bluuebunny/crossref_metadata_2025 huggingface dataset:
Archaeal chromatin and transcription
Authors: Reeve John N.
Published: December 2004

Archaea contain a variety of sequence‐independent DNA binding proteins consistent with the evolution of several different, sometimes overlapping and exchangeable solutions to the problem of genome compaction. Some of these proteins undergo residue‐specific post‐translational lysine acetylation or methylation, hinting at analogues of the histone modifications that regulate eukaryotic chromatin structure and transcription. Archaeal transcription initiation most closely resembles the eukaryotic RNA polymerase II (RNAPII) system, but Archaea do not appear to have homologues of the multisubunit complexes that remodel eukaryotic chromatin and activate RNAPII initiation. In contrast, they have sequence‐specific regulators that repress and perhaps activate archaeal transcription by mechanisms superficially similar to the bacterial paradigm of regulating promoter binding by RNAP. Repressors compete with archaeal TATA‐box binding protein (TBP) and TFB for the TATA‐box and TFB‐recognition elements (BRE) of the archaeal promoter, or with archaeal RNAP for the site of transcription initiation. Transcript‐specific regulation by repressors binding to sites of transcript initiation is consistent with such sites having very little sequence conservation. However, most Archaea have only one TBP and/or TFB that presumably must therefore bind to similar TATA‐box and BRE sequences upstream of most genes. Repressors that function by competing with TBP and/or TFB binding must therefore also make additional contacts with transcript‐specific regulatory sites adjacent or remote from the TATA‐box/BRE region. The fate of the archaeal TBP and TFB following transcription initiation remains to be determined. Based on functional homology with their eukaryotic RNAPII‐system counterparts, archaeal TBP and possibly also TFB should remain bound to the TATA‐box/BRE region after transcription initiation. However, this seems unlikely as it might limit repressor competition at this site to only the first round of transcription initiation.
CLNR1, the AREA/NIT2‐like global nitrogen regulator of the plant fungal pathogen Colletotrichum lindemuthianum is required for the infection cycle
Authors: Pellier Anne‐Laure , Laugé Richard , Veneault‐Fourrey Claire , Langin Thierry
Published: December 2004

Nitrogen starvation is generally assumed to be encountered by biotrophic and hemibiotrophic plant fungal pathogens at the beginning of their infection cycle. We tested whether nitrogen starvation constitutes a cue regulating genes that are required for pathogenicity of Colletotrichum lindemuthianum, a fungal pathogen of common bean. The clnr1 (C. lindemuthianumnitrogen regulator 1) gene, the areA/nit‐2 orthologue of C. lindemuthianum, was isolated. The predicted CLNR1 protein exhibits high amino acid sequence similarities with the AREA and NIT2 global fungal nitrogen regulators. Targeted clnr1– mutants are unable to use a wide array of nitrogen sources, indicating that clnr1 is the C. lindemuthianum major nitrogen regulatory gene. The clnr1– mutants are non‐pathogenic, although few anthracnose lesions seldom occur on whole plantlets. Surprisingly, cytological analysis reveals that the clnr1– mutants are not disturbed from the penetration stage until the end of the biotrophic phase, but that they are impaired during the setting up of the necrotrophic phase. Thus, through CLNR1, nitrogen starvation constitutes a cue for the regulation of genes that are compulsory for this stage of the C. lindemuthianum infection process. Additionally, clnr1– mutants complemented with the Aspergillus nidulans areA gene are fully pathogenic, indicating that areA is able to activate the C. lindemuthianum suited genes, normally under the control of clnr1.
Opi1p, Ume6p and Sin3p control expression from the promoter of the INO2 regulatory gene via a novel regulatory cascade
Authors: Kaadige Mohan R. , Lopes John M.
Published: December 2004

The INO2 gene of Saccharomyces cerevisiae is required for expression of most of the phospholipid biosynthetic genes. INO2 expression is regulated by a complex cascade that includes autoregulation, Opi1p‐mediated repression and Ume6p‐mediated activation. To screen for mutants with altered INO2 expression directly, we constructed an INO2‐HIS3 reporter that provides a plate assay for INO2 promoter activity. This reporter was used to isolate mutants (dim1) that fail to repress expression of the INO2 gene in an otherwise wild‐type strain. The dim1 mutants contain mutations in the OPI1 gene. To define further the mechanism for Ume6p regulation of INO2 expression, we isolated suppressors (rum1, 2, 3) of the ume6Δ mutation that overexpress the INO2‐HIS3 gene. Two of the rum mutant groups contain mutations in the OPI1 and SIN3 genes showing that opi1 and sin3 mutations are epistatic to the ume6Δ mutation. These results are surprising given that Ume6p, Sin3p and Rpd3p are known to form a complex that represses the expression of a diverse set of yeast genes. This prompted us to examine the effect of sin3Δ and rpd3Δ mutants on INO2‐cat expression. Surprisingly, the sin3Δ allele overexpressed INO2‐cat, whereas the rpd3Δ mutant had no effect. We also show that the UME6 gene does not affect the expression of an OPI1‐cat reporter. This suggests that Ume6p does not regulate INO2 expression indirectly by regulating OPI1 expression.
Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis
Authors: Park Heui‐Dong , Guinn Kristi M. , Harrell Maria I. , Liao Reiling , Voskuil Martin I. , Tompa Martin , Schoolnik Gary K. , Sherman David R.
Published: December 2004

