bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lon protease functions as a negative regulator of type III protein secretion in Pseudomonas syringae
Authors: Bretz James , Losada Liliana , Lisboa Keilla , Hutcheson Steven W. 
Published: March 2003

SummaryThe central conserved region of the Pseudomonas syringae hrp pathogenicity island encodes a type III protein secretion system (TTSS) that is required for pathogenicity in plants. Expression of the hrp TTSS is controlled by the alternative sigma factor, HrpL, whose expression, in turn, is positively controlled by two truncated enhancer binding proteins, HrpR and HrpS. Although a number of environmental conditions are known to modulate hrp TTSS expression, such as stringent conditions and pathogenesis, the mechanism by which the activities of these transcriptional factors are modulated had not been established. In this study, HrpR and HrpS were found to be constitutively expressed under conditions in which the hrpL promoter was inactive. To identify a postulated negative regulator of hrpL expression, transposome (Tz) mutagenesis was used to isolate hrp constitutive mutants. P. syringae Pss61 and DC3000 hrp constitutive mutants were identified that carried lon::Tz insertions and exhibited increased cell length and UV sensitivity typical of Δlon mutants. The P. syringae Lon protease retained structural features of its homologues found in other bacteria and was capable of complementing an Escherichia coliΔlon mutant. P. syringae lon::Tz mutants exhibited enhanced expression of the hrpL promoter, suggesting an effect on HrpR and/or HrpS. HrpR was observed to be unstable in wild‐type P. syringae strains grown in non‐inductive media. However, the apparent half‐life increased more than 10‐fold in the P. syringae lon::Tz mutants or upon transfer to an inductive medium. The P. syringae lon mutants elicited rapidly developing plant responses and were shown to hypersecrete effector proteins, such as AvrPto. These results indicate that expression of the hrp regulon and type III secretion are negatively regulated by Lon‐mediated degradation of HrpR.
Protein interactions within Saccharomyces cerevisiae Elongator, a complex essential for Kluyveromyces lactis zymocicity
Authors: Fichtner Lars , Frohloff Frank , Jablonowski Daniel , Stark Michael J. R. , Schaffrath Raffael 
Published: March 2003

SummarymTn3‐tagging identified Kluyveromyces lactis zymocin target genes from Saccharomyces cerevisiae as TOT1–3/ELP1–3 coding for the RNA polymerase II (pol II) Elongator histone acetyltransferase (HAT) complex. tot phenotypes resulting from mTn3 tagging were similar to totΔ null alleles, suggesting loss of Elongator's integrity. Consistently, the Tot1–3/Elp1–3 proteins expressed from the mTn3‐tagged genes were all predicted to be C‐terminally truncated, lacking ≈ 80% of Tot1p, five WD40 Tot2p repeats and two HAT motifs of Tot3p. Besides its role as a HAT, Tot3p assists subunit communication within Elongator by mediating Tot2–Tot4, Tot2–Tot5, Tot2–Tot1 and Tot4–Tot5 protein–protein interactions. TOT1 and TOT2 are essential for Tot4–Tot2 and Tot4–Tot3 interactions respectively. The latter was lost with a C‐terminal Tot2p truncation; the former was affected by progressively truncating TOT1. Despite being dispensable for Tot4–Tot2 interaction, the extreme C‐terminus of Tot1p may play a role in TOT/Elongator function, as its truncation confers zymocin resistance. Tot4p/Kti12p, an Elongator‐associated factor, also interacted with pol II and could be immunoprecipitated while being bound to the ADH1 promoter. Two‐hybrid analysis showed that Tot4p also interacts with Cdc19p, suggesting that Tot4p plays an additional role in concert with Cdc19p, perhaps co‐ordinating cell growth with carbon source metabolism.
Polyadenylation of Escherichia coli transcripts plays an integral role in regulating intracellular levels of polynucleotide phosphorylase and RNase E
Authors: Mohanty Bijoy K. , Kushner Sidney R. 
Published: March 2003

