Erin K. Heiniger

Redirection of Metabolism for Biological Hydrogen Production

ABSTRACT A major route for hydrogen production by purple photosynthetic bacteria is biological nitrogen fixation. Nitrogenases reduce atmospheric nitrogen to ammonia with the concomitant obligate production of molecular hydrogen. However, hydrogen production in the context of nitrogen fixation is a rather inefficient process because about 75% of the reductant consumed by the nitrogenase is used to generate ammonia. In this study we describe a selection strategy to isolate strains of purple photosynthetic bacteria in which hydrogen production is necessary for growth and independent of nitrogen fixation. We obtained four mutant strains of the photosynthetic bacterium Rhodopseudomonas palustris that produce hydrogen constitutively, even in the presence of ammonium, a condition where wild-type cells do not accumulate detectable amounts of hydrogen. Some of these strains produced up to five times more hydrogen than did wild-type cells growing under nitrogen-fixing conditions. Transcriptome analyses of the hydrogen-producing mutant strains revealed that in addition to the nitrogenase genes, 18 other genes are potentially required to produce hydrogen. The mutations that caused constitutive hydrogen production mapped to four different sites in the NifA transcriptional regulator in the four different strains. The strategy presented here can be applied to the large number of diverse species of anoxygenic photosynthetic bacteria that are known to exist in nature to identify strains for which there are fitness incentives to produce hydrogen.

Reversible Nε‐lysine acetylation regulates the activity of acyl‐CoA synthetases involved in anaerobic benzoate catabolism in Rhodopseudomonas palustris

Rhodopseudomonas palustris grows photoheterotrophically on aromatic compounds available in aquatic environments rich in plant‐derived lignin. Benzoate degradation is regulated at the transcriptional level in R. palustris in response to anoxia and the presence of benzoate and/or benzoyl‐CoA (Bz‐CoA). Here, we report evidence that anaerobic benzoate catabolism in this bacterium is also regulated at the post‐translational level. In this pathway, benzoate is activated to Bz‐CoA by the AMP‐forming Bz‐CoA synthetase (BadA) enzyme. Mass spectrometry and mutational analysis data indicate that residue Lys512 is critical to BadA activity. Acetylation of Lys512 inactivated BadA; deacetylation reactivated BadA. Likewise, 4‐hydroxybenzoyl‐CoA (HbaA) and cyclohexanecarboxyl‐CoA (AliA) synthetases were also reversibly acetylated. We identified one acetyltransferase that modified BadA, Hba and AliA in vitro. The acetyltransferase enzyme is homologous to the protein acetyltransferase (Pat) enzyme of Salmonella enterica sv Typhimurium LT2, thus we refer to it as RpPat. RpPat also modified acetyl‐CoA (Ac‐CoA) synthetase (Acs) from R. palustris. In vivo data indicate that at least two deacetylases reactivate BadAAc. One is SrtN (encoded by srtN, formerly rpa2524), a sirtuin‐type NAD+‐dependent deacetylase (O‐acetyl‐ADPribose‐forming); the other deacetylase is LdaA (encoded by ldaA, for lysine deacetylase A; formerly rpa0954), an acetate‐forming protein deacetylase. LdaA reactivated HbaAc and AliAAcin vitro.

Production of Hydrogen Gas from Light and the Inorganic Electron Donor Thiosulfate by Rhodopseudomonas palustris

ABSTRACT A challenge for photobiological production of hydrogen gas (H2) as a potential biofuel is to find suitable electron-donating feedstocks. Here, we examined the inorganic compound thiosulfate as a possible electron donor for nitrogenase-catalyzed H2 production by the purple nonsulfur phototrophic bacterium (PNSB) Rhodopseudomonas palustris. Thiosulfate is an intermediate of microbial sulfur metabolism in nature and is also generated in industrial processes. We found that R. palustris grew photoautotrophically with thiosulfate and bicarbonate and produced H2 when nitrogen gas was the sole nitrogen source (nitrogen-fixing conditions). In addition, illuminated nongrowing R. palustris cells converted about 80% of available electrons from thiosulfate to H2. H2 production with acetate and succinate as electron donors was less efficient (40 to 60%), partly because nongrowing cells excreted the intermediary metabolite α-ketoglutarate into the culture medium. The fixABCX operon (RPA4602 to RPA4605) encoding a predicted electron-transfer complex is necessary for growth using thiosulfate under nitrogen-fixing conditions and may serve as a point of engineering to control rates of H2 production. The possibility to use thiosulfate expands the range of electron-donating compounds for H2 production by PNSBs beyond biomass-based electron donors.

