YtkA (CtaK) and YozB (CtaM) function in the biogenesis of cytochrome c oxidase in Bacillus subtilis.

Cytochrome c oxidase in the respiratory chain of bacteria and mitochondria couples the reduction of molecular oxygen to form water with the generation of a transmembrane proton gradient. Bacillus subtilis has two heme A-containing heme-copper oxidases: the menaquinol oxidase cytochrome aa3 and the cytochrome c oxidase cytochrome caa3 . By screening three collections of mutants for defective cytochrome c oxidase, we found the genes for two, new membrane-bound assembly factors in B. subtilis: ytkA and yozB (renamed ctaK and ctaM, respectively). CtaK is a lipoprotein without sequence similarity to any protein of known function. We show that CtaK functions together with Sco1 (YpmQ) in a pathway, leading to the assembly of the CuA center in cytochrome caa3 and seems to be a functional analogue to proteins of the periplasmic CuA chaperone family (PCuA C). CtaM is required for the activity of both cytochrome caa3 and cytochrome aa3 and dispensable for the insertion of heme A into these oxidases. The orthologous Bacillus anthracis protein and the distantly related Staphylococcus aureus CtaM complemented CtaM deficiency in B. subtilis, establishing a common function of CtaM in these bacteria. As the overall result of our work, 12 different proteins are known to function in the biosynthesis of cytochrome c oxidase in B. subtilis.

Molecular Biology of Bacillus subtilis Cytochromes anno 2020.

Bacillus subtilis serves as a model Gram-positive bacterium and an experimental system for research on respiratory enzymes. This review presents the heme proteins currently known for the well-characterized laboratory strain B. subtilis 168. It focuses on advances in research made during the last three decades concerning the function and composition of the cytochrome bc complex, terminal oxidases, and succinate:menaquinone oxidoreductase. The aerobic respiratory system of strain 168 seems representative for the species B. subtilis, as determined by the cytochrome composition of the undomesticated strain B. subtilis NCIB 3610 and a set of constructed cytochrome-deficient mutants of this strain. Unexplained and unsettled aspects of the molecular biology of respiratory cytochromes in B. subtilis are highlighted in the review.

Two Routes for Extracellular Electron Transfer in Enterococcus faecalis.

Enterococcus faecalis cells are known to have ferric reductase activity and the ability to transfer electrons generated in metabolism to the external environment. We have isolated mutants defective in ferric reductase activity and studied their electron transfer properties to electrodes mediated by ferric ions and an osmium complex-modified redox polymer (OsRP). Electron transfer mediated with ferric ions and ferric reductase activity were both found to be dependent on the membrane-associated Ndh3 and EetA proteins, consistent with findings in Listeria monocytogenes In contrast, electron transfer mediated with OsRP was independent of these two proteins. Quinone in the cell membrane was required for the electron transfer with both mediators. The combined results demonstrate that extracellular electron transfer from reduced quinone to ferric ions and to OsRP occurs via different routes in the cell envelope of E. faecalisIMPORTANCE The transfer of reducing power in the form of electrons, generated in the catabolism of nutrients, from a bacterium to an extracellular acceptor appears to be common in nature. The electron acceptor can be another cell or abiotic material. Such extracellular electron transfer contributes to syntrophic metabolism and is of wide environmental, industrial, and medical importance. Electron transfer between microorganisms and electrodes is fundamental in microbial fuel cells for energy production and for electricity-driven synthesis of chemical compounds in cells. In contrast to the much-studied extracellular electron transfer mediated by cell surface exposed cytochromes, little is known about components and mechanisms for such electron transfer in organisms without these cytochromes and in Gram-positive bacteria such as E. faecalis, which is a commensal gut lactic acid bacterium and opportunistic pathogen.

Forespore Targeting of SpoVD in Bacillus subtilis Is Mediated by the N-Terminal Part of the Protein.