SummaryPolyadenylation in Escherichia coli has been implicated in the destabilization of a variety of transcripts. However, transiently increasing intracellular poly(A) levels has also been shown to stabilize the pnp and rne transcripts, leading to increased polynucleotide phosphorylase (PNPase) and RNase E levels respectively. Here, we show that the half‐lives of both the pnp and rne transcripts are dependent on the intracellular level of polyadenylated transcripts. In addition, experiments using pnp–lacZ and rne–lacZ translational fusions demonstrate that the variations in transcript stability and protein levels arise from alterations in the autoregulation of both genes. Further support for this conclusion is provided by the fact that, in an rne mutant in which autoregulation is inactivated by deletion of most of the 5′ untranslated region, variations in the level of polyadenylated transcripts no longer affect RNase E protein expression. Of even more interest is the fact that the presence of a functional degradosome is essential for RNase E to detect increased levels of poly(A). Thus, it appears that polyadenylation of transcripts in E. coli serves as a sensing mechanism by which the cell adjusts the levels of both RNase E and PNPase.
Multiple bacteria encode metallothioneins and SmtA‐like zinc fingers
Authors: Blindauer Claudia A. , Harrison Mark D. , Robinson Andrea K. , Parkinson John A. , Bowness Peter W. , Sadler Peter J. , Robinson Nigel J. 
Published: March 2003

SummaryZinc is essential but toxic in excess. Bacterial metallothionein, SmtA from Synechococcus PCC 7942, sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA. The dearth of other reported bacterial metallothioneins has been surprising. Here we describe related bacterial metallothioneins (BmtA) from Anabaena PCC 7120, Pseudomonas aeruginosa and Pseudomonas putida that bind multiple zinc ions with high stability towards protons. Thiol modification demonstrates that cysteine coordinates zinc in all of these proteins. Additionally, 111Cd‐NMR, and 111Cd‐edited 1H‐NMR, identified histidine ligands in Anabaena PCC 7120 BmtA, analogous to SmtA. A related Escherichia coli protein bound only a single zinc ion, via four cysteine residues, with low stability towards protons; 111Cd‐NMR and 111Cd‐edited 1H‐NMR confirmed exclusive cysteine‐coordination, and these cysteine residues reacted rapidly with 5,5′‐dithiobis‐(2‐nitrobenzoic acid). 1H‐NMR of proteins from P. aeruginosa, Anabaena PCC 7120 and E. coli generated fingerprints diagnostic for the GATA‐like zinc finger fold of SmtA. These studies reveal first the existence of multiple bacterial metallothioneins, and second proteins with SmtA‐like lone zinc fingers, devoid of a cluster, and designated GatA. We have identified 12 smtA‐like genes in sequence databases including four of the gatA type.
Novel Mycobacterium tuberculosis anti‐σ factor antagonists control σF activity by distinct mechanisms
Authors: Beaucher Jocelyn , Rodrigue Sébastien , Jacques Pierre‐Étienne , Smith Issar , Brzezinski Ryszard , Gaudreau Luc 
Published: March 2003

SummaryThe aetiological agent of tuberculosis, Mycobacterium tuberculosis, encodes 13 σ factors, as well as several putative anti‐, and anti‐anti‐ σ factors. Here we show that a σ factor that has been previously shown to be involved in virulence and persistence processes, σF, can be specifically inhibited by the anti‐σ factor UsfX. Importantly, the inhibitory activity of UsfX, in turn, can be negatively regulated by two novel anti‐anti‐σ factors. The first anti‐anti‐σ factor seems to be regulated by redox potential, and the second may be regulated by phosphorylation as it is rendered non‐functional by the introduction of a mutation that is believed to mimic phosphorylation of the anti‐anti‐σ factor. These results suggest that σF activity might be post‐translationally modulated by at least two distinct pathways in response to different possible physiological cues, the outcome being consistent with the bacteria's ability to adapt to diverse host environments during disease progression, latency and reactivation.
Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity
Authors: DiGiandomenico Antonio , Matewish Mauricia J. , Bisaillon Amy , Stehle John R. , Lam Joseph S. , Castric Peter 
Published: March 2003

