Matthew G. Hicks

The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster

The regulatory protein NsrR, a member of the Rrf2 family of transcription repressors, is specifically dedicated to sensing nitric oxide (NO) in a variety of pathogenic and non-pathogenic bacteria. It has been proposed that NO directly modulates NsrR activity by interacting with a predicted [Fe-S] cluster in the NsrR protein, but no experimental evidence has been published to support this hypothesis. Here we report the purification of NsrR from the obligate aerobe Streptomyces coelicolor. We demonstrate using UV-visible, near UV CD and EPR spectroscopy that the protein contains an NO-sensitive [2Fe-2S] cluster when purified from E. coli. Upon exposure of NsrR to NO, the cluster is nitrosylated, which results in the loss of DNA binding activity as detected by bandshift assays. Removal of the [2Fe-2S] cluster to generate apo-NsrR also resulted in loss of DNA binding activity. This is the first demonstration that NsrR contains an NO-sensitive [2Fe-2S] cluster that is required for DNA binding activity.

The Escherichia coli twin-arginine translocase: conserved residues of TatA and TatB family components involved in protein transport

The Escherichia coli Tat system serves to export folded proteins harbouring an N‐terminal twin‐arginine signal peptide across the cytoplasmic membrane. In this report we have studied the functions of conserved residues within the structurally related TatA and TatB proteins. Our results demonstrate that there are two regions within each protein of high sequence conservation that are critical for efficient Tat translocase function. The first region is the interdomain hinge between the transmembrane and the amphipathic α‐helices of TatA and TatB proteins. The second region is within the amphipathic helices of TatA and TatB. In particular an invariant phenylalanine residue within TatA proteins is essential for activity, whereas a string of glutamic acid residues on the same face of the amphipathic helix of TatB is important for function.

Cysteine Scanning Mutagenesis and Disulfide Mapping Studies of the TatA Component of the Bacterial Twin Arginine Translocase

The Tat (twin arginine translocation) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The integral membrane proteins TatA, TatB, and TatC are essential components of the Tat pathway. TatA forms high order oligomers and is thought to constitute the protein-translocating unit of the Tat system. Cysteine scanning mutagenesis was used to systematically investigate the functional importance of residues in the essential N-terminal transmembrane and amphipathic helices of Escherichia coli TatA. Cysteine substitutions of most residues in the amphipathic helix, including all the residues on the hydrophobic face of the helix, severely compromise Tat function. Glutamine 8 was identified as the only residue in the transmembrane helix that is critical for TatA function. The cysteine variants in the transmembrane helix were used in disulfide mapping experiments to probe the oligomeric arrangement of TatA protomers within the larger TatA complex. Residues in the center of the transmembrane helix (including residues 10–16) show a distinct pattern of cross-linking indicating that this region of the protein forms well defined interactions with other protomers. At least two interacting faces were detected. The results of our TatA studies are compared with analogous data for the homologous, but functionally distinct, TatB protein. This comparison reveals that it is only in TatA that the amphipathic helix is sensitive to amino acid substitutions. The TatA amphipathic helix may play a role in forming and controlling the path of substrate movement across the membrane.

Investigating lipoprotein biogenesis and function in the model Gram‐positive bacterium Streptomyces coelicolor

Lipoproteins are a distinct class of bacterial membrane proteins that are translocated across the cytoplasmic membrane primarily by the Sec general secretory pathway and then lipidated on a conserved cysteine by the enzyme lipoprotein diacylglycerol transferase (Lgt). The signal peptide is cleaved by lipoprotein signal peptidase (Lsp) to leave the lipid‐modified cysteine at the N‐terminus of the mature lipoprotein. In all Gram‐positive bacteria tested to date this pathway is non‐essential and the lipid attaches the protein to the outer leaflet of the cytoplasmic membrane. Here we identify lipoproteins in the model Gram‐positive bacterium Streptomyces coelicolor using bioinformatics coupled with proteomic and downstream analysis. We report that Streptomyces species translocate large numbers of lipoproteins out via the Tat (twin arginine translocase) pathway and we present evidence that lipoprotein biogenesis might be an essential pathway in S. coelicolor. This is the first analysis of lipoproteins and lipoprotein biogenesis in Streptomyces and provides the first evidence that lipoprotein biogenesis could be essential in a Gram‐positive bacterium. This report also provides the first experimental evidence that Tat plays a major role in the translocation of lipoproteins in a specific bacterium.

