scholarly journals Contribution of Pentose Catabolism to Molecular Hydrogen Formation by Targeted Disruption of Arabinose Isomerase (araA) in the Hyperthermophilic Bacterium Thermotoga maritima

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Derrick White ◽  
Raghuveer Singh ◽  
Deepak Rudrappa ◽  
Jackie Mateo ◽  
Levi Kramer ◽  
...  
Keyword(s):  
Molecular Hydrogen ◽  
Thermotoga Maritima ◽  
Mutant Cell ◽  
Complex Medium ◽  
Content Type ◽  
Genetic Method ◽  
Hyperthermophilic Bacterium ◽  
Recombination System ◽  
Targeted Recombination ◽  
Arabinose Isomerase

ABSTRACT Thermotoga maritima ferments a broad range of sugars to form acetate, carbon dioxide, traces of lactate, and near theoretic yields of molecular hydrogen (H2). In this organism, the catabolism of pentose sugars such as arabinose depends on the interaction of the pentose phosphate pathway with the Embden-Myerhoff and Entner-Doudoroff pathways. Although the values for H2 yield have been determined using pentose-supplemented complex medium and predicted by metabolic pathway reconstruction, the actual effect of pathway elimination on hydrogen production has not been reported due to the lack of a genetic method for the creation of targeted mutations. Here, a spontaneous and genetically stable pyrE deletion mutant was isolated and used as a recipient to refine transformation methods for its repair by homologous recombination. To verify the occurrence of recombination and to assess the frequency of crossover events flanking the deleted region, a synthetic pyrE allele, encoding synonymous nucleotide substitutions, was used. Targeted inactivation of araA (encoding arabinose isomerase) in the pyrE mutant was accomplished using a divergent, codon-optimized Thermosipho africanus pyrE allele fused to the T. maritima groES promoter as a genetic marker. Mutants lacking araA were unable to catabolize arabinose in a defined medium. The araA mutation was then repaired using targeted recombination. Levels of synthesis of H2 using arabinose-supplemented complex medium by wild-type and araA mutant cell lines were compared. The difference between strains provided a direct measurement of H2 production that was dependent on arabinose consumption. Development of a targeted recombination system for genetic manipulation of T. maritima provides a new strategy to explore H2 formation and life at an extremely high temperature in the bacterial domain. IMPORTANCE We describe here the development of a genetic system for manipulation of Thermotoga maritima. T. maritima is a hyperthermophilic anaerobic bacterium that is well known for its efficient synthesis of molecular hydrogen (H2) from the fermentation of sugars. Despite considerable efforts to advance compatible genetic methods, chromosome manipulation has remained elusive and hindered use of T. maritima or its close relatives as model hyperthermophiles. Lack of a genetic method also prevented efforts to manipulate specific metabolic pathways to measure their contributions to H2 yield. To overcome this barrier, a homologous chromosomal recombination method was developed and used to characterize the contribution of arabinose catabolism to H2 formation. We report here a stable genetic method for a hyperthermophilic bacterium that will advance studies on the basic and synthetic biology of Thermotogales.

scholarly journals Identification of the ATPase Subunit of the Primary Maltose Transporter in the Hyperthermophilic Anaerobe Thermotoga maritima

2017 ◽  
Vol 83 (18) ◽  
Author(s):  
Raghuveer Singh ◽  
Derrick White ◽  
Paul Blum
Keyword(s):  
Thermotoga Maritima ◽  
Mutant Cell ◽  
Sugar Metabolism ◽  
Genetic System ◽  
Gene Replacement ◽  
Atpase Subunit ◽  
Content Type ◽  
Fermentative Hydrogen Production ◽  
Atpase Subunits ◽  
Fermentative Hydrogen