SummaryThe structural similarity between the pilin glycan and the O‐antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O‐antigen and produced only non‐glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O‐antigen and glycosylated pilin. Expression of a cosmid clone containing the O‐antigen biosynthetic gene cluster from P. aeruginosa PA103 (LPS serotype O11) in P. aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens. The presence of the O11 repeating unit was confirmed by matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Expression of the O‐antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O‐antigens. A role for pilO in the glycosylation of pilin in P. aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P. aeruginosa strains. Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin. These results show that the pilin glycan of P. aeruginosa 1244 is a product of the O‐antigen biosynthetic pathway. In addition, the structural diversity of the O‐antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.
Domains in the S‐layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self‐assembly
Authors: Antikainen Jenni , Anton Lena , Sillanpää Jouko , Korhonen Timo K. 
Published: March 2003

SummaryThe protein regions in the S‐layer protein CbsA of Lactobacillus crispatus JCM 5810, needed for binding to collagens and laminin, anchoring to bacterial cell wall, as well as self‐assembly, were mapped by deletion analysis of His‐tagged peptides isolated from Escherichia coli and by heterologous expression on Lactobacillus casei. Mature CbsA is 410 amino acids long, and stepwise genetic truncation at both termini revealed that the region 32–271 carries the infor‐mation for self‐assembly of CbsA into a periodic structure. The lactobacillar S‐layer proteins exhibit sequence variation in their assembly domain, but the border regions 30–34 and 269–274 in CbsA are conserved in valine‐rich short sequences. Short deletions or substitutions at these regions affected the morphology of His‐CbsA polymers, which varied from sheet‐like to cylindrical tubular polymers, and further truncation beyond the DNA encoding residues 32 and 271 leads to a non‐periodic aggregation. The self‐assembly of the truncated peptides, as seen by electron microscopy, was correlated with their behaviour in a cross‐linking study. The shorter peptides not forming a regular polymer were observed by the cross‐linking study and mass spectrometry to form dimers, trimers and tetramers, whereas the other peptides were cross‐linked to large multimers only. Binding of solubilized type I and IV collagens was observed with the His‐CbsA peptides 1–274 and 31–287, but not with the smaller peptides regardless of their ability to form regular polymers. Strain JCM 5810 also adheres to immobilized laminin and, in order to analyse the possible laminin binding by CbsA, cbsA and its fragments were expressed on the surface of L. casei. Expression of the CbsA peptides 1–274, 1–287, 28–287 and 31–287 on L. casei conferred adhesiveness to both laminin and collagen immobilized on glass as well as to laminin‐ and collagen‐containing regions in chicken colon and ileum. The C‐terminal peptides 251–410 and 288–410 bound to L. crispatus JCM 5810 cells from which the S‐layer had been depleted by chemical extraction, whereas no binding was seen with the His‐CbsA peptides 1–250 or 1–269 or to cells with an intact S‐layer. The His‐CbsA peptides 251–410 and 288–410 bound to teichoic acids of several bacterial species. The results show that CbsA is an adhesive complex with an N‐terminal assembly domain exhibiting affinity for pericellular tissue components and a cationic C‐terminal domain binding to negatively charged cell wall components.
The rpf gene of Micrococcus luteus encodes an essential secreted growth factor
Authors: Mukamolova Galina V. , Turapov Obolbek A. , Kazarian Konstantin , Telkov Miroslav , Kaprelyants Arseny S. , Kell Douglas B. , Young Michael 
Published: March 2003

Summary Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non‐productively at the cell surface, as a truncated form of the protein, encompassing only the ‘Rpf domain’ is fully active. The C‐terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti‐Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid‐located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf‐like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease.
The N‐terminus of enteropathogenic Escherichia coli (EPEC) Tir mediates transport across bacterial and eukaryotic cell membranes
Authors: Crawford J. Adam , Kaper James B. 
Published: March 2003

