We present the first functional analysis of the small GTPase, TbRab7, in Trypanosoma brucei. TbRab7 defines discrete late endosomes closely juxtaposed to the terminal p67+ lysosome. RNAi indicates that TbRab7 is essential in bloodstream trypanosomes. Initial rates of endocytosis were unaffected, but lysosomal delivery of cargo, including tomato lectin (TL) and trypanolytic factor (TLF) were blocked. These accumulate in a dispersed internal compartment of elevated pH, likely derived from the late endosome. Surface binding of TL but not TLF was reduced, suggesting that cellular distribution of flagellar pocket receptors is differentially regulated by TbRab7. TLF activity was reduced approximately threefold confirming that lysosomal delivery is critical for trypanotoxicity. Unexpectedly, delivery of endogenous proteins, p67 and TbCatL, were unaffected indicating that TbRab7 does not regulate biosynthetic lysosomal trafficking. Thus, unlike mammalian cells and yeast, lysosomal trafficking of endocytosed and endogenous proteins occur via different routes and/or are regulated differentially. TbRab7 silencing had no effect on a cryptic default pathway to the lysosome, suggesting that the default lysosomal reporters p67ΔTM, p67ΔCD and VSGΔGPI do not utilize the endocytic pathway as previously proposed. Surprisingly, conditional knockout indicates that TbRab7 may be non-essential in procyclic insect form trypanosomes.
Drug resistance in Plasmodium falciparum remains a challenge for the malaria eradication programmes around the world. With the emergence of artemisinin resistance, the efficacy of the partner drugs in the artemisinin combination therapies (ACT) that include quinoline-based drugs is becoming critical. So far only few resistance markers have been identified from which only two transmembrane transporters namely PfMDR1 (an ATP-binding cassette transporter) and PfCRT (a drug-metabolite transporter) have been experimentally verified. Another P. falciparum transporter, the ATP-binding cassette containing multidrug resistance-associated protein (PfMRP2) represents an additional possible factor of drug resistance in P. falciparum. In this study, we identified a parasite clone that is derived from the 3D7 P. falciparum strain and shows increased resistance to chloroquine, mefloquine and quinine through the trophozoite and schizont stages. We demonstrate that the resistance phenotype is caused by a 4.1 kb deletion in the 5′ upstream region of the pfmrp2 gene that leads to an alteration in the pfmrp2 transcription and thus increased level of PfMRP2 protein. These results also suggest the importance of putative promoter elements in regulation of gene expression during the P. falciparum intra-erythrocytic developmental cycle and the potential of genetic polymorphisms within these regions to underlie drug resistance.
Chemoreceptors McpB and McpC in Salmonella enterica have been reported to promote chemotaxis in LB motility-plate assays. Of the chemicals tested as potential effectors of these receptors, the only response was towards L-cysteine and its oxidized form, L-cystine. Although enhanced radial migration in plates suggested positive chemotaxis to both amino acids, capillary assays failed to show an attractant response to either, in cells expressing only these two chemoreceptors. In vivo fluorescence resonance energy transfer (FRET) measurements of kinase activity revealed that in wild-type bacteria, cysteine and cystine are chemoeffectors of opposing sign, the reduced form being a chemoattractant and the oxidized form a repellent. The attractant response to cysteine was mediated primarily by Tsr, as reported earlier for E. coli. The repellent response to cystine was mediated by McpB / C. Adaptive recovery upon cystine exposure required the methyl-transferase/-esterase pair, CheR / CheB, but restoration of kinase activity was never complete (i.e. imperfect adaptation). We provide a plausible explanation for the attractant-like responses to both cystine and cysteine in motility plates, and speculate that the opposing signs of response to this redox pair might afford Salmonella a mechanism to gauge and avoid oxidative environments.
DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons.
We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side chain. Expression of bioJ allows growth of an E. coli bioH strain on biotin-free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl-ACP methyl ester carboxyl-esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl-ACP to pimeloyl-ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel sub-clade of the α/β-hydrolase family. Structure-guided mapping combined with site-directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence.
