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 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 Hph1 and Hph2 Are Novel Components of the Sec63/Sec62 Posttranslational Translocation Complex That Aid in Vacuolar Proton ATPase Biogenesis

2010 ◽  
Vol 10 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Francisco J. Piña ◽  
Allyson F. O'Donnell ◽  
Silvere Pagant ◽  
Hai Lan Piao ◽  
John P. Miller ◽  
...  
Keyword(s):  
Environmental Stress ◽  
Complex Function ◽  
Content Type ◽  
Growth Defects ◽  
Proton Atpase ◽  
Vacuolar Acidification ◽  
Atpase Subunits ◽  
Specific Proteins ◽  
A Subunit ◽  
Split Ubiquitin

ABSTRACT Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1 Δ hph2 Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71 Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1 Δ hph2 Δ and hph1 Δ hph2 Δ sec71 Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1 Δ hph2 Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.

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