bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamic control of Dps protein levels by ClpXP and ClpAP proteases in Escherichia coli
Authors: Stephani Kunigunde , Weichart Dieter , Hengge Regine 
Published: September 2003

SummaryThe Escherichia coli starvation‐induced DNA protection protein Dps was observed to be degraded rapidly during exponential growth. This turnover is dependent on the clpP and clpX genes. The clpA gene is not required for Dps proteolysis, suggesting that Dps is a substrate for ClpXP protease but not for ClpAP protease. Dps proteolysis was found to be highly regulated. Upon carbon starvation, Dps is stabilized, which together with increased Dps synthesis allows strong accumulation of Dps in the stationary phase. The addition of glucose to starving cells results in rapid resumption of Dps proteolysis by ClpXP. Oxidative stress also leads to efficient stabilization of Dps. After hyperosmotic shift, however, proteolysis remains unaffected. Thus, regulated proteolysis of Dps strongly contributes to controlling Dps levels under very specific stress conditions. In contrast to the regulated degradation of RpoS by ClpXP, Dps proteolysis is independent of the recognition factor RssB. In addition, during starvation, clpP and, to a somewhat lesser extent, clpA are involved in maintaining ongoing Dps synthesis (acting at the level of Dps translation), which is required for strong Dps accumulation in long‐term stationary phase cells. In summary, both ClpXP and ClpAP exert significant control of Dps levels by affecting log phase stability and stationary phase synthesis of Dps respectively.
The global transcriptional response of Bacillus subtilis to manganese involves the MntR, Fur, TnrA and σB regulons
Authors: Guedon Emmanuel , Moore Charles M. , Que Qiang , Wang Tao , Ye Rick W. , Helmann John D. 
Published: September 2003

SummaryWe have used DNA microarrays to monitor the global transcriptional response of Bacillus subtilis to changes in manganese availability. Mn(II) leads to the MntR‐dependent repression of both the mntH and mntABCD operons encoding Mn(II) uptake systems. Mn(II) also represses the Fur regulon. This repression is unlikely to be a direct effect of Mn(II) on Fur as repression is sensitive to 2,2′‐dipyridyl, an iron‐selective chelator. We suggest that elevated Mn(II) displaces iron from cellular‐binding sites and the resulting rise in free iron levels leads to repression of the Fur regulon. Many of the genes induced by Mn(II) are activated by σB or TnrA. Both of these regulators are controlled by Mn(II)‐dependent enzymes. Induction of the σB‐dependent general stress response by Mn(II) is largely dependent on RsbU, a Mn(II)‐dependent phosphatase that dephosphorylates RsbV, ultimately leading to release of active σB from its antisigma, RsbW. The activity of TnrA is inhibited when it forms an inactive complex with feedback‐inhibited glutamine synthetase. Elevated Mn(II) reduces the sensitivity of glutamine synthetase to feedback inhibitors, and we suggest that this leads to the observed increase in TnrA activity. In sum, three distinct mechanisms can account for most of the transcriptional effects elicited by manganese: (i) direct binding of Mn(II) to metalloregulators such as MntR, (ii) perturbation of cellular iron pools leading to increased Fur activity and (iii) altered activity of Mn(II)‐dependent enzymes that regulate the activity of σB and TnrA.
Attenuated virulence of Streptococcus agalactiae deficient in D‐alanyl‐lipoteichoic acid is due to an increased susceptibility to defensins and phagocytic cells
Authors: Poyart Claire , Pellegrini Elisabeth , Marceau Michael , Baptista Marina , Jaubert Francis , Lamy Marie‐Cécile , Trieu‐Cuot Patrick 
Published: September 2003