Francisella; biotin; biotin synthesis; BioJ; nutritional virulence factor
Strains of Pseudomonas aeruginosa (PA) isolated from the airways of cystic fibrosis patients constitutively add palmitate to lipid A, the membrane anchor of lipopolysaccharide. The PhoPQ regulated enzyme PagP is responsible for the transfer of palmitate from outer membrane phospholipids to lipid A. This enzyme had previously been identified in many pathogenic Gram-negative bacteria, but in PA had remained elusive, despite abundant evidence that its lipid A contains palmitate. Using a combined genetic and biochemical approach, we identified PA1343 as the PA gene encoding PagP. Although PA1343 lacks obvious primary structural similarity with known PagP enzymes, the β-barrel tertiary structure with an interior hydrocarbon ruler appears to be conserved. PA PagP transfers palmitate to the 3’ position of lipid A, in contrast to the 2 position seen with the enterobacterial PagP. Palmitoylated PA lipid A alters host innate immune responses, including increased resistance to some antimicrobial peptides and an elevated pro-inflammatory response, consistent with the synthesis of a hexa-acylated structure preferentially recognized by the TLR4/MD2 complex. Palmitoylation commonly confers resistance to cationic antimicrobial peptides, however, increased cytokine production resulting in inflammation is not seen with other palmitoylated lipid A, indicating a unique role for this modification in PA pathogenesis.
Lipopolysaccharide; lipid A; Pseudomonas aeruginosa; PagP; innate immunity; palmitate
Since its inception, Bacterial Locomotion and Signal Transduction (BLAST) meetings have been the place to exchange and share the latest developments in the field of bacterial signal transduction and motility. At the 12th BLAST meeting, held last January in Tucson AZ, researchers from all over the world met to report and discuss progress in diverse aspects of the field. The majority of these advances, however, came at the level of atomic level structures and their associated mechanisms. This was especially true of the biological machines that sense and respond to environmental changes.
Degron binding regulates the activities of the AAA+ Lon protease in addition to targeting proteins for degradation. The sul20 degron from the cell-division inhibitor SulA is shown here to bind to the N domain of E. coli Lon, and the recognition site is identified by crosslinking and scanning for mutations that prevent sul20-peptide binding. These N-domain mutations limit the rates of proteolysis of model sul20-tagged substrates and ATP hydrolysis by an allosteric mechanism. Lon inactivation of SulA in vivo requires binding to the N domain and robust ATP hydrolysis but does not require degradation or translocation into the proteolytic chamber. Lon-mediated relief of proteotoxic stress and protein aggregation in vivo can also occur without degradation but is not dependent on robust ATP hydrolysis. In combination, these results demonstrate that Lon can function as a protease or a chaperone and reveal that some of its ATP-dependent biological activities do not require translocation.
ATP-dependent protease; degradation tag; allostery; protein aggregation
The genome of Mycobacterium tuberculosis encodes two paralogous P1B4-ATPases, CtpD (Rv1469) and CtpJ (Rv3743). Both proteins showed ATPase activation by Co2+ and Ni2+, and both appear to be required for metal efflux from the cell. However, using a combination of biochemical and genetic studies we found that these proteins play nonredundant roles in virulence and metal efflux. CtpJ expression is induced by Co2+ and this protein possesses a relatively high turnover rate. A ctpJ deletion mutant accumulated Co2+, indicating that this ATPase controls cytoplasmic metal levels. In contrast, CtpD expression is induced by redox stressors and this protein displays a relatively low turnover rate. A ctpD mutant failed to accumulate metal, suggesting an alternative cellular function. ctpD is co-transcribed with two thioredoxin genes trxA (Rv1470), trxB (Rv1471), and an enoyl-coA hydratase (Rv1472), indicating a possible role for CtpD in the metallation of these redox-active proteins. Supporting this, in vitro metal binding assays showed that TrxA binds Co2+ and Ni2+. Mutation of ctpD, but not ctpJ, reduced bacterial fitness in the mouse lung, suggesting that redox maintenance, but not Co+2 accumulation, is important for growth in vivo.
