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Proteins and peptides are amongst the most widely used research reagents but often their quality is inadequate and can result in poor data reproducibility. Here we propose a simple set of guidelines that, when correctly applied to protein reagents should provide more reliable experimental data.

Structural and biophysical characterization of molecular mechanisms of disease‐causing pathogens, such as Mycobacterium tuberculosis, often requires recombinant expression of large amounts highly pure protein. For the production of mycobacterial proteins, overexpression in the fast‐growing and non‐pathogenic species Mycobacterium smegmatis has several benefits over the standard Escherichia coli expression strains. However, unlike for E. coli, the range of expression vectors currently available is limited. Here we describe the development of the pMy vector series, a set of expression plasmids for recombinant production of single proteins and protein complexes in M. smegmatis. By incorporating an alternative selection marker, we show that these plasmids can also be used for co‐expression studies. All vectors in the pMy vector series are available in the Addgene repository (www.addgene.com).

A variety of methods to create specific constructs for protein expression, in a broad range of organisms, are available nowadays. Restriction enzyme-free, ligation-independent and recombinase-based cloning methods have enabled high-throughput protein expression for structural and functional studies. These methods are also instrumental for modification of target genes including gene truncations, site-specific mutagenesis and domain swapping. Here, we describe the most common cloning techniques that are currently at hand for recombinant protein expression studies, including a brief overview of techniques associated with co-expression experiments. We also provide an inventory of many of the available reagents for the various cloning methods, and an overview for some computational tools that can help with the design of expression constructs.

Gene synthesis enables the exploitation of the degeneracy of the genetic code to boost expression of recombinant protein targets for structural studies. This has created new opportunities to obtain structural information on proteins that are normally present in low abundance. Unfortunately, synthetic gene expression occasionally leads to insoluble or misfolded proteins. This could be remedied by recent insights in the effect of codon usage on translation initiation and elongation. In this review, we discuss the interplay between optimal gene and vector design to enhance expression in a particular host and highlight the benefits and potential pitfalls associated with protein expression from synthetic genes.

Regulatory pathways that drive early hematogenous dissemination of tumor cells are insufficiently defined. Here, we used the presence of disseminated tumor cells (DTC) in the bone marrow to define patients with early disseminated breast cancer and identified low retinoic acid–induced 2 (RAI2) expression to be significantly associated with DTC status. Low RAI2 expression was also shown to be an independent poor prognostic factor in 10 different cancer datasets. Depletion of RAI2 protein in luminal breast cancer cell lines resulted in dedifferentiation marked by downregulation of ERα, FOXA1, and GATA3, together with increased invasiveness and activation of AKT signaling. Functional analysis of the previously uncharacterized RAI2 protein revealed molecular interaction with CtBP transcriptional regulators and an overlapping function in controlling the expression of a number of key target genes involved in breast cancer. These results suggest that RAI2 is a new metastasis-associated protein that sustains differentiation of luminal breast epithelial cells. Significance: We identified downregulation of RAI2 as a novel metastasis-associated genetic alteration especially associated with early occurring bone metastasis in ERα-positive breast tumors. We specified the role of the RAI2 protein to function as a transcriptional regulator that controls the expression of several key regulators of breast epithelial integrity and cancer. Cancer Discov; 5(5); 506–19. ©2015 AACR. See related commentary by Esposito and Kang, p. 466 This article is highlighted in the In This Issue feature, p. 453

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central β-sheet and two α-helical bundles. Additionally, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3 and EspG5 chaperones using small-angle X-ray scattering (SAXS) reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3-PE5-PPE4 complex based on the SAXS analysis.HighlightsThe crystal structure of EspG1 reveals the common architecture of the type VII secretion system chaperonesStructures of EspG3 chaperones display a number of conformations that could reflect alternative substrate binding modesEspG3 chaperones dimerize in solutionA model of EspG3 in complex with its substrate PE-PPE dimer is proposed based on SAXS data

