Mapping of the Denitrification Pathway in Burkholderia thailandensis by Genome-Wide Mutant Profiling

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Alessandra Vitale ◽  
Sarah Paszti ◽  
Kohei Takahashi ◽  
Masanori Toyofuku ◽  
Gabriella Pessi ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Burkholderia Pseudomallei ◽  
Anaerobic Growth ◽  
Gene Encoding ◽  
Terminal Electron Acceptor ◽  
Burkholderia Thailandensis ◽  
Content Type ◽  
Sufficient Energy ◽  
Genes Encoding ◽  
Human Infections

ABSTRACT Burkholderia thailandensis is a soil saprophyte that is closely related to the pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans. The environmental niches and infection sites occupied by these bacteria are thought to contain only limited concentrations of oxygen, where they can generate energy via denitrification. However, knowledge of the underlying molecular basis of the denitrification pathway in these bacteria is scarce. In this study, we employed a transposon sequencing (Tn-Seq) approach to identify genes conferring a fitness benefit for anaerobic growth of B. thailandensis. Of the 180 determinants identified, several genes were shown to be required for growth under denitrifying conditions: the nitrate reductase operon narIJHGK2K1, the aniA gene encoding a previously unknown nitrite reductase, and the petABC genes encoding a cytochrome bc1, as well as three novel regulators that control denitrification. Our Tn-Seq data allowed us to reconstruct the entire denitrification pathway of B. thailandensis and shed light on its regulation. Analyses of growth behaviors combined with measurements of denitrification metabolites of various mutants revealed that nitrate reduction provides sufficient energy for anaerobic growth, an important finding in light of the fact that some pathogenic Burkholderia species can use nitrate as a terminal electron acceptor but are unable to complete denitrification. Finally, we demonstrated that a nitrous oxide reductase mutant is not affected for anaerobic growth but is defective in biofilm formation and accumulates N2O, which may play a role in the dispersal of B. thailandensis biofilms. IMPORTANCE Burkholderia thailandensis is a soil-dwelling saprophyte that is often used as surrogate of the closely related pathogen Burkholderia pseudomallei, the causative agent of melioidosis and a classified biowarfare agent. Both organisms are adapted to grow under oxygen-limited conditions in rice fields by generating energy through denitrification. Microoxic growth of B. pseudomallei is also considered essential for human infections. Here, we have used a Tn-Seq approach to identify the genes encoding the enzymes and regulators required for growth under denitrifying conditions. We show that a mutant that is defective in the conversion of N2O to N2, the last step in the denitrification process, is unaffected in microoxic growth but is severely impaired in biofilm formation, suggesting that N2O may play a role in biofilm dispersal. Our study identified novel targets for the development of therapeutic agents to treat meliodiosis.

scholarly journals The Catabolite Repressor/Activator Cra Is a Bridge Connecting Carbon Metabolism and Host Colonization in the Plant Drought Resistance-Promoting Bacterium Pantoea alhagi LTYR-11Z

2018 ◽  
Vol 84 (13) ◽  
Author(s):  
Lei Zhang ◽  
Muhang Li ◽  
Qiqi Li ◽  
Chaoqiong Chen ◽  
Meng Qu ◽  
...  
Keyword(s):  
Carbon Source ◽  
Carbon Metabolism ◽  
Carbon Sources ◽  
Endophytic Bacterium ◽  
Bacterial Attachment ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Host Colonization ◽  
Content Type

ABSTRACT Efficient root colonization is a prerequisite for application of plant growth-promoting (PGP) bacteria in improving health and yield of agricultural crops. We have recently identified an endophytic bacterium, Pantoea alhagi LTYR-11Z, with multiple PGP properties that effectively colonizes the root system of wheat and improves its growth and drought tolerance. To identify novel regulatory genes required for wheat colonization, we screened an LTYR-11Z transposon (Tn) insertion library and found cra to be a colonization-related gene. By using transcriptome (RNA-seq) analysis, we found that transcriptional levels of an eps operon, the ydiV gene encoding an anti-FlhD 4 C 2 factor, and the yedQ gene encoding an enzyme for synthesis of cyclic dimeric GMP (c-di-GMP) were significantly downregulated in the Δ cra mutant. Further studies demonstrated that Cra directly binds to the promoters of the eps operon, ydiV , and yedQ and activates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production and biofilm formation. Consistent with previous findings that Cra plays a role in transcriptional regulation in response to carbon source availability, the activating effects of Cra were much more pronounced when LTYR-11Z was grown within a gluconeogenic environment than when it was grown within a glycolytic environment. We further demonstrate that the ability of LTYR-11Z to colonize wheat roots is modulated by the availability of carbon sources. Altogether, these results uncover a novel strategy utilized by LTYR-11Z to achieve host colonization in response to carbon nutrition in the environment, in which Cra bridges a connection between carbon metabolism and colonization capacity of LTYR-11Z. IMPORTANCE Rapid and appropriate response to environmental signals is crucial for bacteria to adapt to competitive environments and to establish interactions with their hosts. Efficient colonization and persistence within the host are controlled by various regulatory factors that respond to specific environmental cues. The most common is nutrient availability. In this work, we unraveled the pivotal role of Cra in regulation of colonization ability of Pantoea alhagi LTYR-11Z in response to carbon source availability. Moreover, we identified three novel members of the Cra regulon involved in EPS synthesis, regulation of flagellar biosynthesis, and synthesis of c-di-GMP and propose a working model to explain the Cra-mediated regulatory mechanism that links carbon metabolism to host colonization. This study elucidates the regulatory role of Cra in bacterial attachment and colonization of plants, which raises the possibility of extending our studies to other bacteria associated with plant and human health.