SummaryEnteropathogenic Escherichia coli (EPEC) uses a type III secretion system to translocate into host cells several effector molecules that are required for virulence. One of these, the translocated intimin receptor, Tir, inserts into the host cell cytoplasmic membrane, where it functions as the receptor for intimin, an outer membrane adhesin expressed by EPEC. A chaperone for Tir, called CesT, is required for stability of Tir in the EPEC cytoplasm. In this study, the cyaA gene reporter system was used to identify domains in Tir that mediate secretion into the culture supernatant and translocation into host cells. A Tir–CyaA fusion containing the first 15 N‐terminal residues of Tir was secreted and translocated into HeLa cells by a ΔtirΔcesT mutant; however, maximal secretion and translocation was observed with the first 26 N‐terminal residues of Tir. Fusions containing progressively larger N‐terminal sequences of Tir were also efficiently secreted and translocated into HeLa cells by the ΔtirΔcesT strain. However, in a Δtir mutant that expresses CesT, Tir26–CyaA and an additional fusion containing the first 69 N‐terminal residues of Tir were not secreted or translocated, but fusions containing larger N‐terminal Tir sequences were secreted and translocated by the Δtir mutant. Wild‐type EPEC secreted and translocated the Tir15–CyaA fusion, whereas longer fusions, such as Tir26–CyaA and Tir69–CyaA, were translocated to higher levels, similar to what was observed with the ΔtirΔcesT mutant. A Tir–CyaA fusion containing the CesT binding domain was translocated into HeLa cells more rapidly in the presence of CesT compared with translocation in the absence of CesT. Collectively, these results suggest that an N‐terminal domain of 26 amino acids functions as a CesT‐independent signal that is capable of delivering Tir into both the culture supernatant and the cytosol of host cells. Furthermore, in addition to its role in the stability of Tir, CesT may function in translocation by mediating rapid delivery of Tir into host cells.
Rga5p is a specific Rho1p GTPase‐activating protein that regulates cell integrity in Schizosaccharomyces pombe
Authors: Calonge Teresa M. , Arellano Manuel , Coll Pedro M. , Perez Pilar 
Published: March 2003

Summary Schizosaccharomyces pombe
Rho1p regulates (1,3)β‐
d
‐glucan synthesis and is required for cell integrity maintenance and actin cytoskeleton organization, but nothing is known about the regulation of this protein. At least nine different
S. pombe
genes code for proteins predicted to act as Rho GTPase‐activating proteins (GAPs). The results shown in this paper demonstrate that the protein encoded by the gene named
rga5
+
is a GAP specific for Rho1p.
rga5
+
overexpression is lethal and causes morphological alterations similar to those reported for Rho1p inactivation.
rga5
+
deletion is not lethal and causes a mild general increase in cell wall biosynthesis and morphological alterations when cells are grown at 37°C. Upon mild overexpression, Rga5p localizes to growth areas and possesses both
in vivo
and
in vitro
GAP activity specific for Rho1p. Overexpression of
rho1
+
in
rga5Δ
cells is lethal, with a morphological phenotype resembling that of the overexpression of the constitutively active allele
rho1G15V.
In addition (1,3)β‐
d
‐glucan synthase activity, regulated by Rho1p, is increased in
rga5Δ
cells and decreased in
rga5
‐overexpressing cells. Moreover, the increase in (1,3)β‐
d
‐glucan synthase activity caused by
rho1
+
overexpression is considerably higher in
rga5Δ than in wild‐type cells. Genetic interactions suggest that Rga5p is also important for the regulation of the other known Rho1p effectors, Pck1p and Pck2p.
The crk1 gene encodes an Ime2‐related protein that is required for morphogenesis in the plant pathogen Ustilago maydis
Authors: Garrido Elia , Pérez‐Martín José 
Published: March 2003

SummaryThe fungal pathogen Ustilago maydis alternates between budding and filamentous growth during its life cycle. This dimorphic transition is regulated by environmental factors and mating. We cloned a new gene, crk1, which encodes a protein with sequence similarity to Ime2, a kinase involved in developmental choices in S. cerevisiae. Disruption of the crk1 gene in U. maydis resulted in cells that are unable to respond in an appropriate manner to environmental stimuli and show defects in morphogenesis and cell cycle adjustment to changing conditions. We have analysed the regulation of the crk1 gene and demonstrated that cAMP and MAPK pathways have opposite influences on the transcript levels for crk1. Furthermore, we have shown that alterations in the components of these pathways impair the ability of the cellular machinery to adapt to changing conditions. These results demonstrate an important role for the crk1‐ encoded protein in the morphogenesis and environmental adaptation in Ustilago maydis.
Domestication of wild Saccharomyces cerevisiae is accompanied by changes in gene expression and colony morphology
Authors: Kuthan Martin , Devaux Frédéric , Janderová Blanka , Slaninová Iva , Jacq Claude , Palková Zdena 
Published: March 2003