Nature microbiology 2, 17047 (2017). doi:10.1038/nmicrobiol.2017.47, Mycobacteria are characterized by their impermeable outer membrane, which is rich in mycolic acids$^1$. To transport substrates across this complex cell envelope, mycobacteria rely on type VII (also known as ESX) secretion systems$^2$. In Mycobacterium tuberculosis, these ESX systems are essential for growth and full virulence and therefore represent an attractive target for anti-tuberculosis drugs$^3$. However, the molecular details underlying type VII secretion are largely unknown, due to a lack of structural information. Here, we report the molecular architecture of the ESX-5 membrane complex from Mycobacterium xenopi determined at 13 Å resolution by electron microscopy. The four core proteins of the ESX-5 complex (EccB$_5$, EccC$_5$, EccD$_5$ and EccE$_5$) assemble with equimolar stoichiometry into an oligomeric assembly that displays six-fold symmetry. This membrane-associated complex seems to be embedded exclusively in the inner membrane, which indicates that additional components are required to translocate substrates across the mycobacterial outer membrane. Furthermore, the extended cytosolic domains of the EccC ATPase, which interact with secretion effectors, are highly flexible, suggesting an as yet unseen mode of substrate interaction. Comparison of our results with known structures of other bacterial secretion systems demonstrates that the architecture of type VII secretion system is fundamentally different, suggesting an alternative secretion mechanism., Published by Springer Nature, London

The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX-1, ESX-3 and ESX-5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system-specific cytoplasmic chaperones for secretion. Here, we report the crystal structure of M. tuberculosis ESX-5-secreted PE25–PPE41 heterodimer in complex with the cytoplasmic chaperone EspG5. EspG5 represents a novel fold that is unrelated to previously characterized secretion chaperones. Functional analysis of the EspG5-binding region uncovered a hydrophobic patch on PPE41 that promotes dimer aggregation, and the chaperone effectively abolishes this process. We show that PPE41 contains a characteristic chaperone-binding sequence, the hh motif, which is highly conserved among ESX-1-, ESX-3- and ESX-5-specific PPE proteins. Disrupting the interaction between EspG5 and three different PPE target proteins by introducing different point mutations generally affected protein secretion. We further demonstrate that the EspG5 chaperone plays an important role in the ESX secretion mechanism by keeping aggregation-prone PE–PPE proteins in their soluble state.

Summary Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases., Graphical Abstract, Highlights • MbcTA is a RES-Xre toxin-antitoxin system in M. tuberculosis (Mtb) • MbcT is a NAD+ phosphorylase • MbcT-catalyzed NAD+ depletion leads to Mtb cell death • MbcT activity synergizes with antibiotics to reduce Mtb burden in infected mice, Toxin-antitoxin systems regulate bacterial growth in response to stress through modification of macromolecules, including proteins, RNA, and DNA. Freire et al. show that MbcT, a toxin produced by the tubercle bacillus, induces bacterial cell death through NAD+ phosphorolysis, an unprecedented enzymatic activity.

SummaryLoss-of-function mutations in the NF1 tumor suppressor result in deregulated Ras signaling and drive tumorigenesis in the familial cancer syndrome neurofibromatosis type I. However, the extent to which NF1 inactivation promotes sporadic tumorigenesis is unknown. Here we report that NF1 is inactivated in sporadic gliomas via two mechanisms: excessive proteasomal degradation and genetic loss. NF1 protein destabilization is triggered by the hyperactivation of protein kinase C (PKC) and confers sensitivity to PKC inhibitors. However, complete genetic loss, which only occurs when p53 is inactivated, mediates sensitivity to mTOR inhibitors. These studies reveal an expanding role for NF1 inactivation in sporadic gliomagenesis and illustrate how different mechanisms of inactivation are utilized in genetically distinct tumors, which consequently impacts therapeutic sensitivity.

The brain-specific synaptic guanosine triphosphatase (GTPase)-activating protein (SynGAP) is important in synaptic plasticity. It shows dual specificity for the small guanine nucleotide-binding proteins Rap and Ras. Here, we show that RapGAP activity of SynGAP requires its C2 domain. In contrast to the isolated GAP domain, which does not show any detectable RapGAP activity, a fragment comprising the C2 and GAP domains (C2-GAP) stimulates the intrinsic GTPase reaction of Rap by approximately 1 x 10(4). The C2-GAP crystal structure, complemented by modelling and biochemical analyses, favours a concerted movement of the C2 domain towards the switch II region of Rap to assist in GTPase stimulation. Our data support a catalytic mechanism similar to that of canonical RasGAPs and distinct from the canonical RapGAPs. SynGAP presents the first example, to our knowledge, of a GAP that uses a second domain for catalytic activity, thus pointing to a new function of C2 domains.