CtpD; CtpJ; Co2+-ATPase; redox stress; metal loading; virulence
The two-component system PhoP/PhoQ controls a large number of genes responsible for a variety of physiological and virulence functions in Salmonella enterica serovar Typhimurium. Here we describe a mechanism whereby the transcriptional activator PhoP elicits expression of dissimilar gene sets when its cognate sensor PhoQ is activated by different signals in the periplasm. We determine that transcription of over half of the genes directly activated by PhoP require the Mg2+ transporter MgtA when the PhoQ inducing signal is low Mg2+, but not when PhoQ is activated by mildly acidic pH or the antimicrobial peptide C18G. MgtA promotes the active (i.e., phosphorylated) form of PhoP by removing Mg2+ from the periplasm where it functions as a repressing signal for PhoQ. MgtA-dependent expression enhances resistance to the cationic antibiotic polymyxin B. Production of the MgtA protein requires cytoplasmic Mg2+ levels to drop below a certain threshold, thereby creating a two-tiered temporal response among PhoP-dependent genes.
The chromosomal passenger complex (CPC) in animals, consisting of Aurora B kinase and three evolutionarily conserved proteins, plays crucial roles in mitosis and cytokinesis. However, Trypanosoma brucei expresses an unusual CPC consisting of an Aurora-like kinase, TbAUK1, and two kinetoplastid-specific proteins, TbCPC1 and TbCPC2. Despite their essential functions, little is known about the regulation of TbAUK1 and the roles of TbCPC1 and TbCPC2. Here, we investigate the effect of post-translational modification on the activity and spatiotemporal control of TbAUK1, and demonstrate that phosphorylation of two conserved threonine residues in the activation loop of the kinase domain contributes to TbAUK1 activation and function. TbAUK1 is SUMOylated in vivo, and mutation of the SUMO-conjugation site compromises TbAUK1 function. Degradation of TbAUK1 requires two destruction boxes and is mediated by the anaphase-promoting complex/cyclosome (APC/C), whereas degradation of TbCPC1 and TbCPC2 is not dependent on the predicted destruction boxes and is APC/C-independent. Moreover, we determine the domains in CPC subunits that mediate the pairwise interactions, and show that disruption of the interaction impairs the localization of TbAUK1 and TbCPC2 but not TbCPC1. Our results demonstrate the requirement of post-translational modifications for TbAUK1 function and a crucial role of TbCPC1 in mediating TbAUK1 localization.
Through cooperative interactions, bacteria can build multicellular communities. To ensure that productive interactions occur, bacteria must recognize their neighbors and respond accordingly. Molecular recognition between cells is thus a fundamental behavior, and in bacteria important discoveries have been made. This MicroReview focuses on a recently described recognition system in myxobacteria that is governed by a polymorphic cell surface receptor called TraA. TraA regulates outer membrane exchange (OME), whereby myxobacterial cells transiently fuse their OMs to efficiently transfer proteins and lipids between cells. Unlike other transport systems, OME is rather indiscriminate in what OM goods are transferred. In contrast, the recognition of partnering cells is discriminatory and only occurs between cells that bear identical or closely related TraA proteins. Therefore TraA functions in kin recognition and, in turn, OME helps regulate social interactions between myxobacteria. Here, I discuss and speculate on the social and evolutionary implications of OME and suggest it helps to guide their transition from free-living cells into coherent and functional populations.
Recognition; membrane fusion; Myxococcus xanthus
Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source-specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a co-expression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat-1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass.
pectin; xylan; cellulose; systems analysis; Neurospora crassa; polysaccharide perception
Iron is an essential but potentially harmful nutrient, poorly soluble in aerobic conditions, and not-freely available in the human host. To acquire iron, bacteria have evolved high affinity iron acquisition systems that are expressed under iron limitation often in conjunction with virulence determinants. Because excess iron can be dangerous, intracellular iron must be tightly controlled. In mycobacteria, IdeR functions as a global iron dependent transcriptional regulator, but because inactivation of ideR is lethal for Mycobacterium tuberculosis, it has not been possible to use genetics to fully characterize this protein’s function or examine the requirement of iron regulation during tuberculosis infection. In this work, a conditional M. tuberculosis ideR mutant was generated and used to study the basis of IdeR’s essentiality. This investigation uncovered positive regulation of iron storage as a critical aspect of IdeR’s function in regular culture and a prominent factor for survival under stresses associated with life in macrophages. Moreover, this study demonstrates that IdeR is indispensable in the mouse model of tuberculosis, thereby linking iron homeostasis to virulence in M. tuberculosis.