SummaryAlthough colonies from Saccharomyces cerevisiae laboratory strains are smooth, those isolated from nature exhibit a structured fluffy pattern. Environmental scanning electron microscopy shows that the cells within wild fluffy colonies are connected by extracellular matrix (ECM) material. This material contains a protein of about 200 kDa unrelated to the flocculins, proteins involved in cell–cell adhesion in liquid media. The matrix material binds to concanavalin A. Within a few passages on rich agar medium, the wild strains switch from the fluffy to the smooth colony morphology. This domestication is accompanied by loss of the ECM and by extensive changes in gene expression as detected by DNA microarrays. The expression of about 320 genes was changed in smooth colonies. The major changes comprise carbohydrate metabolism, cell wall, water channels, Ty‐transposons and subtelomeric genes, iron homeostasis, vitamin metabolism and cell cycle and polarity. The growth in fluffy colonies may represent a metabolic strategy for survival of yeast under unfavourable conditions that is switched off under felicitous laboratory conditions.
Streptococcus pyogenes recruits collagen via surface‐bound fibronectin: a novel colonization and immune evasion mechanism
Authors: Dinkla Katrin , Rohde Manfred , Jansen Wouter T. M. , Carapetis Jonathan R. , Chhatwal Gursharan S. , Talay Susanne R. 
Published: March 2003

SummaryThis study aimed to characterize matrix assembly mechanisms on the surface of the human pathogen Streptococcus pyogenes. Among 125 S. pyogenes isolates, 61% were able to recruit collagen type IV via surface‐bound fibronectin. Streptococcus gordonii expressing the fibronectin‐binding repeat domain of S. pyogenes SfbI protein was equally potent in recruiting collagen, indicating that this domain was sufficient to promote fibronectin‐mediated collagen recruitment. Electron microscopic analysis of streptococci revealed that fibronectin‐mediated collagen recruitment led to matrix deposition on and between streptococcal cells, which induced the formation of large bacterial aggregates. Furthermore, collagen‐recruiting streptococci were able to colonize collagen fibres and were protected from adhering to human polymorphonuclear cells in the presence of op‐sonizing antibodies. Fibronectin‐mediated collagen recruitment thus represents a novel aggregation, colonization and immune evasion mechanism of S. pyogenes.
Activation mechanisms of the HACI‐mediated unfolded protein response in filamentous fungi
Authors: Saloheimo Markku , Valkonen Mari , Penttilä Merja 
Published: March 2003

SummaryThe unfolded protein response (UPR) is a regulatory pathway activating genes involved in multiple functions related to folding, quality control and transport of secreted proteins. Characterization of the hac1/hacA genes encoding the UPR transcription factors from the filamentous fungi Trichoderma reesei and Aspergillus nidulans is described in this article. The corresponding gene in Saccharomyces cerevisiae is activated through a non‐spliceosomal intron‐splicing reaction. The T. reesei hac1 and A. nidulans hacA mRNAs undergo an analogous splicing reaction of a 20‐nt‐long intron during UPR induction. This splicing changes the reading frame of the mRNA and thus could bring in an activation domain to the HACI/HACA proteins. In addition to the non‐spliceosomal splicing, the hac1/A mRNAs of the filamentous fungi are truncated at the 5′‐flanking region upon UPR induction. An upstream open reading frame is omitted from the mRNAs due to the truncation, and evidence is presented showing that the truncated T. reesei hac1 mRNA is translated more efficiently than a full‐length mRNA. This paper reports a novel combination of two different regulatory mechanisms of a transcription factor gene, both operational at the mRNA level.