Characterisation of anhydro-sialic acid transporters from mucosa-associated bacteria.

Sialic acid (Sia) transporters are critical to the capacity of host-associated bacteria to utilise Sia for growth and/or cell surface modification. While N-acetyl-neuraminic acid (Neu5Ac)-specific transporters have been studied extensively, little is known on transporters dedicated to anhydro-Sia forms such as 2,7-anhydro-Neu5Ac (2,7-AN) or 2,3-dehydro-2-deoxy-Neu5Ac (Neu5Ac2en). Here, we used a Sia-transport-null strain of Escherichia coli to investigate the function of members of anhydro-Sia transporter families previously identified by computational studies. First, we showed that the transporter NanG, from the Glycoside-Pentoside-Hexuronide:cation symporter family, is a specific 2,7-AN transporter, and identified by mutagenesis a crucial functional residue within the putative substrate-binding site. We then demonstrated that NanX transporters, of the Major Facilitator Superfamily, also only transport 2,7-AN and not Neu5Ac2en nor Neu5Ac. Finally, we provided evidence that SiaX transporters, of the Sodium-Solute Symporter superfamily, are promiscuous Neu5Ac/Neu5Ac2en transporters able to acquire either substrate equally well. The characterisation of anhydro-Sia transporters expands our current understanding of prokaryotic Sia metabolism within host-associated microbial communities.

A holin/peptidoglycan hydrolase-dependent protein secretion system.

Gram-negative bacteria have evolved numerous pathways to secrete proteins across their complex cell envelopes. Here, we describe a protein secretion system that uses a holin membrane protein in tandem with a cell wall-editing enzyme to mediate the secretion of substrate proteins from the periplasm to the cell exterior. The identity of the cell wall-editing enzymes involved was found to vary across biological systems. For instance, the chitinase secretion pathway of Serratia marcescens uses an endopeptidase to facilitate secretion, whereas the secretion of Typhoid toxin in Salmonella enterica serovar Typhi relies on a muramidase. Various families of holins are also predicted to be involved. Genomic analysis indicates that this pathway is conserved and implicated in the secretion of hydrolytic enzymes and toxins for a range of bacteria. The pairing of holins from different families with various types of peptidoglycan hydrolases suggests that this secretion pathway evolved multiple times. We suggest that the complementary bodies of evidence presented is sufficient to propose that the pathway be named the Type 10 Secretion System (TXSS).

A paean to the ineffable Marjory Stephenson.

It is now 75 years since Marjory Stephenson became the second President of the Society for General Microbiology (SGM). Around the time of her death at the end of 1948 many articles appeared extolling Marjory Stephenson's contribution to the fields of Biochemistry and Microbiology. Not that much has been written about her since that time, which is unfortunate. Therefore, this brief review is intended as a form of redress and aims to highlight the role of this remarkable scientist in establishing the Society and in promoting Microbiology as a discipline. Notwithstanding the significance of these achievements, however, it is her overall impact on the field of 'Chemical Microbiology' and what she achieved through her research that are extraordinary, even by today's standards. Marjory Stephenson recognized that in order to understand a biological system, the 'whole' organism must be considered and this can only be achieved by adopting an interdisciplinary approach: inorganic and organic chemistry, biochemistry, genetics, metabolism and ultimately physiology. Her scientific ethos serves today as a beacon for how scientific research should be conducted, and what we as scientists can learn about how to inspire and mentor the next generation. It is impossible to overstate Marjory Stephenson's scientific legacy, or her overall contribution to Microbiology.

Hydrogen production in the presence of oxygen by Escherichia coli K-12.