Summary D‐alanylation of lipoteichoic acid (LTA), allows Gram‐positive bacteria to modulate their surface charge, regulate ligand binding and control the electromechanical properties of the cell wall. In this study, the role of D‐alanyl LTA in the virulence of the extracellular pathogen Streptococcus agalactiae was investigated. We demonstrate that a DltA– isogenic mutant displays an increased susceptibility to host defence peptides such as human defensins and animal‐derived cationic peptides. Accordingly, the mutant strain is more susceptible to killing by mice bone marrow‐derived macrophages and human neutrophils than the wild‐type strain. In addition, the virulence of the DltA– mutant is severely impaired in mouse and neonatal rat models. This mutant was eliminated more rapidly than the wild‐type strain from the lung of three‐week‐old mice inoculated intranasally and, consequently, is unable to induce a pneumonia. Finally, after intravenous injection of three‐week‐old mice, the survival of the DltA– mutant is markedly reduced in the blood in comparison to that of the wild‐type strain. We hypothesize that the decreased virulence of the DltA– mutant is a consequence of its increased susceptibility to cationic antimicrobial peptides and to killing by phagocytes. These results demonstrate that the D‐alanylation of LTA contributes to the virulence of S. agalactiae.
Tetracycline‐aptamer‐mediated translational regulation in yeast
Authors: Hanson Shane , Berthelot Karine , Fink Barbara , McCarthy John E. G. , Suess Beatrix 
Published: September 2003

SummaryWe describe post‐transcriptional gene regulation in yeast based on direct RNA–ligand interaction. Tetracycline‐dependent translational regulation could be imposed via specific aptamers inserted at two different positions in the 5′ untranslated region (5′UTR). Translation in vivo was suppressed up to ninefold upon addition of tetracycline. Repression via an aptamer located near the start codon (cap‐distal) in the 5′UTR was more effective than repression via a cap‐proximal position. On the other hand, suppression in a cell‐free system reached maximally 50‐fold and was most effective via a cap‐proximal aptamer. Examination of the kinetics of tetracycline‐dependent translational inhibition in vitro revealed that preincubation of tetracycline and mRNA before starting translation led not only to the fastest onset of inhibition but also the most effective repression. The differences between the behaviour of the regulatory system in vivo and in vitro are likely to be related to distinct properties of mRNP structure and mRNA accessibility in intact cells as opposed to cell‐extracts. Tetracycline‐dependent regulation was also observed after insertion of an uORF sequence upstream of the aptamer, indicating that our system also targets reinitiating ribosomes. Polysomal gradient analyses provided insight into the mechanism of regulation. Cap‐proximal insertion inhibits binding of the 43S complex to the cap structure whereas start‐codon‐proximal aptamers interfere with formation of the 80S ribosome, probably by blocking the scanning preinitiation complex.
PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho‐Ser/Thr phosphatase, in Mycobacterium tuberculosis
Authors: Boitel Brigitte , Ortiz‐Lombardía Miguel , Durán Rosario , Pompeo Fréderique , Cole Stewart T. , Cerveñansky Carlos , Alzari Pedro M. 
Published: September 2003

SummaryBacterial genomics revealed the widespread presence of eukaryotic‐like protein kinases and phosphatases in prokaryotes, but little is known on their biochemical properties, regulation mechanisms and physiological roles. Here we focus on the catalytic domains of two trans‐membrane enzymes, the Ser/Thr protein kinase PknB and the protein phosphatase PstP from Mycobacterium tuberculosis. PstP was found to specifically dephosphorylate model phospho‐Ser/Thr substrates in a Mn2+‐dependent manner. Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP‐mediated dephosphorylation. Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase, namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation. Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role. These results indicate that, as for eukaryotic homologues, phosphorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth.
TPR‐mediated interaction of RapC with ComA inhibits response regulator‐DNA binding for competence development in Bacillus subtilis
Authors: Core Leighton , Perego Marta 
Published: September 2003

SummaryThe Bacillus subtilis Rap family of proteins are characterized by protein–protein interaction modules containing the so‐called tetratricopeptide repeats (TPRs). The six TPR motifs of RapC mediate its interaction with the pentapeptide inhibitor PhrC (ERGMT) or with its target protein ComA, a phosphorylation‐dependent response regulator transcription factor for genetic competence. Our results show that RapC interaction with ComA inhibits the response regulator's ability to bind its target DNA promoter but does not affect its phosphorylation state. RapC binds equally well to ComA or to ComA∼P. The PhrC pentapeptide binds to RapC and inhibits its interaction with ComA. The D195 residue in TPR3 and the P263 residue in TPR5 of RapC are critical for the interaction with PhrC as their mutation to asparagine or leucine, respectively, prevents peptide inhibitory activity. The RapC mechanism of regulating ComA activity is a new example of how TPR motifs and their structural organization have been adapted for different specific functions within the B. subtilis Rap family.
Essential role of DivIVA in polar growth and morphogenesis in Streptomyces coelicolor A3(2)
Authors: Flärdh Klas 
Published: September 2003