scholarly journals Transferable Resistance Gene optrA in Enterococcus faecalis from Swine in Brazil

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Lara M. Almeida ◽  
François Lebreton ◽  
Anthony Gaca ◽  
Paulo M. Bispo ◽  
Jose T. Saavedra ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Enterococcus Faecalis ◽  
Sequence Comparison ◽  
Hypothetical Protein ◽  
Sequence Type ◽  
Gene Encoding ◽  
Content Type ◽  
Nursery Pigs ◽  
Closed Circle ◽  
Swine Herds

ABSTRACT OptrA is an ATP-binding cassette (ABC)-F protein that confers resistance to oxazolidinones and phenicols and can be either plasmid-encoded or chromosomally encoded. Here, we isolated 13 Enterococcus faecalis strains possessing a linezolid MIC of ≥4 mg/liter from nursery pigs in swine herds located across Brazil. Genome sequence comparison showed that these strains possess optrA in different genetic contexts occurring in 5 different E. faecalis sequence type backgrounds. The optrA gene invariably occurred in association with an araC regulator and a gene encoding a hypothetical protein. In some contexts, this genetic island was able to excise and form a covalently closed circle within the cell; this circle appeared to occur in high abundance and to be transmissible by coresident plasmids.

scholarly journals Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae

2014 ◽  
Vol 80 (21) ◽  
pp. 6677-6684 ◽  
Author(s):  
Youyun Liang ◽  
Tong Si ◽  
Ee Lui Ang ◽  
Huimin Zhao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Phosphoric Acid ◽  
Sole Carbon Source ◽  
Consolidated Bioprocessing ◽  
Content Type ◽  
Yeast Strains ◽  
Recombinant Yeast Strain ◽  
Polysaccharide Monooxygenases ◽  
Simultaneous Saccharification

ABSTRACTSeveral yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases inSaccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.

scholarly journals Biodegradation of Polyester Polyurethane by Endophytic Fungi

2011 ◽  
Vol 77 (17) ◽  
pp. 6076-6084 ◽  
Author(s):  
Jonathan R. Russell ◽  
Jeffrey Huang ◽  
Pria Anand ◽  
Kaury Kucera ◽  
Amanda G. Sandoval ◽  
...  
Keyword(s):  
Carbon Source ◽  
Endophytic Fungi ◽  
Sole Carbon Source ◽  
Anaerobic Growth ◽  
Serine Hydrolase ◽  
Microbial World ◽  
Content Type ◽  
Metabolic Properties ◽  
Liquid Suspensions ◽  
Promising Source

ABSTRACTBioremediation is an important approach to waste reduction that relies on biological processes to break down a variety of pollutants. This is made possible by the vast metabolic diversity of the microbial world. To explore this diversity for the breakdown of plastic, we screened several dozen endophytic fungi for their ability to degrade the synthetic polymer polyester polyurethane (PUR). Several organisms demonstrated the ability to efficiently degrade PUR in both solid and liquid suspensions. Particularly robust activity was observed among several isolates in the genusPestalotiopsis, although it was not a universal feature of this genus. TwoPestalotiopsis microsporaisolates were uniquely able to grow on PUR as the sole carbon source under both aerobic and anaerobic conditions. Molecular characterization of this activity suggests that a serine hydrolase is responsible for degradation of PUR. The broad distribution of activity observed and the unprecedented case of anaerobic growth using PUR as the sole carbon source suggest that endophytes are a promising source of biodiversity from which to screen for metabolic properties useful for bioremediation.

Sign in / Sign up

Export Citation Format

Share Document