Escherichia coli is a facultative anaerobe that can grow in a variety of environmental conditions. In the complete absence of O2, E. coli can perform a mixed-acid fermentation that contains within it an elaborate metabolism of formic acid. In this study, we use cavity-enhanced Raman spectroscopy (CERS), FTIR, liquid Raman spectroscopy, isotopic labelling and molecular genetics to make advances in the understanding of bacterial formate and H2 metabolism. It is shown that, under anaerobic (anoxic) conditions, formic acid is generated endogenously, excreted briefly from the cell, and then taken up again to be disproportionated to H2 and CO2 by formate hydrogenlyase (FHL-1). However, exogenously added D-labelled formate behaves quite differently from the endogenous formate and is taken up immediately, independently, and possibly by a different mechanism, by the cell and converted to H2 and CO2. Our data support an anion-proton symport model for formic acid transport. In addition, when E. coli was grown in a micro-aerobic (micro-oxic) environment it was possible to analyse aspects of formate and O2 respiration occurring alongside anaerobic metabolism. While cells growing under micro-aerobic conditions generated endogenous formic acid, no H2 was produced. However, addition of exogenous formate at the outset of cell growth did induce FHL-1 biosynthesis and resulted in formate-dependent H2 production in the presence of O2.

Harnessing Escherichia coli for Bio-Based Production of Formate under Pressurized H2 and CO2 Gases.

Escherichia coli is a Gram-negative bacterium that is a workhorse for biotechnology. The organism naturally performs a mixed-acid fermentation under anaerobic conditions where it synthesizes formate hydrogenlyase (FHL-1). The physiological role of the enzyme is the disproportionation of formate into H2 and CO2. However, the enzyme has been observed to catalyze hydrogenation of CO2 given the correct conditions, and so it has possibilities in bio-based carbon capture and storage if it can be harnessed as a hydrogen-dependent CO2 reductase (HDCR). In this study, an E. coli host strain was engineered for the continuous production of formic acid from H2 and CO2 during bacterial growth in a pressurized batch bioreactor. Incorporation of tungsten, in place of molybdenum, in FHL-1 helped to impose a degree of catalytic bias on the enzyme. This work demonstrates that it is possible to couple cell growth to simultaneous, unidirectional formate production from carbon dioxide and develops a process for growth under pressurized gases. IMPORTANCE Greenhouse gas emissions, including waste carbon dioxide, are contributing to global climate change. A basket of solutions is needed to steadily reduce emissions, and one approach is bio-based carbon capture and storage. Here, we present our latest work on harnessing a novel biological solution for carbon capture. The Escherichia coli formate hydrogenlyase (FHL-1) was engineered to be constitutively expressed. Anaerobic growth under pressurized H2 and CO2 gases was established, and aqueous formic acid was produced as a result. Incorporation of tungsten into the enzyme in place of molybdenum proved useful in poising FHL-1 as a hydrogen-dependent CO2 reductase (HDCR).

The plant pathogen Pectobacterium atrosepticum contains a functional formate hydrogenlyase-2 complex.

Pectobacterium atrosepticum SCRI1043 is a phytopathogenic Gram-negative enterobacterium. Genomic analysis has identified that genes required for both respiration and fermentation are expressed under anaerobic conditions. One set of anaerobically expressed genes is predicted to encode an important but poorly understood membrane-bound enzyme termed formate hydrogenlyase-2 (FHL-2), which has fascinating evolutionary links to the mitochondrial NADH dehydrogenase (Complex I). In this work, molecular genetic and biochemical approaches were taken to establish that FHL-2 is fully functional in P. atrosepticum and is the major source of molecular hydrogen gas generated by this bacterium. The FHL-2 complex was shown to comprise a rare example of an active [NiFe]-hydrogenase-4 (Hyd-4) isoenzyme, itself linked to an unusual selenium-free formate dehydrogenase in the final complex. In addition, further genetic dissection of the genes encoding the predicted membrane arm of FHL-2 established surprisingly that the majority of genes encoding this domain are not required for physiological hydrogen production activity. Overall, this study presents P. atrosepticum as a new model bacterial system for understanding anaerobic formate and hydrogen metabolism in general, and FHL-2 function and structure in particular.

Activation of a [NiFe]-hydrogenase-4 isoenzyme by maturation proteases.

