Aerobic marine bacteria can use H2 for growth.

Molecular hydrogen (H2) and carbon monoxide (CO) are ubiquitously available in the Earth's oceans, yet their low dissolved concentrations seemed unlikely to support microbial growth. Lappan, Shelley, Islam et al. now report that dissolved H2 supports the growth of diverse aerobic marine bacteria in the oceans.

Tandem repeats in giant archaeal Borg elements undergo rapid evolution and create new intrinsically disordered regions in proteins.

Borgs are huge, linear extrachromosomal elements associated with anaerobic methane-oxidizing archaea. Striking features of Borg genomes are pervasive tandem direct repeat (TR) regions. Here, we present six new Borg genomes and investigate the characteristics of TRs in all ten complete Borg genomes. We find that TR regions are rapidly evolving, recently formed, arise independently, and are virtually absent in host Methanoperedens genomes. Flanking partial repeats and A-enriched character constrain the TR formation mechanism. TRs can be in intergenic regions, where they might serve as regulatory RNAs, or in open reading frames (ORFs). TRs in ORFs are under very strong selective pressure, leading to perfect amino acid TRs (aaTRs) that are commonly intrinsically disordered regions. Proteins with aaTRs are often extracellular or membrane proteins, and functionally similar or homologous proteins often have aaTRs composed of the same amino acids. We propose that Borg aaTR-proteins functionally diversify Methanoperedens and all TRs are crucial for specific Borg-host associations and possibly cospeciation.

Lactate metabolism in strictly anaerobic microorganisms with a soluble NAD+ -dependent l-lactate dehydrogenase.

Lactate is a universal metabolite and energy source, yet the mode of lactate metabolism in many strictly anaerobic microorganisms is still enigmatic. This sparked us to investigate the biochemistry and bioenergetics of lactate metabolism in the model acetogenic bacterium Moorella thermoacetica. Growth and metabolism were dependent on CO2 and the chemiosmotic gradient. We discovered a l-lactate:NAD+ oxidoreductase (LDH) in cell-free extracts, exhibiting an average specific activity of 362.8 ± 22.9 mU mg-1 . The enzyme was reversible, most active at 65°C and pH 9, with Km values of 23.1 ± 3.7 mM for l-lactate and 273.3 ± 39.1 µM for NAD+ . In-gel activity assays and mass spectrometric proteomics revealed that the ldh gene encoded the characterized LDH. Transcriptomic and genomic analyses showed that ldh expression was induced by lactate and there was a single nucleotide polymorphism near the predicted NAD+ binding site. Genes encoding central redox and energy metabolism complexes, such as, the energetic coupling site Ech2, menaquinone, and the electron bifurcating EtfABCX and MTHFR were also upregulated in cells grown on lactate. These findings ultimately lead to a redox-balanced metabolic model that shows how growth on lactate can proceed in a microorganism that only has a conventional NAD+ -reducing LDH.

The pyruvate:ferredoxin oxidoreductase of the thermophilic acetogen, Thermoanaerobacter kivui.

Pyruvate:ferredoxin oxidoreductase (PFOR) is a key enzyme in bacterial anaerobic metabolism. Since a low-potential ferredoxin (Fd2- ) is used as electron carrier, PFOR allows for hydrogen evolution during heterotrophic growth as well as pyruvate synthesis during lithoautotrophic growth. The thermophilic acetogenic model bacterium Thermoanaerobacter kivui can use both modes of lifestyle, but the nature of the PFOR in this organism was previously unestablished. Here, we have isolated PFOR to apparent homogeneity from cells grown on glucose. Peptide mass fingerprinting revealed that it is encoded by pfor1. PFOR uses pyruvate as an electron donor and methylene blue (1.8 U·mg-1 ) and ferredoxin (Fd; 27.2 U·mg-1 ) as electron acceptors, and the reaction is dependent on thiamine pyrophosphate, pyruvate, coenzyme A, and Fd. The pH and temperature optima were 7.5 and 66 °C, respectively. We detected 13.6 mol of iron·mol of protein-1 , consistent with the presence of three predicted [4Fe-4S] clusters. The ability to provide reduced Fd makes PFOR an interesting auxiliary enzyme for enzyme assays. To simplify and speed up the purification procedure, we established a protocol for homologous protein production in T. kivui. Therefore, pfor1 was cloned and expressed in T. kivui and the encoded protein containing a genetically engineered His-tag was purified in only two steps to apparent homogeneity. The homologously produced PFOR1 had the same properties as the enzyme from T. kivui. The enzyme can be used as auxiliary enzyme in enzymatic assays that require reduced Fd as electron donor, such as electron-bifurcating enzymes, to keep a constant level of reduced Fd.