Positive Selection for Loss-of-Function tat Mutations Identifies Critical Residues Required for TatA Activity

The Tat system, found in the cytoplasmic membrane of many bacteria, is a general export pathway for folded proteins. Here we describe the development of a method, based on the transport of chloramphenicol acetyltransferase, that allows positive selection of mutants defective in Tat function. We have demonstrated the utility of this method by selecting novel loss-of-function alleles of tatA from a pool of random tatA mutations. Most of the mutations that were isolated fall in the amphipathic region of TatA, emphasizing the pivotal role that this part of the protein plays in TatA function.

Dissecting the complete lipoprotein biogenesis pathway in Streptomyces scabies

Following translocation, bacterial lipoproteins are lipidated by lipoprotein diacylglycerol transferase (Lgt) and cleaved of their signal peptides by lipoprotein signal peptidase (Lsp). In Gram‐negative bacteria and mycobacteria, lipoproteins are further lipidated by lipoprotein N‐acyl transferase (Lnt), to give triacylated lipoproteins. Streptomyces are unusual amongst Gram‐positive bacteria because they export large numbers of lipoproteins via the twin arginine protein transport (Tat) pathway. Furthermore, some Streptomyces species encode two Lgt homologues and all Streptomyces species encode two homologues of Lnt. Here we characterize lipoprotein biogenesis in the plant pathogen Streptomyces scabies and report that lgt and lsp mutants are defective in growth and development while only moderately affected in virulence. Lipoproteins are lost from the membrane in an S. scabies lgt mutant but restored by expression of Streptomyces coelicolor lgt1 or lgt2 confirming that both encode functional Lgt enzymes. Furthermore, lipoproteins are N‐acylated in Streptomyces with efficient N‐acylation dependent on Lnt1 and Lnt2. However, deletion of lnt1 and lnt2 has no effect on growth, development or virulence. We thus present a detailed study of lipoprotein biogenesis in Streptomyces, the first study of Lnt function in a monoderm bacterium and the first study of bacterial lipoproteins as virulence factors in a plant pathogen.

Crystal Structure of the Acid Induced Arginine Decarboxylase from Escherichia Coli: Reversible Decamer Assembly Controls Enzyme Activity.

The acid-induced arginine decarboxylase is part of an enzymatic system in Escherichia coli that contributes to making this organism acid resistant. The arginine decarboxylase is a vitamin B(6)-dependent enzyme that is active at acidic pH. It consumes a proton in the decarboxylation of arginine to agmatine, and by working in tandem with an arginine-agmatine antiporter, this enzymatic cycle protects the organism by preventing the accumulation of protons inside the cell. We have determined the structure of the acid-induced arginine decarboxylase by X-ray crystallography to 2.4 A resolution. The arginine decarboxylase structure revealed a ca. 800 kDa decamer composed as a pentamer of five homodimers. Each homodimer has an abundance of acidic surface residues, which at neutral pH prevents inactive homodimers from associating into active decamers. Conversely, acidic conditions favor the assembly of active decamers. Therefore, the structure of arginine decarboxylase presents a mechanism by which its activity is modulated by external pH.

The c-Type Cytochrome OmcA Localizes to the Outer Membrane upon Heterologous Expression in Escherichia coli

We have functionally produced the outer membrane cytochrome OmcA from Shewanella oneidensis in Escherichia coli. Substrate accessibility experiments indicate that OmcA is surface exposed in an E. coli B strain but not in a K-12 strain. We show that a functional type II secretion system is required for surface localization.

Formation of functional Tat translocases from heterologous components

BackgroundThe Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plants. In Eschericha coli, Tat transport requires the integral membrane proteins TatA, TatB and TatC. In this study we have tested the ability of tat genes from the eubacterial species Pseudomonas syringae, Streptomyces coelicolor and Aquifex aeolicus, to compensate for the absence of the cognate E. coli tat gene, and thus to form functional Tat translocases with E. coli Tat components.ResultsAll three subunits of the Tat system from the Gram positive organism Streptomyces coelicolor were able to form heterologous translocases with substantive Tat transport activity. However, only the TatA and TatB proteins of Pseudomonas syringae were able to functionally interact with the E. coli Tat system even though the two organisms are closely related. Of the Tat components from the phylogenetically distant hyperthermophillic bacterium Aquifex aeolicus only the TatA proteins showed any detectable level of heterologous functionality. The heterologously expressed TatA proteins of S. coelicolor and A. aeolicus were found exclusively in the membrane fraction.ConclusionOur results show that of the three Tat proteins, TatA is most likely to show cross-species complementation. By contrast, TatB and TatC do not always show cross-complementation, probably because they must recognise heterologous signal peptides. Since heterologously-expressed S. coelicolor TatA protein was functional and found only in the membrane fraction, it suggests that soluble forms of Streptomyces TatA reported by others do not play a role in protein export.