SummaryStreptomycetes grow by cell wall extension at hyphal tips. The molecular basis for such polar growth in prokaryotes is largely unknown. It is reported here that DivIVASC, the Streptomyces coelicolor homologue of the Bacillus subtilis protein DivIVA, is essential and directly involved in hyphal tip growth and morphogenesis. A DivIVASC‐EGFP hybrid was distinctively localized to hyphal tips and lateral branches. Reduction of divIVASC expression to about 10% of the normal level produced a phenotype strikingly similar to that of many tip growth mutants in fungi, including irregular curly hyphae and apical branching. Overexpression of the gene dramatically perturbed determination of cell shape at the growing tips. Furthermore, staining of nascent peptidoglycan with a fluorescent vancomycin conjugate revealed that induction of overexpression in normal hyphae disturbed tip growth, and gave rise to several new sites of cell wall assembly, effectively causing hyperbranching. The results show that DivIVASC is a novel bacterial morphogene, and it is localized at or very close to the apical sites of peptidoglycan assembly in Streptomyces hyphae.
Genes controlled by the essential YycG/YycF two‐component system of Bacillus subtilis revealed through a novel hybrid regulator approach
Authors: Howell Alistair , Dubrac Sarah , Andersen Kasper Krogh , Noone David , Fert Juliette , Msadek Tarek , Devine Kevin 
Published: September 2003

SummaryThe YycG/YycF two‐component system, originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low G + C Gram‐positive bacteria. This system is required for cell viability, although the basis for this and the nature of the YycF regulon remained elusive. Using a combined hybrid regulator/transcriptome approach involving the inducible expression of a PhoP′‐′YycF chimerical protein in B. subtilis, we have shown that expression of yocH, which encodes a potential autolysin, is specifically activated by YycF. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the regulatory regions of yocH as well as ftsAZ, previously reported to be controlled by YycF. Nucleotide sequence analysis and site‐directed mutagenesis allowed us to define a potential consensus recognition sequence for the YycF response regulator, composed of two direct repeats: 5′‐TGT A/T A A/T/C‐N5‐TGT A/T A A/T/C‐3′. A DNA‐motif analysis indicates that there are potentially up to 10 genes within the B. subtilis YycG/YycF regulon, mainly involved in cell wall metabolism and membrane protein synthesis. Among these, YycF was shown to bind directly to the region upstream from the ykvT gene, encoding a potential cell wall hydrolase, and the intergenic region of the tagAB/tagDEF divergon, encoding essential components of teichoic acid biosynthesis. Definition of a potential YycF recognition sequence allowed us to identify likely members of the YycF regulon in other low G + C Gram‐positive bacteria, including several pathogens such as Listeria monocytogenes, Staphylococcus aureus and Streptococcus pneumoniae.
A supramolecular complex in the environmental stress signalling pathway ofBacillus subtilis
Authors: Chen Chien‐Cheng , Lewis Richard J. , Harris Robin , Yudkin Michael D. , Delumeau Olivier 
Published: September 2003