Cysteine: an overlooked energy and carbon source.

Biohybrids composed of microorganisms and nanoparticles have emerged as potential systems for bioenergy and high-value compound production from CO2 and light energy, yet the cellular and metabolic processes within the biological component of this system are still elusive. Here we dissect the biohybrid composed of the anaerobic acetogenic bacterium Moorella thermoacetica and cadmium sulphide nanoparticles (CdS) in terms of physiology, metabolism, enzymatics and transcriptomic profiling. Our analyses show that while the organism does not grow on L-cysteine, it is metabolized to acetate in the biohybrid system and this metabolism is independent of CdS or light. CdS cells have higher metabolic activity, despite an inhibitory effect of Cd2+ on key enzymes, because of an intracellular storage compound linked to arginine metabolism. We identify different routes how cysteine and its oxidized form can be innately metabolized by the model acetogen and what intracellular mechanisms are triggered by cysteine, cadmium or blue light.

Phenotypic and Transcriptomic Analyses of Seven Clinical Stenotrophomonas maltophilia Isolates Identify a Small Set of Shared and Commonly Regulated Genes Involved in the Biofilm Lifestyle.

Stenotrophomonas maltophilia is one of the most frequently isolated multidrug-resistant nosocomial opportunistic pathogens. It contributes to disease progression in cystic fibrosis (CF) patients and is frequently isolated from wounds, infected tissues, and catheter surfaces. On these diverse surfaces S. maltophilia lives in single-species or multispecies biofilms. Since very little is known about common processes in biofilms of different S. maltophilia isolates, we analyzed the biofilm profiles of 300 clinical and environmental isolates from Europe of the recently identified main lineages Sgn3, Sgn4, and Sm2 to Sm18. The analysis of the biofilm architecture of 40 clinical isolates revealed the presence of multicellular structures and high phenotypic variability at a strain-specific level. Further, transcriptome analyses of biofilm cells of seven clinical isolates identified a set of 106 shared strongly expressed genes and 33 strain-specifically expressed genes. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that just 9.43% ± 1.36% of all genes were differentially regulated. This implies that just a small set of shared and commonly regulated genes is involved in the biofilm lifestyle. Strikingly, iron uptake appears to be a key factor involved in this metabolic shift. Further, metabolic analyses implied that S. maltophilia employs a mostly fermentative growth mode under biofilm conditions. The transcriptome data of this study together with the phenotypic and metabolic analyses represent so far the largest data set on S. maltophilia biofilm versus planktonic cells. This study will lay the foundation for the identification of strategies for fighting S. maltophilia biofilms in clinical and industrial settings.IMPORTANCE Microorganisms living in a biofilm are much more tolerant to antibiotics and antimicrobial substances than planktonic cells are. Thus, the treatment of infections caused by microorganisms living in biofilms is extremely difficult. Nosocomial infections (among others) caused by S. maltophilia, particularly lung infection among CF patients, have increased in prevalence in recent years. The intrinsic multidrug resistance of S. maltophilia and the increased tolerance to antimicrobial agents of its biofilm cells make the treatment of S. maltophilia infection difficult. The significance of our research is based on understanding the common mechanisms involved in biofilm formation of different S. maltophilia isolates, understanding the diversity of biofilm architectures among strains of this species, and identifying the differently regulated processes in biofilm versus planktonic cells. These results will lay the foundation for the treatment of S. maltophilia biofilms.

Energy-converting hydrogenases: the link between H2 metabolism and energy conservation.

The reversible interconversion of molecular hydrogen and protons is one of the most ancient microbial metabolic reactions and catalyzed by hydrogenases. A widespread yet largely enigmatic group comprises multisubunit [NiFe] hydrogenases, that directly couple H2 metabolism to the electrochemical ion gradient across the membranes of bacteria and of archaea. These complexes are collectively referred to as energy-converting hydrogenases (Ech), as they reversibly transform redox energy into physicochemical energy. Redox energy is typically provided by a low potential electron donor such as reduced ferredoxin to fuel H2 evolution and the establishment of a transmembrane electrochemical ion gradient ([Formula: see text]). The [Formula: see text] is then utilized by an ATP synthase for energy conservation by generating ATP. This review describes the modular structure/function of Ech complexes, focuses on insights into the energy-converting mechanisms, describes the evolutionary context and delves into the implications of relying on an Ech complex as respiratory enzyme for microbial metabolism.

