When 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.

The antibiotic, fosmidomycin (FSM) targets the methylerythritol phosphate (MEP) pathway of isoprenoid synthesis by inhibiting the essential enzyme, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) and is lethal to intracellular parasites and bacteria. The obligate intracellular bacterial pathogen, Chlamydia trachomatis, alternates between two developmental forms: the extracellular, infectious elementary body (EB), and the intracellular, replicative form called the reticulate body (RB). Several stressful growth conditions including iron deprivation halt chlamydial cell division and cause development of a morphologically enlarged, but viable form termed an aberrant body (AB). This phenotype constitutes the chlamydial developmental state known as persistence. This state is reversible as removal of the stressor allows the chlamydiae to re-enter and complete the normal developmental cycle. Bioinformatic analysis indicates that C. trachomatis encodes a homolog of Dxr, but its function and the requirement for isoprenoid synthesis in chlamydial development is not fully understood. We hypothesized that chlamydial Dxr (DxrCT) is functional and that the methylerythritol phosphate (MEP) pathway is required for normal chlamydial development. Thus, FSM exposure should be lethal to C. trachomatis. Overexpression of chlamydial Dxr (DxrCT) in Escherichia coli under FSM exposure and in a conditionally lethal dxr mutant demonstrated that DxrCT functions similarly to E. coli Dxr. When Chlamydia-infected cultures were exposed to FSM, EB production was significantly reduced. However, titer recovery assays, electron microscopy, and peptidoglycan labeling revealed that FSM inhibition of isoprenoid synthesis is not lethal to C. trachomatis, but instead induces persistence. Bactoprenol is a critical isoprenoid required for peptidoglycan precursor assembly. We therefore conclude that FSM induces persistence in Chlamydia by preventing bactoprenol production necessary for peptidoglycan precursor assembly and subsequent cell division.

Peptidoglycan is a sugar/amino acid polymer unique to bacteria and essential for division and cell shape maintenance. The d-amino acids that make up its cross-linked stem peptides are not abundant in nature and must be synthesized by bacteria de novo. d-Glutamate is present at the second position of the pentapeptide stem and is strictly conserved in all bacterial species. In Gram-negative bacteria, d-glutamate is generated via the racemization of l-glutamate by glutamate racemase (MurI). Chlamydia trachomatis is the leading cause of infectious blindness and sexually transmitted bacterial infections worldwide. While its genome encodes a majority of the enzymes involved in peptidoglycan synthesis, no murI homologue has ever been annotated. Recent studies have revealed the presence of peptidoglycan in C. trachomatis and confirmed that its pentapeptide includes d-glutamate. In this study, we show that C. trachomatis synthesizes d-glutamate by utilizing a novel, bifunctional homologue of diaminopimelate epimerase (DapF). DapF catalyzes the final step in the synthesis of meso-diaminopimelate, another amino acid unique to peptidoglycan. Genetic complementation of an Escherichia coli murI mutant demonstrated that Chlamydia DapF can generate d-glutamate. Biochemical analysis showed robust activity, but unlike canonical glutamate racemases, activity was dependent on the cofactor pyridoxal phosphate. Genetic complementation, enzymatic characterization, and bioinformatic analyses indicate that chlamydial DapF shares characteristics with other promiscuous/primordial enzymes, presenting a potential mechanism for d-glutamate synthesis not only in Chlamydia but also numerous other genera within the Planctomycetes-Verrucomicrobiae-Chlamydiae superphylum that lack recognized glutamate racemases.

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Thermotoga maritima is an anaerobic hyperthermophilic bacterium known for its high amounts of hydrogen (H 2) production. In the current study, the kinetic modeling was applied on the engineered strains of T. maritima that surpassed the natural H 2 production limit. The study generated a kinetic model explaining H 2 overproduction and predicted a continuous fermentation system. A Leudking-Piret equationbased model predicted that H 2 production by Tma200 (0.217 mol-H 2 g-1-biomass) and Tma100 (0.147 mol-H 2 g-1-biomass) were higher than wild type (0.096 mol-H 2 g-1-biomass) with reduced rates of maltose utilization. Sensitivity analysis confirmed satisfactory fitting of the experimental data. The slow growth rates of Tma200 (0.550 h-1) and Tma100 (0.495 h-1) are compared with the wild type (0.663 h-1). A higher 1 digitalcommons.unl.edu

Peptidoglycan, the sugar-amino acid polymer that composes the bacterial cell wall, requires a significant expenditure of energy to synthesize and is highly immunogenic. To minimize the loss of an energetically expensive metabolite and avoid host detection, bacteria often recycle their peptidoglycan, transporting its components back into the cytoplasm, where they can be used for subsequent rounds of new synthesis. The peptidoglycan-recycling substrate binding protein (SBP) MppA, which is responsible for recycling peptidoglycan fragments in Escherichia coli, has not been annotated for most intracellular pathogens. One such pathogen, Chlamydia trachomatis, has a limited capacity to synthesize amino acids de novo and therefore must obtain oligopeptides from its host cell for growth. Bioinformatics analysis suggests that the putative C. trachomatis oligopeptide transporter OppABCDF (OppABCDFCt) encodes multiple SBPs (OppA1Ct, OppA2Ct, and OppA3Ct). Intracellular pathogens often encode multiple SBPs, while only one, OppA, is encoded in the E. coli opp operon. We hypothesized that the putative OppABCDF transporter of C. trachomatis functions in both oligopeptide transport and peptidoglycan recycling. We coexpressed the putative SBP genes (oppA1Ct, oppA2Ct, oppA3Ct) along with oppBCDFCt in an E. coli mutant lacking the Opp transporter and determined that all three chlamydial OppA subunits supported oligopeptide transport. We also demonstrated the in vivo functionality of the chlamydial Opp transporter in C. trachomatis. Importantly, we found that one chlamydial SBP, OppA3Ct, possessed dual substrate recognition properties and is capable of transporting peptidoglycan fragments (tri-diaminopimelic acid) in E. coli and in C. trachomatis. These findings suggest that Chlamydia evolved an oligopeptide transporter to facilitate the acquisition of oligopeptides for growth while simultaneously reducing the accumulation of immunostimulatory peptidoglycan fragments in the host cell cytosol. The latter property reflects bacterial pathoadaptation that dampens the host innate immune response to Chlamydia infection.

Thermotoga maritima ferments a broad range of sugars to form acetate, carbon dioxide, traces of lactate, and near theoretic yields of molecular hydrogen (H 2 ). 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 H 2 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 H 2 using arabinosesupplemented complex medium by wild-type and araA mutant cell lines were compared. The difference between strains provided a direct measurement of H 2 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 H 2 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 (H 2 ) 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 H 2 yield. To overcome this barrier, a homologous chromosomal recombination method was developed and used to characterize the contribution of arabinose catabolism to H 2 formation. We report here a stable genetic

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