Prediction of twin-arginine signal peptides

BACKGROUND: Proteins carrying twin-arginine (Tat) signal peptides are exported into the periplasmic compartment or extracellular environment independently of the classical Sec-dependent translocation pathway. To complement other methods for classical signal peptide prediction we here present a publicly available method, TatP, for prediction of bacterial Tat signal peptides. RESULTS: We have retrieved sequence data for Tat substrates in order to train a computational method for discrimination of Sec and Tat signal peptides. The TatP method is able to positively classify 91% of 35 known Tat signal peptides and 84% of the annotated cleavage sites of these Tat signal peptides were correctly predicted. This method generates far less false positive predictions on various datasets than using simple pattern matching. Moreover, on the same datasets TatP generates less false positive predictions than a complementary rule based prediction method. CONCLUSION: The method developed here is able to discriminate Tat signal peptides from cytoplasmic proteins carrying a similar motif, as well as from Sec signal peptides, with high accuracy. The method allows filtering of input sequences based on Perl syntax regular expressions, whereas hydrophobicity discrimination of Tat- and Sec-signal peptides is carried out by an artificial neural network. A potential cleavage site of the predicted Tat signal peptide is also reported. The TatP prediction server is available as a public web server at

Cosmid based mutagenesis causes genetic instability in Streptomyces coelicolor, as shown by targeting of the lipoprotein signal peptidase gene

Bacterial lipoproteins are extracellular proteins tethered to cell membranes by covalently attached lipids. Deleting the lipoprotein signal peptidase (lsp) gene in Streptomyces coelicolor results in growth and developmental defects that cannot be restored by reintroducing lsp. This led us to hypothesise that lsp is essential and that the lsp mutant we isolated previously had acquired compensatory secondary mutations. Here we report resequencing of the genomes of wild-type M145 and the cis-complemented ∆lsp mutant (BJT1004) to map and identify these secondary mutations but we show that they do not increase the efficiency of disrupting lsp and are not lsp suppressors. We provide evidence that they are induced by introducing the cosmid St4A10∆lsp, as part of ReDirect PCR mutagenesis protocol, which transiently duplicates a number of important cell division genes. Disruption of lsp using a suicide vector (which does not result in gene duplication) still results in growth and developmental delays and we conclude that loss of Lsp function results in developmental defects due to the loss of all lipoproteins from the cell membrane. Significantly, our results also indicate the use of cosmid libraries for the genetic manipulation of bacteria can lead to phenotypes not necessarily linked to the gene(s) of interest

In Situ Activation and Heterologous Production of a Cryptic Lantibiotic from an African Plant Ant-Derived Saccharopolyspora Species.

Most clinical antibiotics are derived from actinomycete natural products discovered at least 60 years ago. However, the repeated rediscovery of known compounds led the pharmaceutical industry to largely discard microbial natural products (NPs) as a source of new chemical diversity. Recent advances in genome sequencing have revealed that these organisms have the potential to make many more NPs than previously thought. Approaches to unlock NP biosynthesis by genetic manipulation of strains, by the application of chemical genetics, or by microbial cocultivation have resulted in the identification of new antibacterial compounds. Concomitantly, intensive exploration of coevolved ecological niches, such as insect-microbe defensive symbioses, has revealed these to be a rich source of chemical novelty. Here, we report the new lanthipeptide antibiotic kyamicin, which was generated through the activation of a cryptic biosynthetic gene cluster identified by genome mining Saccharopolyspora species found in the obligate domatium-dwelling ant Tetraponera penzigi of the ant plant Vachellia drepanolobium Transcriptional activation of this silent gene cluster was achieved by ectopic expression of a pathway-specific activator under the control of a constitutive promoter. Subsequently, a heterologous production platform was developed which enabled the purification of kyamicin for structural characterization and bioactivity determination. This strategy was also successful for the production of lantibiotics from other genera, paving the way for a synthetic heterologous expression platform for the discovery of lanthipeptides that are not detected under laboratory conditions or that are new to nature.IMPORTANCE The discovery of novel antibiotics to tackle the growing threat of antimicrobial resistance is impeded by difficulties in accessing the full biosynthetic potential of microorganisms. The development of new tools to unlock the biosynthesis of cryptic bacterial natural products will greatly increase the repertoire of natural product scaffolds. Here, we report a strategy for the ectopic expression of pathway-specific positive regulators that can be rapidly applied to activate the biosynthesis of cryptic lanthipeptide biosynthetic gene clusters. This allowed the discovery of a new lanthipeptide antibiotic directly from the native host and via heterologous expression

Analysis of the tunicamycin biosynthetic gene cluster of streptomyces chartreusis reveals new insights into tunicamycin production and immunity