SpoVD and PBP4b are structurally very similar high-molecular-weight, class B penicillin-binding proteins produced early during sporulation in Bacillus subtilis SpoVD is known to be essential for endospore cortex synthesis and thereby the production of heat-resistant spores. The role of PBP4b is still enigmatic. Both proteins are synthesized in the cytoplasm of the mother cell. PBP4b remains in the cytoplasmic membrane of the mother cell, whereas SpoVD accumulates in the forespore outer membrane. By the use of SpoVD/PBP4b chimeras with swapped protein domains, we show that the N-terminal part of SpoVD, containing the single transmembrane region, determines the forespore targeting of the protein.IMPORTANCE Beta-lactam-type antibiotics target penicillin-binding proteins (PBPs), which function in cell wall peptidoglycan synthesis. Bacteria of a subset of genera, including Bacillus and Clostridium species, can form endospores. The extreme resistance of endospores against harsh physicochemical conditions is of concern in clinical microbiology and the food industry. Endospore cortex layer biogenesis constitutes an experimental model system for research on peptidoglycan synthesis. The differentiation of a vegetative bacterial cell into an endospore involves the formation of a forespore within the cytoplasm of the sporulating cell. A number of proteins, including some PBPs, accumulate in the forespore. An understanding of the molecular mechanisms behind such subcellular targeting of proteins in bacterial cells can, for example, lead to a means of blocking the process of sporulation.

Extracellular Electron Transfer by the Gram-Positive Bacterium Enterococcus faecalis.

Extracellular electron transfer (EET) in microbial cells is essential for certain biotechnological applications and contributes to the biogeochemical cycling of elements and syntrophic microbial metabolism in complex natural environments. The Gram-positive lactic acid bacterium Enterococcus faecalis, an opportunistic human pathogen, is shown to be able to transfer electrons generated in fermentation metabolism to electrodes directly and indirectly via mediators. By exploiting E. faecalis wild-type and mutant cells, we demonstrate that reduced demethylmenaquinone in the respiratory chain in the bacterial cytoplasmic membrane is crucial for the EET. Heme proteins are not involved, and cytochrome bd oxidase activity was found to attenuate EET. These results are significant for the mechanistic understanding of EET in bacteria and for the design of microbial electrochemical systems. The basic findings infer that in dense microbial communities, such as in biofilm and in the large intestine, metabolism in E. faecalis and similar Gram-positive lactic acid bacteria might be electrically connected to other microbes. Such a transcellular electron transfer might confer syntrophic metabolism that promotes growth and other activities of bacteria in the microbiota of humans and animals.

Transpeptidase activity of penicillin-binding protein SpoVD in peptidoglycan synthesis conditionally depends on the disulfide reductase StoA.

Endospore cortex peptidoglycan synthesis is not required for bacterial growth but essential for endospore heat resistance. It therefore constitutes an amenable system for research on peptidoglycan biogenesis. The Bacillus subtilis sporulation-specific class B penicillin-binding protein (PBP) SpoVD and many homologous PBPs contain two conserved cysteine residues of unknown function in the transpeptidase domain - one as residue x in the SxN catalytic site motif and the other in a flexible loop near the catalytic site. A disulfide bond between these residues blocks the function of SpoVD in cortex synthesis. With a combination of experiments with purified proteins and B. subtilis mutant cells, it was shown that in active SpoVD the two cysteine residues most probably interact by hydrogen bonding and that this is important for peptidoglycan synthesis in vivo. It was furthermore demonstrated that the sporulation-specific thiol-disulfide oxidoreductase StoA reduces SpoVD and that requirement of StoA for cortex synthesis can be suppressed by two completely different types of structural alterations in SpoVD. It is concluded that StoA plays a critical role mainly during maturation of SpoVD in the forespore outer membrane. The findings advance our understanding of essential PBPs and redox control of extra-cytoplasmic protein disulfides in bacterial cells.

Heme A synthase in bacteria depends on one pair of cysteinyls for activity.

Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O.

In vitro assembly of catalase.

Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process.

The PASTA domain of penicillin-binding protein SpoVD is dispensable for endospore cortex peptidoglycan assembly in Bacillus subtilis.