Maturation of [NiFe]-hydrogenases often involves specific proteases responsible for cleavage of the catalytic subunits. Escherichia coli HycI is the protease dedicated to maturation of the Hydrogenase-3 isoenzyme, a component of formate hydrogenlyase-1. In this work, it is demonstrated that a Pectobacterium atrosepticum HycI homologue, HyfK, is required for hydrogenase-4 activity, a component of formate hydrogenlyase-2, in that bacterium. The P. atrosepticum ΔhyfK mutant phenotype could be rescued by either P. atrosepticum hyfK or E. coli hycI on a plasmid. Conversely, an E. coli ΔhycI mutant was complemented by either E. coli hycI or P. atrosepticum hyfK in trans. E. coli is a rare example of a bacterium containing both hydrogenase-3 and hydrogenase-4, however the operon encoding hydrogenase-4 has no maturation protease gene. This work suggests HycI should be sufficient for maturation of both E. coli formate hydrogenlyases, however no formate hydrogenlyase-2 activity was detected in any E. coli strains tested here.

Controlling and co-ordinating chitinase secretion in a Serratia marcescens population.

Serratia marcescens is a γ-Proteobacterium and an opportunistic animal and insect pathogen. The bacterium exhibits a complex extracellular protein 'secretome' comprising numerous enzymes, toxins and effector molecules. One component of the secretome is the 'chitinolytic machinery', which is a set of at least four chitinases that allow the use of insoluble extracellular chitin as sole carbon source. Secretion of the chitinases across the outer membrane is governed by the chiWXYZ operon encoding a holin/endopeptidase pair. Expression of the chiWXYZ operon is co-ordinated with the chitinase genes and is also bimodal, as normally only 1% of the population expresses the chitinolytic machinery. In this study, the role of the ChiR protein in chitinase production has been explored. Using live cell imaging and flow cytometry, ChiR was shown to govern the co-ordinated regulation of chiWXYZ with both chiA and chiC. Moreover, overexpression of chiR alone was able to increase the proportion of the cell population expressing chitinase genes to >60 %. In addition, quantitative label-free proteomic analysis of cells overexpressing chiR established that ChiR regulates the entire chitinolytic machinery. The proteomic experiments also revealed a surprising link between the regulation of the chitinolytic machinery and the production of proteins involved in the metabolism of nitrogen compounds such as nitrate and nitrite. The research demonstrates for the first time that ChiR plays a critical role in controlling bimodal gene expression in S. marcescens, and provides new evidence of a clear link between chitin breakdown and nitrogen metabolism.

Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens.

The Gram-negative bacterium Serratia marcescens secretes many proteins that are involved in extracellular chitin degradation. This so-called chitinolytic machinery includes three types of chitinase enzymes and a lytic polysaccharide monooxygenase. An operon has been identified in S. marcescens, chiWXYZ, that is thought to be involved in the secretion of the chitinolytic machinery. Genetic evidence points to the ChiX protein being a key player in the secretion mechanism, since deletion of the chiX gene in S. marcescens led to a mutant strain blocked for secretion of all members of the chitinolytic machinery. In this work, a detailed structural and biochemical characterisation of ChiX is presented. The high-resolution crystal structure of ChiX reveals the protein to be a member of the LAS family of peptidases. ChiX is shown to be a zinc-containing metalloenzyme, and in vitro assays demonstrate that ChiX is an l-Ala d-Glu endopeptidase that cleaves the cross-links in bacterial peptidoglycan. This catalytic activity is shown to be intimately linked with the secretion of the chitinolytic machinery, since substitution of the ChiX Asp-120 residue results in a variant protein that is both unable to digest peptidoglycan and cannot rescue the phenoytype of a chiX mutant strain.

The structure of hydrogenase-2 from Escherichia coli: implications for H2-driven proton pumping.