The tunicamycin biosynthetic gene cluster of Streptomyces chartreusis consists of 14 genes (tunA to tunN) with a high degree of apparent translational coupling. Transcriptional analysis revealed that all of these genes are likely to be transcribed as a single operon from two promoters, tunp1 and tunp2. In-frame deletion analysis revealed that just six of these genes (tunABCDEH) are essential for tunicamycin production in the heterologous host Streptomyces coelicolor, while five (tunFGKLN) with likely counterparts in primary metabolism are not necessary, but presumably ensure efficient production of the antibiotic at the onset of tunicamycin biosynthesis. Three genes are implicated in immunity, namely, tunI and tunJ, which encode a two-component ABC transporter presumably required for export of the antibiotic, and tunM, which encodes a putative S-adenosylmethionine (SAM)-dependent methyltransferase. Expression of tunIJ or tunM in S. coelicolor conferred resistance to exogenous tunicamycin. The results presented here provide new insights into tunicamycin biosynthesis and immunity

Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design

Summary: Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic “lipid-altered” tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug

The effect of bioreducible cytotoxic drugs upon the SOS response of Escherichia coli

The DNA damaging activity of RSU 1069 and seven of its analogues (RSU 1131, RSU 1164, RSU 1150, RB 7040, RSU 1172, RSU 1137 and RSU 1170) plus misonidazole and CB 1954 were investigated using the SOS-Chromotest The SOS-Chromotest is a genotoxicity assay that monitors the induction of the SOS response, which is induced in response to DNA damage. The strains used were PQ37, which possesses a uvrA mutation and is deficient in UvrA excinuclease activity, and PQ35, which is uvr and UvrABC excinuclease conpetent. These strains were exposed to the compounds being investigated under both oxic and hypoxic conditions. The results showed that RSU 1069 and some of its analogues were more active than misonidazole under both oxic and hypoxic conditions. This increase was due to their aziridine side-chains. With the exception of RSU 1137 and RSU 1170 all of the compounds showed altered SOS induction activities between oxic and hypoxic conditions. This alteration was shown to correlate with increased reduction of their nitro-groups under hypoxia. There was a difference in the hypoxic activities of RSU 1(J69 and some of its analogues between the uvrA-strain and the uvr -strain. With the uvrA-strain RSU 1069 showed decrease activity under hypoxia compared to oxia, whereas, the converse applied with the uvr -strain. This was interpreted to mean that RSU 1069 caused some damage that required an active UvrABC excinuclease to produce an SOS response. It has been proposed that this damage takes the form of DNA crosslinks. RSU 1137 showed insignificant SOS induction and this was demonstrated to be due to its nitro-group not being reduced. It was suggested that the ring opened aziridine side chain of RSU 1137 in some way inhibited its bioreduction. The order of activity of the drugs for SOS induction activities did not correlate with that for their toxicities. This indicated that DNA lesions other than, or in addition to, those responsible for cytotoxicity induced the SOS response. The DNA damaging activity and mutagenicity of RSU 1069 was also investigated using Ml3 phage rfDNA. Radiation reduced RSU 1069 was shown to produce some relatively long lived product that was more active towards DNA than unreduced RSU 1069, as judged by phage survival. Unreduced RSU 1069 was shown to be non-mutagenic, producing mutation rates under 1.5 times background level. The effect of strict hypoxic conditions upon the SOS response was investigated using the SOS-Chromotest with the uvrA tester strain. The results showed that the SOS response was induced under strictly anaerobic conditions in E. coli but that the response was altered compared to that obtained aerobically. The nature of the alteration was not determined as six different compounds, with five different modes of action, were used as SOS-inducers and all showed different types of response under hypoxic conditions

Formation of functional Tat translocases from heterologous components

<p>Abstract</p> <p>Background</p> <p>The Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plants. In <it>Eschericha coli</it>, Tat transport requires the integral membrane proteins TatA, TatB and TatC. In this study we have tested the ability of <it>tat </it>genes from the eubacterial species <it>Pseudomonas syringae</it>, <it>Streptomyces coelicolor </it>and <it>Aquifex aeolicus</it>, to compensate for the absence of the cognate <it>E. coli tat </it>gene, and thus to form functional Tat translocases with <it>E. coli </it>Tat components.</p> <p>Results</p> <p>All three subunits of the Tat system from the Gram positive organism <it>Streptomyces coelicolor </it>were able to form heterologous translocases with substantive Tat transport activity. However, only the TatA and TatB proteins of <it>Pseudomonas syringae </it>were able to functionally interact with the <it>E. coli </it>Tat system even though the two organisms are closely related. Of the Tat components from the phylogenetically distant hyperthermophillic bacterium <it>Aquifex aeolicus </it>only the TatA proteins showed any detectable level of heterologous functionality. The heterologously expressed TatA proteins of <it>S. coelicolor </it>and <it>A. aeolicus </it>were found exclusively in the membrane fraction.</p> <p>Conclusion</p> <p>Our 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 <it>S. coelicolor </it>TatA protein was functional and found only in the membrane fraction, it suggests that soluble forms of <it>Streptomyces </it>TatA reported by others do not play a role in protein export.</p

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

Summary 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

Specificity Determinants of Proteolytic Processing of Aspergillus PacC Transcription Factor Are Remote from the Processing Site, and Processing Occurs in Yeast If pH Signalling Is Bypassed

The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of “neutrality-mimicking” pacC mutations having aspects of both acidity- and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues ∼252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur in Saccharomyces cerevisiae