How Posttranslational Modification of Nitrogenase Is Circumvented in Rhodopseudomonas palustris Strains That Produce Hydrogen Gas Constitutively

ABSTRACT Nitrogenase catalyzes the conversion of dinitrogen gas (N2) and protons to ammonia and hydrogen gas (H2). This is a catalytically difficult reaction that requires large amounts of ATP and reducing power. Thus, nitrogenase is not normally expressed or active in bacteria grown with a readily utilized nitrogen source like ammonium. nifA* mutants of the purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris have been described that express nitrogenase genes constitutively and produce H2 when grown with ammonium as a nitrogen source. This raised the regulatory paradox of why these mutants are apparently resistant to a known posttranslational modification system that should switch off the activity of nitrogenase. Microarray, mutation analysis, and gene expression studies showed that posttranslational regulation of nitrogenase activity in R. palustris depends on two proteins: DraT2, an ADP-ribosyltransferase, and GlnK2, an NtrC-regulated PII protein. GlnK2 was not well expressed in ammonium-grown NifA* cells and thus not available to activate the DraT2 nitrogenase modification enzyme. In addition, the NifA* strain had elevated nitrogenase activity due to overexpression of the nif genes, and this increased amount of expression overwhelmed a basal level of activity of DraT2 in ammonium-grown cells. Thus, insufficient levels of both GlnK2 and DraT2 allow H2 production by an nifA* mutant grown with ammonium. Inactivation of the nitrogenase posttranslational modification system by mutation of draT2 resulted in increased H2 production by ammonium-grown NifA* cells.

A disposable chemical heater and dry enzyme preparation for lysis and extraction of DNA and RNA from microorganisms

Sample preparation, including bacterial lysis, remains a hurdle in the realization of complete point-of-care tests for many pathogens. Here, we developed a sample preparation methodology for enzymatic lysis and sample heating for low-resource, point-of-care applications. We show an instrument-free chemical heater system for rapid lysis of a Gram-positive bacterium (Staphylococcus aureus) and an RNA virus (human respiratory syncytial virus) using a dried lysis enzyme mixture (achromopeptidase) for S. aureus. After a lysis step (<1 minute), lysis enzymes are heat deactivated (<5 minutes) using a simple disposable chemical heater. We demonstrated that both DNA and RNA in the heat-treated sample could be directly amplified without purification, even in the presence of a clinically-obtained human nasal sample. This simple approach to dry enzyme storage and sample heating is adaptable to many applications where samples need to be lysed, including use in low-resource laboratories and in single-use or cartridge-based point-of-care diagnostic devices.

Comparison of point-of-care-compatible lysis methods for bacteria and viruses

Nucleic acid sample preparation has been an especially challenging barrier to point-of-care nucleic acid amplification tests in low-resource settings. Here we provide a head-to-head comparison of methods for lysis of, and nucleic acid release from, several pathogenic bacteria and viruses-methods that are adaptable to point-of-care usage in low-resource settings. Digestion with achromopeptidase, a mixture of proteases and peptidoglycan-specific hydrolases, followed by thermal deactivation in a boiling water bath, effectively released amplifiable nucleic acid from Staphylococcus aureus, Bordetella pertussis, respiratory syncytial virus, and influenza virus. Achromopeptidase was functional after dehydration and reconstitution, even after eleven months of dry storage without refrigeration. Mechanical lysis methods proved to be effective against a hard-to-lyse Mycobacterium species, and a miniature bead-mill, the AudioLyse, is shown to be capable of releasing amplifiable DNA and RNA from this species. We conclude that point-of-care-compatible sample preparation methods for nucleic acid tests need not introduce amplification inhibitors, and can provide amplification-ready lysates from a wide range of bacterial and viral pathogens.

Posttranslational modification of a vanadium nitrogenase

In microbes that fix nitrogen, nitrogenase catalyzes the conversion of N2 to ammonia in an ATP‐demanding reaction. To help conserve energy some bacteria inhibit nitrogenase activity upon exposure to ammonium. The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris strain CGA009 can synthesize three functional nitrogenase isoenzymes: a molybdenum nitrogenase, a vanadium nitrogenase, and an iron nitrogenase. Previous studies showed that in some alphaproteobacteria, including R. palustris, molybdenum nitrogenase activity is inhibited by ADP‐ribosylation when cells are exposed to ammonium. Some iron nitrogenases are also posttranslationally modified. However, the posttranslational modification of vanadium nitrogenase has not been reported. Here, we investigated the regulation of the alternative nitrogenases of R. palustris and determined that both its vanadium nitrogenase and its iron nitrogenase activities were inhibited and posttranslationally modified when cells are exposed to ammonium. Vanadium nitrogenase is not found in all strains of R. palustris, suggesting that it may have been acquired by horizontal gene transfer. Also, phylogenetic analyses of the three nitrogenases suggest that VnfH, the target of ADP‐ribosylation, may be the product of a gene duplication of nifH, the molybdenum nitrogenase homolog.

Electromechanical cell lysis using a portable audio device

Audio sources are ubiquitously available on portable electronic devices, including cell phones. Here we demonstrate lysis of Mycobacterium marinum and Staphylococcus epidermidis bacteria utilizing a portable audio device coupled with a simple and inexpensive electromagnetic coil. The resulting alternating magnetic field rotates a magnet in a tube with the sample and glass beads, lysing the cells and enabling sample preparation for these bacteria anywhere there is a cell phone, mp3 player, laptop, or other device with a headphone jack.