Purpose Tuberculosis is still a major threat to global health. New tools and strategies to produce disease-related proteins are quintessential for the development of novel vaccines and diagnostic markers. Experimental design To obtain recombinant proteins from Mycobacterium tuberculosis (Mtb) for use in clinical applications, a standardized procedure was developed that includes subcloning, protein expression in Mycobacterium smegmatis and protein purification using chromatography. The potential for the different protein targets to serve as diagnostic markers for tuberculosis was established using multiplex immunoassays. Results Twelve soluble proteins from Mtb, including one protein complex, were purified to near-homogeneity following recombinant expression in M. smegmatis. Protein purity was assessed both by size exclusion chromatography and mass spectrometry. Multiplex serological testing of the final protein preparations showed that all but one protein displayed a clear antibody response in serum samples from 278 tuberculosis patients. Conclusion and clinical relevance The established workflow comprises a simple, cost-effective and scalable pipeline for production of soluble proteins from Mtb and can be used to prioritize immunogenic proteins suitable for use as diagnostic markers. This article is protected by copyright. All rights reserved

Pseudomonas sp. BW11M1 produces a novel type of bacteriocin that inhibits the growth of Pseudomonas putida GR12-2R3 and some phytopathogenic fluorescent Pseudomonas. A collection of mutants was screened for altered bacteriocin production phenotypes. Strongly reduced bacteriocin production was found to be caused by inactivation of the recA gene or the spoT gene. Conversely, in a recJ mutant, the bacteriocin was constitutively overproduced. The same phenotype was observed for a mutant hit in a gene of unknown function. The predicted gene product belongs to a distinct subgroup of prokaryotic helicase-like proteins within the SWI/SNF family of regulatory proteins. One mutant that also exhibited a bacteriocin overproducer phenotype was deficient in the production of the peptidoglycan-associated lipoprotein OprL. This study shows that various environmental stress response pathways are involved in controlling expression of the Pseudomonas sp. BW11M1 bacteriocin.

Purified thiocarbamate-inducible ThcF of Rhodococcus erythropolis NI86/21, overexpressed in Escherichia coli, displayed several characteristics of the HASH family of enzymes that groups prokaryotic proteins of the alpha/beta hydrolase superfamily possessing serine-dependent hydrolase and/or haloperoxidase activity. Kinetic analysis of bromination and ester hydrolysis revealed a low affinity of ThcF for model substrates. Sulfoxidation of thiocarbamates was demonstrated but probably represents a side activity due to peroxoacid generation by the enzyme. The thcF-linked thcG gene, encoding a LAL-type regulator, triggers expression of thcF in Rhodococcus. The tandem gene organization thcG-thcF is conserved in the thiocarbamate-degrading strain Rhodococcus sp. B30. It is proposed that HASH enzymes may be involved in the metabolism of plant-derived compounds.

The Bacillus cytochrome P450 BM3 integrates an entire P450 system in one polypeptide and represents a convenient prokaryotic model for microsomal P450s. This self-sufficient class II P450 is also present in actinomycetes and fungi. By genome analysis we have identified additional homologues in the pathogenic species Bacillus anthracis and Bacillus cereus, and in Ralstonia metallidurans. This analysis also revealed a novel class of putative self-sufficient P450s, P450 PFOR, comprising a class I P450 that is related to Rhodococcus erythropolis CYP116, and a phthalate family oxygenase reductase (PFOR) module. P450 PFOR genes are found in a Rhodococcus strain, three pathogenic Burkholderia species and in the R. metallidurans strain that possesses a P450 BM3 homologue. Co-evolution of P450 and reductase domains is apparent in both types of self-sufficient enzymes. The new class of P450 enzymes is of potential interest for various biotechnological applications.

Pseudomonas aeruginosa strains frequently produce proteinaceous, narrow-spectrum antibacterial bacteriocins known as pyocins. The majority of these compounds exert their toxicity through non-specific DNA degradation inside sensitive cells. Here, we describe the identification of novel bacteriocins of different types in related bacteria inhabiting diverse niches. The abundance of bacteriocin genes in bacteria such as Pseudomonas , Klebsiella and Photorhabdus suggests that these systems play an important role in the competition between rival bacteria. The chimeric nature of these proteins implies that extensive domain swapping has contributed to the diversification of bacteriocins in γ-proteobacteria.

The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX-1, ESX-3 and ESX-5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system-specific cytoplasmic chaperones for secretion. Here, we report the crystal structure of M. tuberculosis ESX-5-secreted PE25-PPE41 heterodimer in complex with the cytoplasmic chaperone EspG(5). EspG(5) represents a novel fold that is unrelated to previously characterized secretion chaperones. Functional analysis of the EspG(5) -binding region uncovered a hydrophobic patch on PPE41 that promotes dimer aggregation, and the chaperone effectively abolishes this process. We show that PPE41 contains a characteristic chaperone-binding sequence, the hh motif, which is highly conserved among ESX-1-, ESX-3- and ESX-5-specific PPE proteins. Disrupting the interaction between EspG(5) and three different PPE target proteins by introducing different point mutations generally affected protein secretion. We further demonstrate that the EspG(5) chaperone plays an important role in the ESX secretion mechanism by keeping aggregation-prone PE-PPE proteins in their soluble state.