Energy conservation involving 2 respiratory circuits.

Chemiosmosis and substrate-level phosphorylation are the 2 mechanisms employed to form the biological energy currency adenosine triphosphate (ATP). During chemiosmosis, a transmembrane electrochemical ion gradient is harnessed by a rotary ATP synthase to phosphorylate adenosine diphosphate to ATP. In microorganisms, this ion gradient is usually composed of [Formula: see text], but it can also be composed of Na+ Here, we show that the strictly anaerobic rumen bacterium Pseudobutyrivibrio ruminis possesses 2 ATP synthases and 2 distinct respiratory enzymes, the ferredoxin:[Formula: see text] oxidoreductase (Rnf complex) and the energy-converting hydrogenase (Ech complex). In silico analyses revealed that 1 ATP synthase is [Formula: see text]-dependent and the other Na+-dependent, which was validated by biochemical analyses. Rnf and Ech activity was also biochemically identified and investigated in membranes of P. ruminis Furthermore, the physiology of the rumen bacterium and the role of the energy-conserving systems was investigated in dependence of 2 different catabolic pathways (the Embden-Meyerhof-Parnas or the pentose-phosphate pathway) and in dependence of Na+ availability. Growth of P. ruminis was greatly stimulated by Na+, and a combination of physiological, biochemical, and transcriptional analyses revealed the role of the energy conserving systems in P. ruminis under different metabolic scenarios. These data demonstrate the use of a 2-component ion circuit for [Formula: see text] bioenergetics and a 2nd 2-component ion circuit for Na+ bioenergetics in a strictly anaerobic rumen bacterium. In silico analyses infer that these 2 circuits are prevalent in a number of other strictly anaerobic microorganisms.

Energy conservation by a hydrogenase-dependent chemiosmotic mechanism in an ancient metabolic pathway.

The ancient reductive acetyl-CoA pathway is employed by acetogenic bacteria to form acetate from inorganic energy sources. Since the central pathway does not gain net ATP by substrate-level phosphorylation, chemolithoautotrophic growth relies on the additional formation of ATP via a chemiosmotic mechanism. Genome analyses indicated that some acetogens only have an energy-converting, ion-translocating hydrogenase (Ech) as a potential respiratory enzyme. Although the Ech-encoding genes are widely distributed in nature, the proposed function of Ech as an ion-translocating chemiosmotic coupling site has neither been demonstrated in bacteria nor has it been demonstrated that it can be the only energetic coupling sites in microorganisms that depend on a chemiosmotic mechanism for energy conservation. Here, we show that the Ech complex of the thermophilic acetogenic bacterium Thermoanaerobacter kivui is indeed a respiratory enzyme. Experiments with resting cells prepared from T. kivui cultures grown on carbon monoxide (CO) revealed CO oxidation coupled to H2 formation and the generation of a transmembrane electrochemical ion gradient ([Formula: see text]). Inverted membrane vesicles (IMVs) prepared from CO-grown cells also produced H2 and ATP from CO (via a loosely attached CO dehydrogenase) or a chemical reductant. Finally, we show that Ech activity led to the translocation of both H+ and Na+ across the membrane of the IMVs. The H+ gradient was then used by the ATP synthase for energy conservation. These data demonstrate that the energy-converting hydrogenase in concert with an ATP synthase may be the simplest form of respiration; it combines carbon dioxide fixation with the synthesis of ATP in an ancient pathway.

Regulation of lactate metabolism in the acetogenic bacterium Acetobacterium woodii.

Acetogenic bacteria compete in an energy-limited environment by coupling different metabolic routes to their central metabolism of CO2 fixation. The underlying regulatory mechanisms are often still not understood. In this work, we analysed how lactate metabolism is regulated in the model acetogen Acetobacterium woodii. Construction of a ΔlctCDEF mutant and growth analyses demonstrated that the genes are essential for growth on lactate. Subsequent bridging PCR and quantitative PCR analyses revealed that the lctBCDEF genes form an operon that was expressed only during lactate metabolism. The lctA gene was cloned, expressed in Escherichia coli and purified. LctA bound to the intergenic DNA region between lctA and the lct operon in electromobility shift assays, and binding was revoked in the presence of lactate. Further restriction site protection analyses consolidated the lactate-dependent binding of LctA and identified the binding site within the DNA. Cells grew mixotrophically on lactate and another energy source and showed no diauxic growth. From these data, we conclude that the catabolic lactate metabolism is encoded by the lct operon and its expression is negatively regulated by the DNA-binding repressor LctA.