ABSTRACT Thermotoga maritima is a hyperthermophilic anaerobic bacterium that produces molecular hydrogen (H2) by fermentation. It catabolizes a broad range of carbohydrates through the action of diverse ABC transporters. However, in T. maritima and related species, highly similar genes with ambiguous annotation obscure a precise understanding of genome function. In T. maritima, three putative malK genes, all annotated as ATPase subunits, exhibited high identity to each other. To distinguish between these genes, malK disruption mutants were constructed by gene replacement, and the resulting mutant cell lines were characterized. Only a disruption of malK3 produced a defect in maltose catabolism. To verify that the mutant phenotype arose specifically from malK3 inactivation, the malK3 mutation was repaired by recombination, and maltose catabolism was restored. This study demonstrates the importance of a maltose ABC-type transporter and its relationship to sugar metabolism in T. maritima. IMPORTANCE The application and further development of a genetic system was used here to investigate gene paralogs in the hyperthermophile Thermotoga maritima. The occurrence of three ABC transporter ATPase subunits all annotated as malK was evaluated using a combination of genetic and bioinformatic approaches. The results clarify the role of only one malK gene in maltose catabolism in a nonmodel organism noted for fermentative hydrogen production.

scholarly journals Stationary Phase and Nutrient Levels Trigger Transcription of a Genomic Locus Containing a Novel Peptide (TM1316) in the Hyperthermophilic Bacterium Thermotoga maritima

2013 ◽  
Vol 79 (21) ◽  
pp. 6637-6646
Author(s):  
Andrew D. Frock ◽  
Clemente I. Montero ◽  
Sara E. Blumer-Schuette ◽  
Robert M. Kelly
Keyword(s):  
Amino Acids ◽  
Stationary Phase ◽  
Antimicrobial Agents ◽  
Cyclic Peptide ◽  
Morphological Changes ◽  
Thermotoga Maritima ◽  
Cell Envelope ◽  
Genomic Locus ◽  
Content Type ◽  
Hyperthermophilic Bacterium

ABSTRACTThe genome of the hyperthermophilic bacteriumThermotoga maritimaencodes numerous putative peptides/proteins of 100 amino acids or less. While most of these open reading frames (ORFs) are transcribed during growth, their corresponding physiological roles are largely unknown. The onset of stationary phase inT. maritimawas accompanied by significant morphological changes and upregulation of several ORFs located in the TM1298-TM1336 genome locus. This region contains putative HicAB toxin-antitoxin pairs, hypothetical proteins, radicalS-adenosylmethionine (SAM) enzymes, and ABC transporters. Of particular note was the TM1315-TM1319 operon, which includes a putative 31-amino-acid peptide (TM1316) that was the most highly transcribed gene in the transcriptome during stationary phase. Antibodies directed against a synthetic version of TM1316 were used to track its production, which correlated closely with transcriptomic data. Immunofluorescence microscopy revealed that TM1316 was localized to the cell envelope and prominent in cell aggregates formed during stationary phase. The only functionally characterized locus with an organization similar to that of TM1315-TM1319 is inBacillus subtilis, which contains subtilosin A, a cyclic peptide with Cys–to–α-carbon linkages that functions as an antilisterial bacteriocin. While the organization of TM1316 resembled that of theBacilluspeptide (e.g., in its number of amino acids and spacing of Cys residues), preparations containing high levels of TM1316 affected the growth of neitherThermotogaspecies norPyrococcus furiosus, a hyperthermophilic archaeon isolated from the same locale asT. maritima. Several other putative Cys-rich peptides could be identified in the TM1298-TM1336 locus, and while their roles are also unclear, they merit examination as potential antimicrobial agents in hyperthermophilic biotopes.

scholarly journals Uncoupling Fermentative Synthesis of Molecular Hydrogen from Biomass Formation inThermotoga maritima