Under anaerobic conditions, Escherichia coli is able to metabolize molecular hydrogen via the action of several [NiFe]-hydrogenase enzymes. Hydrogenase-2, which is typically present in cells at low levels during anaerobic respiration, is a periplasmic-facing membrane-bound complex that functions as a proton pump to convert energy from hydrogen (H2) oxidation into a proton gradient; consequently, its structure is of great interest. Empirically, the complex consists of a tightly bound core catalytic module, comprising large (HybC) and small (HybO) subunits, which is attached to an Fe-S protein (HybA) and an integral membrane protein (HybB). To date, efforts to gain a more detailed picture have been thwarted by low native expression levels of Hydrogenase-2 and the labile interaction between HybOC and HybA/HybB subunits. In the present paper, we describe a new overexpression system that has facilitated the determination of high-resolution crystal structures of HybOC and, hence, a prediction of the quaternary structure of the HybOCAB complex.

Mechanism of hydrogen activation by [NiFe] hydrogenases.

The active site of [NiFe] hydrogenases contains a strictly conserved arginine that suspends a guanidine nitrogen atom <4.5 Å above the nickel and iron atoms. The guanidine headgroup interacts with the side chains of two conserved aspartic acid residues to complete an outer-shell canopy that has thus far proved intractable to investigation by site-directed mutagenesis. Using hydrogenase-1 from Escherichia coli, the strictly conserved residues R509 and D574 have been replaced by lysine (R509K) and asparagine (D574N) and the highly conserved D118 has been replaced by alanine (D118A) or asparagine (D118N/D574N). Each enzyme variant is stable, and their [(RS)2Niµ(SR)2Fe(CO)(CN)2] inner coordination shells are virtually unchanged. The R509K variant had >100-fold lower activity than native enzyme. Conversely, the variants D574N, D118A and D118N/D574N, in which the position of the guanidine headgroup is retained, showed 83%, 26% and 20% activity, respectively. The special kinetic requirement for R509 implicates the suspended guanidine group as the general base in H2 activation by [NiFe] hydrogenases.

The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex.

The assembly of multi-protein complexes requires the concerted synthesis and maturation of its components and subsequently their co-ordinated interaction. The membrane-bound formate hydrogenlyase (FHL) complex is the primary hydrogen-producing enzyme in Escherichia coli and is composed of seven subunits mostly encoded within the hycA-I operon for [NiFe]-hydrogenase-3 (Hyd-3). The HycH protein is predicted to have an accessory function and is not part of the final structural FHL complex. In this work, a mutant strain devoid of HycH was characterised and found to have significantly reduced FHL activity due to the instability of the electron transfer subunits. HycH was shown to interact specifically with the unprocessed species of HycE, the catalytic hydrogenase subunit of the FHL complex, at different stages during the maturation and assembly of the complex. Variants of HycH were generated with the aim of identifying interacting residues and those that influence activity. The R70/71/K72, the Y79, the E81 and the Y128 variant exchanges interrupt the interaction with HycE without influencing the FHL activity. In contrast, FHL activity, but not the interaction with HycE, was negatively influenced by H37 exchanges with polar residues. Finally, a HycH Y30 variant was unstable. Surprisingly, an overlapping function between HycH with its homologous counterpart HyfJ from the operon encoding [NiFe]-hydrogenase-4 (Hyd-4) was identified and this is the first example of sharing maturation machinery components between Hyd-3 and Hyd-4 complexes. The data presented here show that HycH has a novel dual role as an assembly chaperone for a cytoplasmic [NiFe]-hydrogenase.

Expanding the substrates for a bacterial hydrogenlyase reaction.

Escherichia coli produces enzymes dedicated to hydrogen metabolism under anaerobic conditions. In particular, a formate hydrogenlyase (FHL) enzyme is responsible for the majority of hydrogen gas produced under fermentative conditions. FHL comprises a formate dehydrogenase (encoded by fdhF) linked directly to [NiFe]-hydrogenase-3 (Hyd-3), and formate is the only natural substrate known for proton reduction by this hydrogenase. In this work, the possibility of engineering an alternative electron donor for hydrogen production has been explored. Rational design and genetic engineering led to the construction of a fusion between Thermotoga maritima ferredoxin (Fd) and Hyd-3. The Fd-Hyd-3 fusion was found to evolve hydrogen when co-produced with T. maritima pyruvate :: ferredoxin oxidoreductase (PFOR), which links pyruvate oxidation to the reduction of ferredoxin. Analysis of the key organic acids produced during fermentation suggested that the PFOR/Fd-Hyd-3 fusion system successfully diverted pyruvate onto a new pathway towards hydrogen production.