Adenylyl cyclases are widely distributed across all kingdoms whereas guanylyl cyclases are generally thought to be restricted to eukaryotes. Here we report that the α–proteobacterium Rhodospirillum centenum secretes cGMP when developing cysts and that a guanylyl cyclase deletion strain fails to synthesize cGMP and is defective in cyst formation. The R. centenum cyclase was purified and shown to effectively synthesize cGMP from GTP in vitro, demonstrating that it is a functional guanylyl cyclase. A homolog of the Escherichia coli cAMP receptor protein (CRP) is linked to the guanylyl cyclase and when deleted is deficient in cyst development. Isothermal calorimetry (ITC) and differential scanning fluorimetry (DSF) analyses demonstrate that the recombinant CRP homolog preferentially binds to, and is stabilized by cGMP, but not cAMP. This study thus provides evidence that cGMP has a crucial role in regulating prokaryotic development. The involvement of cGMP in regulating bacterial development has broader implications as several plant-interacting bacteria contain a similar cyclase coupled by the observation that Azospirillum brasilense also synthesizes cGMP when inducing cysts.
YdiV is an EAL-like protein that acts as a post-transcriptional, negative regulator of the flagellar master transcriptional activator complex, FlhD4C2, in Salmonella enterica to couple flagellar gene expression to nutrient availability. Mutants defective in ClpXP protease no longer exhibit YdiV-dependent inhibition of FlhD4C2-dependent transcription under moderate YdiV expression conditions. ClpXP protease degrades FlhD4C2, and this degradation is accelerated in the presence of YdiV. YdiV complexed with both free and DNA-bound FlhD4C2; and stripped FlhD4C2 from DNA. A L22H substitution in FlhD was isolated as insensitive to YdiV inhibition. The FlhD L22H substitution prevented the interaction of YdiV with free FlhD4C2 and the ability of YdiV to release FlhD4C2 bound to DNA. These results demonstrate that YdiV prevents FlhD4C2-dependent flagellar gene transcription and acts as a putative adaptor to target FlhD4C2 for ClpXP-dependent proteolysis. Our results suggest that YdiV is an EAL-like protein that has evolved from a dicyclic-GMP phosphodiesterase into a dual-function regulatory protein that connects flagellar gene expression to nutrient starvation.
ClpXP adaptor; EAL-like protein; flagella; FlhD4C2 regulator; proteolysis
Mismatch repair (MMR) corrects DNA polymerase errors occurring during genome replication. MMR is critical for genome maintenance, and its loss increases mutation rates several hundredfold. Recent work has shown that the interaction between the mismatch recognition protein MutS and the replication processivity clamp is important for MMR in Bacillus subtilis. To further understand how MMR is coupled to DNA replication, we examined the subcellular localization of MMR and DNA replication proteins fused to green fluorescent protein (GFP) in live cells, following an increase in DNA replication errors. We demonstrate that foci of the essential DNA polymerase DnaE-GFP decreases following mismatch incorporation and that loss of DnaE-GFP foci requires MutS. Furthermore, we show that MutS and MutL bind DnaE in vitro, suggesting that DnaE is coupled to repair. We also found that DnaE-GFP foci decrease in vivo following a DNA damage-independent arrest of DNA synthesis showing that loss of DnaE-GFP foci is caused by perturbations to DNA replication. We propose that MutS directly contacts the DNA replication machinery, causing a dynamic change in the organization of DnaE at the replication fork during MMR. Our results establish a striking and intimate connection between MMR and the replicating DNA polymerase complex in vivo.
fluorescence; localization; mismatch repair; MutS; MutL; DnaE
Although mycoplasmas have a paucity of glycosyltransferases and nucleotidyltransferases recognizable by bioinformatics, these bacteria are known to produce polysaccharides and glycolipids. We show here that mycoplasmas also produce glycoproteins and hence have glycomes more complex than previously realized. Proteins from several species of Mycoplasma reacted with a glycoprotein stain, and the murine pathogen Mycoplasma arthritidis was chosen for further study. The presence of M. arthritidis glycoproteins was confirmed by high-resolution mass spectrometry. O-linked glycosylation was clearly identified at both serine and threonine residues. No consensus amino acid sequence was evident for the glycosylation sites of the glycoproteins. A single hexose was identified as the O-linked modification, and glucose was inferred by 13C labeling to be the hexose at several of the glycosylation sites. This is the first study to conclusively identify sites of protein glycosylation in any of the mollicutes.
FT-MS; general glycosylation; glycosyltransferase; lipoprotein; mollicutes