We have previously identified a Saccharomyces cerevisiae mutant that is markedly more resistant than wild-type to Dahlia merckii antimicrobial peptide 1 (DmAMP1), an antifungal plant defensin isolated from seeds of dahlia ( Dahlia merckii ). A complementation approach was followed that consisted of the introduction of a genomic library of DmAMP1-sensitive wild-type yeast into the DmAMP1-resistant yeast mutant and screening for restored sensitivity to DmAMP1. The gene determining sensitivity of S. cerevisiae to DmAMP1 was identified as IPT1 , a gene encoding an enzyme involved in the last step of the synthesis of the sphingolipid mannose-(inositol-phosphate) 2 -ceramide. Strains with a nonfunctional IPT1 allele lacked mannose-(inositol-phosphate) 2 -ceramide in their plasma membranes, bound significantly less DmAMP1 compared with wild-type strains, and were highly resistant to DmAMP1-mediated membrane permeabilization. All of these phenotypic deviations could be restored by reintroduction of a functional IPT1 gene. Our data support a model in which membrane patches containing sphingolipids act as binding sites for DmAMP1 or, alternatively, are required to anchor membrane or cell wall-associated proteins, which themselves interact with DmAMP1.

LlpA is a bacteriocin produced by Pseudomonas sp. BW11M1 that shows remarkable similarity to a family of mannose-binding plant lectins. A His-tagged version of LlpA was recombinantly produced in Escherichia coli and purified to homogeneity. Single crystals were grown by vapour diffusion and belong to space group P2(1)2(1)2, with unit-cell parameters a = 150.5, b = 154.5, c = 34.2 A. The crystals diffract to at least 2.2 A using synchrotron radiation.

Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG(5), which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG(5) interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG(1) interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG(5) and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG(5) and EspG(1) interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.

Context Autosomal dominant inactivating sprouty-related EVH1 domain–containing protein 1 (SPRED1) mutations have recently been described in individuals presenting mainly with cafe au lait macules (CALMs), axillary freckling, and macrocephaly. The extent of the clinical spectrum of this new disorder needs further delineation. Objective To determine the frequency, mutational spectrum, and phenotype of neurofibromatosis type 1–like syndrome (NFLS) in a large cohort of patients. Design, Setting, and Participants In a cross-sectional study, 22 unrelated probands carrying a SPRED1 loss-of-function (LOF) mutation identified through clinical testing participated with their families in a genotype-phenotype study (2007-2008). In a second cross-sectional study, 1318 unrelated anonymous samples collected in 2003-2007 from patients with a broad range of signs typically found in neurofibromatosis type 1 (NF1) but no detectable NF1 germline mutation underwent SPRED1 mutation analysis. Main Outcome Measures Comparison of aggregated clinical features in patients with or without a SPRED1 or NF1 mutation. Functional assays were used to evaluate the pathogenicity of missense mutations. Results Among 40 SPRED1 LOF-positive individuals from the clinical cohort, 20 (50%; 95% confidence interval [CI], 34%-66%) fulfilled National Institutes of Health (NIH) NF1 diagnostic criteria based on the presence of more than 5 CALMs with or without freckling or an NF1-compatible family history. None of the 40 SPRED1 LOF-positive individuals (0%; 95% CI, 0%-7%) had discrete cutaneous or plexiform neurofibromas, typical NF1 osseous lesions, or symptomatic optic pathway gliomas. In the anonymous cohort of 1318 individuals, 34 different SPRED1 mutations in 43 probands were identified: 26 pathogenic mutations in 33 probands and 8 probable nonpathogenic missense or silent mutations in 10 probands. Of 94 probands with familial CALMs with or without freckling and no other NF1 features, 69 (73%; 95% CI, 63%-80%) had an NF1 mutation and 18 (19%; 95% CI, 12%-29%) had a pathogenic SPRED1 mutation. In the anonymous cohort, 1.9% (95% CI, 1.2%-2.9%) of individuals with the clinical diagnosis of NF1 according to the NIH criteria had NFLS. Conclusions A high SPRED1 mutation detection rate was found in NF1 mutation–negative families with an autosomal dominant phenotype of CALMs with or without freckling and no other NF1 features. Among individuals in this study, NFLS was not associated with the peripheral and central nervous system tumors seen in NF1.