SummaryσB, an alternative sigma‐factor ofBacillus subtilis,mediates the response of the cell to a variety of physical insults. Within the environmental stress signalling pathway RsbU, a protein phosphatase, is stimulated by its interaction with the protein kinase RsbT. In the absence of stress RsbT is expected to be trapped by an alternative binding partner, RsbS. Here, we have demonstrated that RsbS alone cannot act as an alternative partner for RsbT, but instead requires the presence of RsbR to create a high molecular mass RsbR:RsbS complex (∼ 1 MDa) able to capture RsbT. In this complex the phosphorylation state of RsbS, and not that of RsbR, controlled the binding to RsbT, whose kinase activity towards RsbS could be counterbalanced by the activity of RsbX, the phosphatase for RsbS‐P. The RsbR:RsbS complex recruited RsbT from a mixture of RsbT and RsbU. The phosphorylated form of RsbR in the complex enhanced the kinase activity of RsbT towards RsbS. This supramolecular complex thus has the functional properties of an alternative partner for RsbT. Electron micrographs of this complex are presented, and the purification of the RsbR:RsbS complex from cellular extracts provides evidence for the existence of such a complexin vivo.
The HfaB and HfaD adhesion proteins of Caulobacter crescentus are localized in the stalk
Authors: Cole Jennifer L. , Hardy Gail G. , Bodenmiller Diane , Toh Evelyn , Hinz Aaron , Brun Yves V. 
Published: September 2003

SummaryThe differentiating bacterium Caulobacter crescentus produces two different cell types at each cell division, a motile swarmer cell and an adhesive stalked cell. The stalked cell harbours a stalk, a thin cylindrical extension of the cell surface. The tip of the stalk is decorated with a holdfast, an adhesive organelle composed at least in part of polysaccharides. The synthesis of the stalk and holdfast occur at the same pole during swarmer cell differentiation. Mutations in the hfaABDC gene cluster had been shown to disrupt the attachment of the holdfast to the tip of the stalk, but the role of individual genes was unknown. We used lacZ fusions of various DNA fragments from the hfaABDC region to show that these genes form an operon. In order to analyse the relative contribution of the different genes to holdfast attachment, mutations were constructed for each gene. hfaC was not required for holdfast attachment or binding to surfaces. The hfaA and hfaD mutants shed some holdfast material into the surrounding medium and were partially deficient in binding to surfaces. Unlike hfaA and hfaB mutants, hfaD mutants were still able to form rosettes efficiently. Cells with insertions in hfaB were unable to bind to surfaces, and lectin binding studies indicated that the hfaB mutants had the strongest holdfast shedding phenotype. We determined that HfaB and HfaD are membrane‐associated proteins and that HfaB is a lipoprotein. Purification of stalks and cell bodies indicated that both HfaB and HfaD are enriched in the stalk as compared to the cell body. These results suggest that HfaB and HfaD, and probably HfaA, serve to anchor the holdfast to the tip of the stalk.
Binding of response regulator DegU to the aprE promoter is inhibited by RapG, which is counteracted by extracellular PhrG in Bacillus subtilis
Authors: Ogura Mitsuo , Shimane Kana , Asai Kei , Ogasawara Naotake , Tanaka Teruo 
Published: September 2003

SummaryWe screened the putative rap‐phr (response regulator aspartyl‐phosphate phosphatase‐phosphatase regulator) systems identified in the Bacillus subtilis genome for a rap gene that affects aprE (alkaline protease gene) expression by using a multicopy plasmid. We found that rapG was involved in the regulation of aprE, which belongs to the regulon of DegU, the response regulator of the DegS‐DegU two‐component system. Disruption of rapG and phrG resulted in enhancement and reduction of aprE‐lacZ expression, respectively, suggesting that PhrG inhibits RapG activity. Addition of 1–30 nM of a synthetic pentapeptide (PhrG; NH2‐EKMIG‐COOH) to the phrG disruptant completely rescued aprE‐lacZ expression, indicating that the PhrG peptide is indeed involved in aprE‐lacZ expression. Surprisingly, either introduction of multicopy phrG or addition of the PhrG peptide at high concentrations (100–300 nM) to the phrG cells decreased aprE‐lacZ expression. These results are reminiscent of the previous observation that at higher concentrations the PhrC peptide inhibits srfA‐lacZ expression directed by ComA, the regulator of the ComP‐ComA two‐component system. Because the Rap proteins belong to a family of aspartyl protein phosphatases, we tried to investigate the possible influence of RapG on dephosphorylation of DegU‐P (phosphorylated DegU) in vitro. RapG, however, did not affect dephosphorylation of DegU‐P under the adopted experimental conditions. Therefore, we hypothesized that RapG might inhibit the binding activity of DegU to the target promoters. We analysed the interaction of DegU and RapG using the aprE promoter and another target, a comK promoter. Gel shift analysis revealed that RapG served as the inhibitor of DegU binding to the promoter regions of aprE and comK and that this inhibition was counteracted by the PhrG peptide.
Genetic and molecular evidence that the Pseudomonas syringae type III effector protein AvrRpt2 is a cysteine protease
Authors: Axtell Michael J. , Chisholm Stephen T. , Dahlbeck Douglas , Staskawicz Brian J. 
Published: September 2003