2018 ◽  
Vol 84 (17) ◽  
Author(s):  
Raghuveer Singh ◽  
Derrick White ◽  
Yaşar Demirel ◽  
Robert Kelly ◽  
Kenneth Noll ◽  
...  
Keyword(s):  
Energy Management ◽  
Cell Lines ◽  
Molecular Hydrogen ◽  
Thermotoga Maritima ◽  
Gene Inactivation ◽  
Sugar Uptake ◽  
Spontaneous Mutant ◽  
Genome Resequencing ◽  
Content Type ◽  
Maltose Transport

ABSTRACTWhen carbohydrates are fermented by the hyperthermophilic anaerobeThermotoga maritima, molecular hydrogen (H2) is formed in strict proportion to substrate availability. Excretion of the organic acids acetate and lactate provide an additional sink for removal of excess reductant. However, mechanisms controlling energy management of these metabolic pathways are largely unexplored. To investigate this topic, transient gene inactivation was used to block lactate production as a strategy to produce spontaneous mutant cell lines that overproduced H2through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded byldh) with a truncatedldhfused to a kanamycin resistance cassette expressed from a native PgroESLpromoter. Passage of the unstable recombinant resulted in loss of the genetic marker and recovery of evolved cell lines, including strain Tma200. Relative to the wild type, and considering the mass balance of fermentation substrate and products, Tma200 grew more slowly, produced H2at levels above the physiologic limit, and simultaneously consumed less maltose while oxidizing it more efficiently. Whole-genome resequencing indicated that the ABC maltose transporter subunit, encoded bymalK3, had undergone repeated mutation, and high-temperature anaerobic [14C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of themalK3mutation into a clean genetic background also conferred increased H2production, confirming that the mutant allele was sufficient for increased H2synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2production, changing fermentation efficiency and shifting energy management.IMPORTANCEBiorenewable energy sources are of growing interest to mitigate climate change, but like other commodities with nominal value, require innovation to maximize yields. Energetic considerations constrain production of many biofuels, such as molecular hydrogen (H2) because of the competing needs for cell mass synthesis and metabolite formation. Here we describe cell lines of the extremophileThermotoga maritimathat exceed the physiologic limits for H2formation arising from genetic changes in fermentative metabolism. These cell lines were produced using a novel method called transient gene inactivation combined with adaptive laboratory evolution. Genome resequencing revealed unexpected changes in a maltose transport protein. Reduced rates of sugar uptake were accompanied by lower rates of growth and enhanced productivity of H2.

scholarly journals Regulation of Coenzyme A Biosynthesis in the Hyperthermophilic Bacterium Thermotoga maritima

2016 ◽  
Vol 198 (14) ◽  
pp. 1993-2000 ◽  
Author(s):  
Takahiro Shimosaka ◽  
Hiroya Tomita ◽  
Haruyuki Atomi
Keyword(s):  
Coenzyme A ◽  
Feedback Inhibition ◽  
Thermotoga Maritima ◽  
Single Type ◽  
Type I ◽  
Type Ii ◽  
Pantothenate Kinase ◽  
Content Type ◽  
Type Iii ◽  
Hyperthermophilic Bacterium