Costello syndrome is a mental retardation syndrome characterized by high birth weight, postnatal growth retardation, coarse face, loose skin, cardiovascular problems, and tumor predisposition. De novo heterozygous missense mutations in HRAS codon 12 and 13 disturbing the intrinsic GTP hydrolysis cause Costello syndrome. We report a patient with typical Costello syndrome and a novel heterozygous missense mutation in codon 117 (c.350A>G, p.Lys117Arg) of the HRAS gene, resulting in constitutive activation of the RAS/MAPK pathway similar to the typical p.Gly12Ser and p.Gly12Ala mutations. Recombinant HRAS p.Lys117Arg demonstrates normal intrinsic GTP hydrolysis and responsiveness to GTPase-activating proteins, but the nucleotide dissociation rate is increased 80-fold. Consistent with the biochemical data, the crystal structure of the p.Lys117Arg mutant indicates an altered interaction pattern of the side chain that is associated with unfavorable nucleotide binding properties. Together, these data show that a RAS mutation that only perturbs guanine nucleotide binding has similar functional consequences as mutations that impair GTP hydrolysis and causes human disease.

Bacteriocin LlpA, produced by Pseudomonas sp. strain BW11M1, is a peculiar antibacterial protein due to its homology to mannose-binding lectins mostly found in monocots (A. H. A. Parret, G. Schoofs, P. Proost, and R. De Mot, J. Bacteriol. 185: 897-908, 2003). Biocontrol strain Pseudomonas fluorescens Pf-5 contains two llpA -like genes, named llpA1 Pf-5 and llpA2 Pf-5 . Recombinant Escherichia coli cells expressing llpA1 Pf-5 or llpA2 Pf-5 acquired bacteriocin activity and secreted a 31-kDa protein cross-reacting with LlpA BW11M1 antibodies. Antibacterial activity of the recombinant proteins was evidenced by gel overlay assays. Analysis of the antimicrobial spectrum indicated that LlpA1 Pf-5 and LlpA2 Pf-5 are able to inhibit P. fluorescens strains, as well as the related mushroom pathogen Pseudomonas tolaasii . LlpA-type bacteriocins are characterized by a domain structure consisting of tandem monocot mannose-binding lectin (MMBL) domains. Molecular phylogeny of these MMBL domains suggests that the individual MMBL domains within an LlpA protein have evolved separately toward a specific, as yet unknown, function or, alternatively, were acquired from different ancestral sources. Our observations are consistent with earlier observations, which hinted that MMBL-like bacteriocins represent a new family of antibacterial proteins, probably with a novel mode of action.

Deletion mutants of the Rhodococcus erythropolis ARC AAA ATPase were generated and characterized by biochemical analysis and electron microscopy. Based on sequence comparisons the ARC protein was divided into three consecutive regions, the N-terminal coiled coil, the central ARC-specific inter domain and the C-terminal AAA domain. When the ARC AAA domain was expressed separately it formed aggregates of undefined structure. However, when the AAA domain was expressed in conjunction with the preceeding inter domain, but without the N-terminal coiled coil, high-molecular weight-complexes were formed (ARC-DeltaCC) which showed an N-ethylmaleimide-sensitive ATPase activity. In 2D crystallization experiments the ARC-DeltaCC particles yielded crystals nearly identical to those formed by the wild-type ARC complexes. Thus, the N-terminal coiled coil, which was proposed to have a role in the assembly of and/or interaction between the eukaryotic AAA ATPases in the 26S proteasome, is neither essential for assembly nor for ATP hydrolysis of the ARC ATPase. The N-terminal domain of related AAA ATPases mediates the interaction with substrates or co-factors, suggesting a regulatory function for the N-terminal coiled coil of the ARC ATPase. Surprisingly, the mutant ARC protein ARC-DeltaAAA consisting of the N-terminal coiled coil and the central inter domain, but deleted for the C-terminal AAA domain, was shown to form a dodecameric complex with sixfold symmetry. This suggests an important role of the inter domain for the ordered assembly of the ARC ATPase.

Rhizosphere isolate Pseudomonas sp. strain BW11M1, which belongs to the Pseudomonas putida cluster, secretes a heat- and protease-sensitive bacteriocin which kills P. putida GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a Tn Mod mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into Escherichia coli cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in E. coli . This gene was designated llp A ( l ectin- l ike p utidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the llpA gene also conveyed bacteriocin production to several Pseudomonas fluorescens strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described Pseudomonas bacteriocins.