SummaryUpon delivery to the plant cell during infection, the Pseudomonas syringae effector protein AvrRpt2 undergoes proteolytic processing, enhances pathogen virulence and causes the elimination of the Arabidopsis RIN4 protein. A structure‐prediction method was employed in order to investigate possible biochemical functions of AvrRpt2. Results of a secondary structure prediction algorithm suggest that the functional C‐terminal portion of AvrRpt2 is a cysteine protease. Mutation of predicted catalytic residues within this portion of AvrRpt2 abolished in planta processing, elimination of Arabidopsis RIN4, and the ability to trigger an RPS2‐specific resistance response. These data indicate that AvrRpt2 is most likely a sequence divergent cysteine protease whose activity is required for elimination of RIN4 during infection.
Requirement for kasB in Mycobacterium mycolic acid biosynthesis, cell wall impermeability and intracellular survival: implications for therapy
Authors: Gao Lian‐Yong , Laval Francoise , Lawson Elise H. , Groger Richard K. , Woodruff Andy , Morisaki J. Hiroshi , Cox Jeffery S. , Daffe Mamadou , Brown Eric J. 
Published: September 2003

Summary Mycobacterium tuberculosis infects one‐third of the world's population and causes two million deaths annually. The unusually low permeability of its cell wall contributes to the ability of M. tuberculosis to grow within host macrophages, a property required for pathogenesis of infection. Mycobacterium marinum is an established model for discovering genes involved in mycobacterial infection. Mycobacterium marinum mutants with transposon insertions in the β‐ketoacyl‐acyl carrier protein synthase B gene (kasB) grew poorly in macrophages, although growth in vitro was unaffected. Detailed analyses by thin‐layer chromatography, nuclear magnetic resonance (NMR), matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, infrared spectroscopy, and chemical degradations showed that the kasB mutants synthesize mycolic acids that are 2–4 carbons shorter than wild type; the defect was localized to the proximal portion of the meromycolate chain. In addition, these mutants showed a significant (∼30%) reduction in the abundance of keto‐mycolates, with a slight compensatory increase of both α‐ and methoxy‐mycolates. Despite these small changes in mycolate length and composition, the kasB mutants exhibited strikingly altered cell wall permeability, leading to a marked increase in susceptibility to lipophilic antibiotics and the host antimicrobial molecules defensin and lysozyme. The abnormalities of the kasB mutants were fully complemented by expressing M. tuberculosis kasB, but not by the closely related gene kasA. These studies identify kasB as a novel target for therapeutic intervention in mycobacterial diseases.
The Salmonella SpiC protein targets the mammalian Hook3 protein function to alter cellular trafficking
Authors: Shotland Yoram , Krämer Helmut , Groisman Eduardo A. 
Published: September 2003

SummaryThe Salmonella SpiC protein is secreted into the cytosol of macrophages via a unique type III secretion system that functions intracellularly to translocate proteins across the phagosomal membrane. The SpiC protein is required for survival within macrophages and inhibition of phagosome‐lysosome fusion in vivo, and it is sufficient to inhibit endosome‐endosome fusion in vitro. Here, we establish that SpiC targets the function of Hook3, a mammalian protein implicated in cellular trafficking. Purified GST‐SpiC pulled down Hook3 from murine macrophages, and anti‐Hook3 antibodies precipitated SpiC from the cytosol of Salmonella‐infected macrophages. Expression of the spiC gene disrupted Golgi morphology in Vero cells and altered the distribution of lysosomes in macrophages, mimicking the phenotype of cells expressing a hook3 dominant‐negative mutant. By inactivating Hook3 function, the SpiC protein may alter the lysosome network and prevent phagosome‐lysosome fusion.