Peptidoglycan is the major structural component of the bacterial cell wall. Penicillin-binding proteins (PBPs), located at the exterior of the cytoplasmic membrane, play a major role in peptidoglycan synthesis and remodelling. A PASTA domain (penicillin-binding protein and serine/threonine kinase associated domain) of about 65 residues is found at the C-terminal end of some PBPs and eukaryotic-like protein serine/threonine kinases in a variety of bacteria. The function of PASTA domains is not understood, but some of them are thought to bind uncross linked peptidoglycan. Bacillus subtilis has 16 different PBPs, but only 2 of them, Pbp2b and SpoVD, contain a PASTA domain. SpoVD is specific for sporulation and essential for endospore cortex peptidoglycan synthesis. We have studied the role of the PASTA domain in SpoVD by deleting this domain and analysing the effects on endospore formation and subcellular localization of SpoVD. Our results demonstrate that the PASTA domain in SpoVD is not essential for cortex synthesis and not important for targeting SpoVD to the forespore outer membrane during sporulation.

Heme A biosynthesis.

Respiration in plants, most animals and many aerobic microbes is dependent on heme A. This is a highly specialized type of heme found as prosthetic group in cytochrome a-containing respiratory oxidases. Heme A differs structurally from heme B (protoheme IX) by the presence of a hydroxyethylfarnesyl group instead of a vinyl side group at the C2 position and a formyl group instead of a methyl side group at position C8 of the porphyrin macrocycle. Heme A synthase catalyzes the formation of the formyl side group and is a poorly understood heme-containing membrane bound atypical monooxygenase. This review presents our current understanding of heme A synthesis at the molecular level in mitochondria and aerobic bacteria. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

The physical state of water in bacterial spores.

The bacterial spore, the hardiest known life form, can survive in a metabolically dormant state for many years and can withstand high temperatures, radiation, and toxic chemicals. The molecular basis of spore dormancy and resistance is not understood, but the physical state of water in the different spore compartments is thought to play a key role. To characterize this water in situ, we recorded the water (2)H and (17)O spin relaxation rates in D(2)O-exchanged Bacillus subtilis spores over a wide frequency range. The data indicate high water mobility throughout the spore, comparable with binary protein-water systems at similar hydration levels. Even in the dense core, the average water rotational correlation time is only 50 ps. Spore dormancy therefore cannot be explained by glass-like quenching of molecular diffusion but may be linked to dehydration-induced conformational changes in key enzymes. The data demonstrate that most spore proteins are rotationally immobilized, which may contribute to heat resistance by preventing heat-denatured proteins from aggregating irreversibly. We also find that the water permeability of the inner membrane is at least 2 orders of magnitude lower than for model membranes, consistent with the reported high degree of lipid immobilization in this membrane and with its proposed role in spore resistance to chemicals that damage DNA. The quantitative results reported here on water mobility and transport provide important clues about the mechanism of spore dormancy and resistance, with relevance to food preservation, disease prevention, and astrobiology.

Diversity of Cytochrome c Oxidase Assembly Proteins in Bacteria.

Cytochrome c oxidase in animals, plants and many aerobic bacteria functions as the terminal enzyme of the respiratory chain where it reduces molecular oxygen to form water in a reaction coupled to energy conservation. The three-subunit core of the enzyme is conserved, whereas several proteins identified to function in the biosynthesis of the common family A1 cytochrome c oxidase show diversity in bacteria. Using the model organisms Bacillus subtilis, Corynebacterium glutamicum, Paracoccus denitrificans, and Rhodobacter sphaeroides, the present review focuses on proteins for assembly of the heme a, heme a3, CuB, and CuA metal centers. The known biosynthesis proteins are, in most cases, discovered through the analysis of mutants. All proteins directly involved in cytochrome c oxidase assembly have likely not been identified in any organism. Limitations in the use of mutants to identify and functionally analyze biosynthesis proteins are discussed in the review. Comparative biochemistry helps to determine the role of assembly factors. This information can, for example, explain the cause of some human mitochondrion-based diseases and be used to find targets for new antimicrobial drugs. It also provides information regarding the evolution of aerobic bacteria.

Enterococcus faecalis NADH Peroxidase-Defective Mutants Stain Falsely in Colony Zymogram Assay for Extracellular Electron Transfer to Ferric Ions.