ABSTRACTRegulation of coenzyme A (CoA) biosynthesis in bacteria and eukaryotes occurs through feedback inhibition targeting type I and type II pantothenate kinase (PanK), respectively. In contrast, the activity of type III PanK is not affected by CoA. As the hyperthermophilic bacteriumThermotoga maritimaharbors only a single type III PanK (Tm-PanK), here we examined the mechanisms that regulate CoA biosynthesis in this organism. We first examined the enzyme responsible for the ketopantoate reductase (KPR) reaction, which is the target of feedback inhibition in archaea. A classical KPR homolog was not present on theT. maritimagenome, but we found a homolog (TM0550) of the ketol-acid reductoisomerase (KARI) fromCorynebacterium glutamicum, which exhibits KPR activity. The purified TM0550 protein displayed both KPR and KARI activities and was designatedTm-KPR/KARI. WhenT. maritimacell extract was subjected to anion-exchange chromatography, the fractions containing high levels of KPR activity also displayed positive signals in a Western blot analysis using polyclonal anti-TM0550 protein antisera, strongly suggesting thatTm-KPR/KARI was the major source of KPR activity in the organism. The KPR activity ofTm-KPR/KARI was not inhibited in the presence of CoA. We thus examined the properties ofTm-PanK and the pantothenate synthetase (Tm-PS) of this organism.Tm-PS was not affected by CoA. Surprisingly however,Tm-PanK was inhibited by CoA, with almost complete inhibition in the presence of 400 μM CoA. Our results suggest that CoA biosynthesis inT. maritimais regulated by feedback inhibition targeting PanK, althoughTm-PanK is a type III enzyme.IMPORTANCEBacteria and eukaryotes regulate the biosynthesis of coenzyme A (CoA) by feedback inhibition targeting type I or type II pantothenate kinase (PanK). The hyperthermophilic bacteriumThermotoga maritimaharbors a single type III PanK (Tm-PanK), previously considered to be unaffected by CoA. By examining the properties of three enzymes involved in CoA biosynthesis in this organism, we found thatTm-PanK, although a type III enzyme, is inhibited by CoA. The results provide a feasible explanation of how CoA biosynthesis is regulated inT. maritima, which may also apply for other bacteria that harbor only type III PanK enzymes.

scholarly journals A Double-Strand Break Does Not PromoteNeisseria gonorrhoeaePilin Antigenic Variation

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Lauren L. Prister ◽  
Jing Xu ◽  
H Steven Seifert
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Dna Structure ◽  
Strand Break ◽  
Double Strand Break ◽  
Content Type ◽  
G4 Dna ◽  
Recombination System ◽  
Guanine Quadruplex ◽  
Pilin Gene

ABSTRACTThe major subunit of the type IV pilus (T4p) ofNeisseria gonorrhoeaeundergoes antigenic variation (AV) dependent on a guanine quadruplex (G4) DNA structure located upstream of the pilin gene. Since the presence of G4 DNA induces genome instability in both eukaryotic and prokaryotic chromosomes, we tested whether a double-strand break (DSB) at the site of thepilEG4 sequence could substitute for G4-directed pilin AV. The G4 motif was replaced by an I-SceI cut site, and the cut site was also introduced to locations near the origin of replication and the terminus. Expression of the I-SceI endonuclease from an irrelevant chromosomal site confirmed that the endonuclease functions to induce double-strand breaks at all three locations. No antigenic variants were detected when the G4 was replaced with the I-SceI cut site, but there was a growth defect from having a DSB in the chromosome, and suppressor mutations that were mainly deletions of the cut site and/or the entirepilEgene accumulated. Thus, thepilEG4 does not act to promote pilin AV by generating a DSB but requires either a different type of break, a nick, or more complex interactions with other factors to stimulate this programmed recombination system.IMPORTANCENeisseria gonorrhoeae, the causative agent of gonorrhea, possesses a DNA recombination system to change one of its surface-exposed antigens. This recombination system, known as antigenic variation, uses an alternate DNA structure to initiate variation. The guanine quadruplex DNA structure is known to cause nicks or breaks in DNA; however, much remains unknown about how this structure functions in cells. We show that inducing a break by different means does not allow antigenic variation, indicating that the DNA structure may have a more complicated role.

A novel bifunctional aspartate kinase-homoserine dehydrogenase from the hyperthermophilic bacterium, Thermotoga maritima

2018 ◽  
Vol 82 (12) ◽  
pp. 2084-2093
Author(s):  
Tatsuya Ohshida ◽  
Kohei Koba ◽  
Junji Hayashi ◽  
Kazunari Yoneda ◽  
Taketo Ohmori ◽  
...  
Keyword(s):  
Thermotoga Maritima ◽  
Aspartate Kinase ◽  
Homoserine Dehydrogenase ◽  
Hyperthermophilic Bacterium
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