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 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 Essential and nonessential histone H2A variants in Tetrahymena thermophila.

1996 ◽  
Vol 16 (8) ◽  
pp. 4305-4311 ◽  
Author(s):  
X Liu ◽  
B Li ◽  
GorovskyMA
Keyword(s):  
Tetrahymena Thermophila ◽  
Essential Gene ◽  
Histone Variants ◽  
Gene Replacement ◽  
Histone H2a ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Simple Mass ◽  
Essential Function ◽  
High Degree

Although variants have been identified for every class of histone, their functions remain unknown. We have been studying the histone H2A variant hv1 in the ciliated protozoan Tetrahymena thermophila. Sequence analysis indicates that hv1 belongs to the H2A.F/Z type of histone variants. On the basis of the high degree of evolutionary conservation of this class of histones, they are proposed to have one or more distinct and essential functions that cannot be performed by their major H2A counterparts. Considerable evidence supports the hypothesis that the hv1 protein in T. thermophila and hv1-like proteins in other eukaryotes are associated with active chromatin. In T. thermophila, simple mass transformation and gene replacement techniques have recently become available. In this report, we demonstrate that either the HTA1 gene or the HTA2 gene, encoding the major H2As, can be completely replaced by disrupted genes in the polyploid, transcriptionally active macronucleus, indicating that neither of the two genes is essential. However, only some of the HTA3 genes encoding hv1 can be replaced by disrupted genes, indicating that the H2A.F/Z type variants have an essential function that cannot be performed by the major H2A genes. Thus, an essential gene in T. thermophila can be defined by the fact that it can be partially, but not completely, eliminated from the polyploid macronucleus. To our knowledge, this study represents the first use of gene disruption technology to study core histone gene function in any organism other than yeast and the first demonstration of an essential gene in T. thermophila using these methods. When a rescuing plasmid carrying a wild-type HTA3 gene was introduced into the T. thermophila cells, the endogenous chromosomal HTA3 could be completely replaced, defining a gene replacement strategy that can be used to analyze the function of essential genes.

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 PhhB, a Pseudomonas aeruginosa Homolog of Mammalian Pterin 4a-Carbinolamine Dehydratase/DCoH, Does Not Regulate Expression of Phenylalanine Hydroxylase at the Transcriptional Level

1999 ◽  
Vol 181 (9) ◽  
pp. 2789-2796 ◽  
Author(s):  
Jian Song ◽  
Tianhui Xia ◽  
Roy A. Jensen
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Phenylalanine Hydroxylase ◽  
Transcriptional Level ◽  
Gene Encoding ◽  
Catalytic Function ◽  
Genes Encoding ◽  
Catalytic Role ◽  
Regulatory Effects

ABSTRACT Pterin 4a-carbinolamine dehydratase is bifunctional in mammals. In addition to playing a catalytic role in pterin recycling in the cytoplasm, it plays a regulatory role in the nucleus, where it acts as a dimerization-cofactor component (called DCoH) for the transcriptional activator HNF-1α. A thus far unique operon in Pseudomonas aeruginosa contains a gene encoding a homolog (PhhB) of the regulatory dehydratase, together with genes encoding phenylalanine hydroxylase (PhhA) and aromatic aminotransferase (PhhC). Using complementation of tyrosine auxotrophy in Escherichia colias a functional test, we have found that the in vivo function of PhhA requires PhhB. Strikingly, mammalian DCoH was an effective substitute for PhhB, and either one was effective in trans. Surprisingly, the required presence of PhhB for complementation did not reflect a critical positive regulatory effect of phhB onphhA expression. Rather, in the absence of PhhB, PhhA was found to be extremely toxic in E. coli, probably due to the nonenzymatic formation of 7-biopterin or a similar derivative. However, bacterial PhhB does appear to exert modest regulatory effects in addition to having a catalytic function. PhhB enhances the level of PhhA two- to threefold, as was demonstrated by gene inactivation ofphhB in P. aeruginosa and by comparison of the levels of expression of PhhA in the presence and absence of PhhB inEscherichia coli. Experiments using constructs having transcriptional and translational fusions with a lacZreporter indicated that PhhB activates PhhA at the posttranscriptional level. Regulation of PhhA and PhhB is semicoordinate; both PhhA and PhhB are induced coordinately in the presence of eitherl-tyrosine or l-phenylalanine, but PhhB exhibits a significant basal level of activity that is lacking for PhhA. Immunoprecipitation and affinity chromatography showed that PhhA and PhhB form a protein-protein complex.

Mannose utilization in Escherichia coli requires cyclic AMP but not an exogenous inducer

1980 ◽  
Vol 26 (12) ◽  
pp. 1508-1511 ◽  
Author(s):  
Ann D. E. Fraser ◽  
Hiroshi Yamazaki
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Sole Carbon Source ◽  
Receptor Protein ◽  
Deficient Mutant ◽  
Phosphotransferase System ◽  
Cyclic Amp Receptor Protein ◽  
Cyclic Monophosphate

It has not been clarified whether the utilization of mannose by Escherichia coli requires adenosine 3′,5′-cyclic monophosphate (cyclic AMP). Using an adenylyl cyclase deficient mutant (CA8306B) and a cyclic AMP receptor protein (CRP) deficient mutant (5333B) we have shown that the utilization of mannose is dependent on the cyclic AMP–CRP complex. 2-Deoxyglucose (DG) is a nonmetabolizable glucose analog specific for the phosphotransferase system (PTS) which transports mannose (termed here PTSM). Growth of CA8306B on glycerol is unaffected by addition of the analog, whereas growth of the strain on glycerol plus cyclic AMP ceases im mediately upon addition of DG. These results suggest that the formation of PTSM is dependent on cyclic AMP. In addition, CA8306B grown on glycerol plus cyclic AMP can immediately utilize mannose when transferred to a medium containing mannose as a sole carbon source, whereas the same strain grown on glycerol without cyclic AMP cannot utilize mannose when so transferred. These results suggest that the formation of PTSM does not require an exogenous inducer.

scholarly journals Heterologous Production of 6-Deoxyerythronolide B in Escherichia coli through the Wood Werkman Cycle

Metabolites ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 228
Author(s):  
R. Axayacatl Gonzalez-Garcia ◽  
Lars K. Nielsen ◽  
Esteban Marcellin
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Structural Diversity ◽  
Heterologous Production ◽  
E Coli ◽  
Anticancer Properties ◽  
Defined Media ◽  
Extender Unit

Polyketides are a remarkable class of natural products with diverse functional and structural diversity. The class includes many medicinally important molecules with antiviral, antimicrobial, antifungal and anticancer properties. Native bacterial, fungal and plant hosts are often difficult to cultivate and coax into producing the desired product. As a result, Escherichia coli has been used for the heterologous production of polyketides, with the production of 6-deoxyerythronolide B (6-dEB) being the first example. Current strategies for production in E. coli require feeding of exogenous propionate as a source for the precursors propionyl-CoA and S-methylmalonyl-CoA. Here, we show that heterologous polyketide production is possible from glucose as the sole carbon source. The heterologous expression of eight genes from the Wood-Werkman cycle found in Propionibacteria, in combination with expression of the 6-dEB synthases DEBS1, DEBS2 and DEBS3 resulted in 6-dEB formation from glucose as the sole carbon source. Our results show that the Wood-Werkman cycle provides the required propionyl-CoA and the extender unit S-methylmalonyl-CoA to produce up to 0.81 mg/L of 6-dEB in a chemically defined media.

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