Enterococcus faecalis cells can reduce ferric ions and other electron acceptors by extracellular electron transfer (EET). To find mutants with enhanced or defective EET, strain OG1RF with random transposon insertions in the chromosome was screened for ferric reductase activity by colony zymogram staining using the chromogenic ferrous-chelating compound Ferrozine. The screen revealed npr, eetB, and ndh3 mutants. The aberrant ferric reductase phenotype of Npr (NADH peroxidase)-defective mutants was found to be a property of colonies and not apparent with washed cells grown in liquid culture. EetB- and Ndh3-defective mutants, in contrast, consistently showed low ferric reductase activity. It is concluded that colony zymogram staining for ferric reductase activity using Ferrozine can be misleading, especially through false negative results. It is suggested that hydrogen peroxide produced in the colony quenches the zymogram staining. In addition, it is demonstrated that the negative effect of heme on EET to ferric ion in E. faecalis is relieved by cytochrome bd deficiency. The findings can help to identify bacteria with EET ability and contribute to our understanding of EET in Gram-positive bacteria and the physiology of E. faecalis.

Penicillin-binding protein SpoVD disulphide is a target for StoA in Bacillus subtilis forespores.

The bacterial endospore is a dormant and heat-resistant form of life. StoA (SpoIVH) in Bacillus subtilis is a membrane-bound thioredoxin-like protein involved in endospore cortex synthesis. It is proposed to reduce disulphide bonds in hitherto unknown proteins in the intermembrane compartment of developing forespores. Starting with a bioinformatic analysis combined with mutant studies we identified the sporulation-specific, high-molecular-weight, class B penicillin-binding protein SpoVD as a putative target for StoA. We then demonstrate that SpoVD is a membrane-bound protein with two exposed redox-active cysteine residues. Structural modelling of SpoVD, based on the well characterized orthologue PBP2x of Streptococcus pneumoniae, confirmed that a disulphide bond can form close to the active site of the penicillin-binding domain restricting access of enzyme substrate or functional association with other cortex biogenic proteins. Finally, by exploiting combinations of mutations in the spoVD, stoA and ccdA genes in B. subtilis cells, we present strong in vivo evidence that supports the conclusion that StoA functions to specifically break the disulphide bond in the SpoVD protein in the forespore envelope. The findings contribute to our understanding of endospore biogenesis and open a new angle to regulation of cell wall synthesis and penicillin-binding protein activity.

Production, purification and detergent exchange of isotopically labeled Bacillussubtilis cytochrome b558 (SdhC).

Cytochrome b558 of the gram-positive bacterium Bacillussubtilis is the membrane anchor subunit of the succinate:quinone oxidoreductase of the citric acid cycle. The cytochrome consists of the SdhC polypeptide (202 residues) and two protoheme IX groups that function in transmembrane electron transfer to menaquinone. The general structure of the cytochrome is known from extensive experimental studies and by comparison to Wolinellasuccinogenes fumarate reductase for which the X-ray crystal structure has been determined. Solution state NMR can potentially be used to identify the quinone binding site(s) and study, e.g. redox-linked, dynamics of cytochrome b558. In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome b558 produced in Escherichia coli. We have also evaluated several detergents suitable for NMR for their effectiveness in maintaining the cytochrome solubilized and intact for days at room temperature.

Respiratory Heme A-Containing Oxidases Originated in the Ancestors of Iron-Oxidizing Bacteria.

Respiration is a major trait shaping the biology of many environments. Cytochrome oxidase containing heme A (COX) is a common terminal oxidase in aerobic bacteria and is the only one in mammalian mitochondria. The synthesis of heme A is catalyzed by heme A synthase (CtaA/Cox15), an enzyme that most likely coevolved with COX. The evolutionary origin of COX in bacteria has remained unknown. Using extensive sequence and phylogenetic analysis, we show that the ancestral type of heme A synthases is present in iron-oxidizing Proteobacteria such as Acidithiobacillus spp. These bacteria also contain a deep branching form of the major COX subunit (COX1) and an ancestral variant of CtaG, a protein that is specifically required for COX biogenesis. Our work thus suggests that the ancestors of extant iron-oxidizers were the first to evolve COX. Consistent with this conclusion, acidophilic iron-oxidizing prokaryotes lived on emerged land around the time for which there is the earliest geochemical evidence of aerobic respiration on earth. Hence, ecological niches of iron oxidation have apparently promoted the evolution of aerobic respiration.