Design and characterisation of synthetic operons for biohydrogen technology.

Biohydrogen is produced by a number of microbial systems and the commonly used host bacterium Escherichia coli naturally produces hydrogen under fermentation conditions. One approach to engineering additional hydrogen production pathways is to introduce non-native hydrogenases into E. coli. An attractive candidate is the soluble [NiFe]-hydrogenase from Ralstonia eutropha, which has been shown to link NADH/NAD+ biochemistry directly to hydrogen metabolism, an activity that E. coli does not perform. In this work, three synthetic operons were designed that code for the soluble hydrogenase and two different enzyme maturase systems. Interestingly, using this system, the recombinant soluble hydrogenase was found to be assembled by the native E. coli [NiFe]-hydrogenase assembly machinery, and, vice versa, the synthetic maturase operons were able to complement E. coli mutants defective in hydrogenase biosynthesis. The heterologously expressed soluble hydrogenase was found to be active and was shown to produce biohydrogen in vivo.

How oxygen reacts with oxygen-tolerant respiratory [NiFe]-hydrogenases.

An oxygen-tolerant respiratory [NiFe]-hydrogenase is proven to be a four-electron hydrogen/oxygen oxidoreductase, catalyzing the reaction 2 H2 + O2 = 2 H2O, equivalent to hydrogen combustion, over a sustained period without inactivating. At least 86% of the H2O produced by Escherichia coli hydrogenase-1 exposed to a mixture of 90% H2 and 10% O2 is accounted for by a direct four-electron pathway, whereas up to 14% arises from slower side reactions proceeding via superoxide and hydrogen peroxide. The direct pathway is assigned to O2 reduction at the [NiFe] active site, whereas the side reactions are an unavoidable consequence of the presence of low-potential relay centers that release electrons derived from H2 oxidation. The oxidase activity is too slow to be useful in removing O2 from the bacterial periplasm; instead, the four-electron reduction of molecular oxygen to harmless water ensures that the active site survives to catalyze sustained hydrogen oxidation.

Bacterial formate hydrogenlyase complex.

Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimately produces molecular hydrogen. The enzyme directly responsible for hydrogen production is the membrane-bound formate hydrogenlyase (FHL) complex, which links formate oxidation to proton reduction and has evolutionary links to Complex I, the NADH:quinone oxidoreductase. Although the genetics, maturation, and some biochemistry of FHL are understood, the protein complex has never been isolated in an intact form to allow biochemical analysis. In this work, genetic tools are reported that allow the facile isolation of FHL in a single chromatographic step. The core complex is shown to comprise HycE (a [NiFe] hydrogenase component termed Hyd-3), FdhF (the molybdenum-dependent formate dehydrogenase-H), and three iron-sulfur proteins: HycB, HycF, and HycG. A proportion of this core complex remains associated with HycC and HycD, which are polytopic integral membrane proteins believed to anchor the core complex to the cytoplasmic side of the membrane. As isolated, the FHL complex retains formate hydrogenlyase activity in vitro. Protein film electrochemistry experiments on Hyd-3 demonstrate that it has a unique ability among [NiFe] hydrogenases to catalyze production of H2 even at high partial pressures of H2. Understanding and harnessing the activity of the FHL complex is critical to advancing future biohydrogen research efforts.

Biosynthesis of selenate reductase in Salmonella enterica: critical roles for the signal peptide and DmsD.