Haem-delivery proteins in cytochrome c maturation System II.

Cytochromes of the c-type function on the outer side of the cytoplasmic membrane in bacteria where they also are assembled from apo-cytochrome polypeptide and haem. Two distinctly different systems for cytochrome c maturation are found in bacteria. System I present in Escherichia coli has eight to nine different Ccm proteins. System II is found in Bacillus subtilis and comprises four proteins: CcdA, ResA, ResB and ResC. ResB and ResC are poorly understood polytopic membrane proteins required for cytochrome c synthesis. We have analysed these two B. subtilis proteins produced in E. coli and in the native organism. ResB is shown to bind protohaem IX and haem is found covalently bound to residue Cys-138. Results in B. subtilis suggest that also ResC can bind haem. Our results complement recent findings made with Helicobacter CcsBA supporting the hypothesis that ResBC as a complex translocates haem by attaching it to ResB on the cytoplasmic side of the membrane and then transferring it to an extra-cytoplasmic location in ResC, from where it is made available to the apo-cytochromes.

Quantification of membrane proteins using nonspecific protease digestions.

We present a mass spectrometry-based method for the identification and quantification of membrane proteins using the low-specificity protease Proteinase K, at very high pH, to digest proteins isolated by a modified SDS-PAGE protocol. The resulting peptides are modified with a fragmentation-directing isotope labeled tag. We apply the method to quantify differences in membrane protein expression of Bacillus subtilis grown in the presence or absence of glucose.

Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA.

The thiol-disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pK(a) values for the CEPC (one-letter code) active-site cysteine residues of ResA are unusual for thioredoxin-like proteins in that they are both high (>8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid-base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pK(a) values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, DeltapK(a) values of -2.5 were measured for both cysteine residues. Increases in midpoint reduction potentials were also observed, although these were comparatively small: CPHC (DsbA-type) ResA exhibited an increase of +40 mV compared with the wild-type protein. Unfolding studies revealed that, despite the observed differences in the properties of the reduced proteins, changes in stability were largely confined to the oxidized state. High-resolution structures of two of the variants (CEHC and CPHC ResA) in their reduced states were determined and are discussed in terms of the observed changes in properties. Finally, the in vivo functional properties of CEHC ResA are shown to be significantly affected compared with those of the wild-type protein.

Extracellular electron transfer features of Gram-positive bacteria.

Electroactive microorganisms possess the unique ability to transfer electrons to or from solid phase electron conductors, e.g., electrodes or minerals, through various physiological mechanisms. The processes are commonly known as extracellular electron transfer and broadly harnessed in microbial electrochemical systems, such as microbial biosensors, microbial electrosynthesis, or microbial fuel cells. Apart from a few model microorganisms, the nature of the microbe-electrode conductive interaction is poorly understood for most of the electroactive species. The interaction determines the efficiency and a potential scaling up of bioelectrochemical systems. Gram-positive bacteria generally have a thick electron non-conductive cell wall and are believed to exhibit weak extracellular electron shuttling activity. This review highlights reported research accomplishments on electroactive Gram-positive bacteria. The use of electron-conducting polymers as mediators is considered as one promising strategy to enhance the electron transfer efficiency up to application scale. In view of the recent progress in understanding the molecular aspects of the extracellular electron transfer mechanisms of Enterococcus faecalis, the electron transfer properties of this bacterium are especially focused on. Fundamental knowledge on the nature of microbial extracellular electron transfer and its possibilities can provide insight in interspecies electron transfer and biogeochemical cycling of elements in nature. Additionally, a comprehensive understanding of cell-electrode interactions may help in overcoming insufficient electron transfer and restricted operational performance of various bioelectrochemical systems and facilitate their practical applications.