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium with a flexible respiratory capability. Under anaerobic conditions, S. enterica can utilize a range of terminal electron acceptors, including selenate, to sustain respiratory electron transport. The S. enterica selenate reductase is a membrane-bound enzyme encoded by the ynfEFGH-dmsD operon. The active enzyme is predicted to comprise at least three subunits where YnfE is a molybdenum-containing catalytic subunit. The YnfE protein is synthesized with an N-terminal twin-arginine signal peptide and biosynthesis of the enzyme is coordinated by a signal peptide binding chaperone called DmsD. In this work, the interaction between S. enterica DmsD and the YnfE signal peptide has been studied by chemical crosslinking. These experiments were complemented by genetic approaches, which identified the DmsD binding epitope within the YnfE signal peptide. YnfE signal peptide residues L24 and A28 were shown to be important for assembly of an active selenate reductase. Conversely, a random genetic screen identified the DmsD V16 residue as being important for signal peptide recognition and selenate reductase assembly.

Hydrogen activation by [NiFe]-hydrogenases.

Hydrogenase-1 (Hyd-1) from Escherichia coli is a membrane-bound enzyme that catalyses the reversible oxidation of molecular H2 The active site contains one Fe and one Ni atom and several conserved amino acids including an arginine (Arg(509)), which interacts with two conserved aspartate residues (Asp(118) and Asp(574)) forming an outer shell canopy over the metals. There is also a highly conserved glutamate (Glu(28)) positioned on the opposite side of the active site to the canopy. The mechanism of hydrogen activation has been dissected by site-directed mutagenesis to identify the catalytic base responsible for splitting molecular hydrogen and possible proton transfer pathways to/from the active site. Previous reported attempts to mutate residues in the canopy were unsuccessful, leading to an assumption of a purely structural role. Recent discoveries, however, suggest a catalytic requirement, for example replacing the arginine with lysine (R509K) leaves the structure virtually unchanged, but catalytic activity falls by more than 100-fold. Variants containing amino acid substitutions at either or both, aspartates retain significant activity. We now propose a new mechanism: heterolytic H2 cleavage is via a mechanism akin to that of a frustrated Lewis pair (FLP), where H2 is polarized by simultaneous binding to the metal(s) (the acid) and a nitrogen from Arg(509) (the base).

How the oxygen tolerance of a [NiFe]-hydrogenase depends on quaternary structure.

'Oxygen-tolerant' [NiFe]-hydrogenases can catalyze H2 oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O2 molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H2 oxidation released at the active site of one partner are immediately transferred to its counterpart when an O2 molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O2-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins.

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins.

A subset of bacterial [NiFe]-hydrogenases have been shown to be capable of activating dihydrogen-catalysis under aerobic conditions; however, it remains relatively unclear how the assembly and activation of these enzymes is carried out in the presence of air. Acquiring this knowledge is important if a generic method for achieving production of O2-resistant [NiFe]-hydrogenases within heterologous hosts is to be developed. Salmonella enterica serovar Typhimurium synthesizes the [NiFe]-hydrogenase-5 (Hyd-5) enzyme under aerobic conditions. As well as structural genes, the Hyd-5 operon also contains several accessory genes that are predicted to be involved in different stages of biosynthesis of the enzyme. In this work, deletions in the hydF, hydG, and hydH genes have been constructed. The hydF gene encodes a protein related to Ralstonia eutropha HoxO, which is known to interact with the small subunit of a [NiFe]-hydrogenase. HydG is predicted to be a fusion of the R. eutropha HoxQ and HoxR proteins, both of which have been implicated in the biosynthesis of an O2-tolerant hydrogenase, and HydH is a homologue of R. eutropha HoxV, which is a scaffold for [NiFe] cofactor assembly. It is shown here that HydG and HydH play essential roles in Hyd-5 biosynthesis. Hyd-5 can be isolated and characterized from a ΔhydF strain, indicating that HydF may not play the same vital role as the orthologous HoxO. This study, therefore, emphasises differences that can be observed when comparing the function of hydrogenase maturases in different biological systems.