Unlike many pathogens that are overtly harmful to their hosts, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. Latency is often linked to hypoxic conditions within the host. Among M. tuberculosis genes induced by hypoxia is a putative transcription factor, Rv3133c/DosR. We performed targeted disruption of this locus followed by transcriptome analysis of wild‐type and mutant bacilli. Nearly all the genes powerfully regulated by hypoxia require Rv3133c/DosR for their induction. Computer analysis identified a consensus motif, a variant of which is located upstream of nearly all M. tuberculosis genes rapidly induced by hypoxia. Further, Rv3133c/DosR binds to the two copies of this motif upstream of the hypoxic response gene alpha‐crystallin. Mutations within the binding sites abolish both Rv3133c/DosR binding as well as hypoxic induction of a downstream reporter gene. Also, mutation experiments with Rv3133c/DosR confirmed sequence‐based predictions that the C‐terminus is responsible for DNA binding and that the aspartate at position 54 is essential for function. Together, these results demonstrate that Rv3133c/DosR is a transcription factor of the two‐component response regulator class, and that it is the primary mediator of a hypoxic signal within M. tuberculosis.
Glucose triggers different global responses in yeast, depending on the strength of the signal, and transiently stabilizes ribosomal protein mRNAs
Authors: Yin Zhikang , Wilson Séan , Hauser Nicole C. , Tournu Helene , Hoheisel Jörg D. , Brown Alistair J. P.
Published: December 2004

Glucose exerts profound effects upon yeast physiology. In general, the effects of high glucose concentrations (>1%) upon Saccharomyces cerevisiae have been studied. In this paper, we have characterized the global responses of yeast cells to very low (0.01%), low (0.1%) and high glucose signals (1.0%) by transcript profiling. We show that yeast is more sensitive to very low glucose signals than was previously thought, and that yeast displays different responses to these different glucose signals. Genes involved in central metabolic pathways respond rapidly to very low glucose signals, whereas genes involved in the biogenesis of cytoplasmic ribosomes generally respond only to glucose concentrations of> 0.1%. We also show that cytoplasmic ribosomal protein mRNAs are transiently stabilized by glucose, indicating that both transcriptional and post‐transcriptional mechanisms combine to accelerate the accumulation of ribosomal protein mRNAs. Presumably, this facilitates rapid ribosome biogenesis after exposure to glucose. However, our data indicate that yeast activates ribosome biogenesis only when sufficient glucose is available to make this metabolic investment worthwhile. In contrast, the regulation of metabolic functions in response to very low glucose signals presumably ensures that yeast can exploit even minute amounts of this preferred nutrient.
Identification of a novel plasmin(ogen)‐binding motif in surface displayed α‐enolase of Streptococcus pneumoniae
Authors: Bergmann Simone , Wild Daniela , Diekmann Oliver , Frank Ronald , Bracht Dagmar , Chhatwal Gursharan S. , Hammerschmidt Sven
Published: December 2004

The interaction of Streptococcus pneumoniae with human plasmin(ogen) represents a mechanism to enhance bacterial virulence by capturing surface‐associated proteolytic activity in the infected host. Plasminogen binds to surface displayed pneumococcal α‐enolase (Eno) and is subsequently activated to the serine protease plasmin by host‐derived tissue plasminogen activator (tPA) or urokinase (uPA). The C‐terminal lysyl residues of Eno at position 433 and 434 were identified as a binding site for the kringle motifs of plasmin(ogen) which contain lysine binding sites. In this report we have identified a novel internal plamin(ogen)‐binding site of Eno by investigating the protein–protein interaction. Plasmin(ogen)‐binding activity of C‐terminal mutated Eno proteins used in binding assays as well as surface plasmon resonance studies suggested that an additional binding motif of Eno is involved in the Eno‐plasmin(ogen) complex formation. The analysis of spot synthesized synthetic peptides representing Eno sequences identified a peptide of nine amino acids located between amino acids 248–256 as the minimal second binding epitope mediating binding of plasminogen to Eno. Binding of radiolabelled plasminogen to viable pneumococci was competitively inhibited by a synthetic peptide FYDKERKVYD representing the novel internal plasmin(ogen)‐binding motif of Eno. In contrast, a synthetic peptide with amino acid substitutions at critical positions in the internal binding motif identified by systematic mutational analysis did not inhibit binding of plasminogen to pneumococci. Pneumococcal mutants expressing α‐enolase with amino acid substitutions in the internal binding motif showed a substantially reduced plasminogen‐binding activity. The virulence of these mutants was also attenuated in a mouse model of intranasal infection indicating the significance of the novel plasminogen‐binding motif in the pathogenesis of pneumococcal diseases.
SOAP, a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development
Authors: Dessens Johannes T. , Sidén‐Kiamos Inga , Mendoza Jacqui , Mahairaki Vassiliki , Khater Emad , Vlachou Dina , Xu Xiao‐Jin , Kafatos Fotis C. , Louis Christos , Dimopoulos George , Sinden Robert E.
Published: December 2004

An essential, but poorly understood part of malaria transmission by mosquitoes is the development of the ookinetes into the sporozoite‐producing oocysts on the mosquito midgut wall. For successful oocyst formation newly formed ookinetes in the midgut lumen must enter, traverse, and exit the midgut epithelium to reach the midgut basal lamina, processes collectively known as midgut invasion. After invasion ookinete‐to‐oocyst transition must occur, a process believed to require ookinete interactions with basal lamina components. Here, we report on a novel extracellular malaria protein expressed in ookinetes and young oocysts, named secreted ookinete adhesive protein (SOAP). The SOAP gene is highly conserved amongst Plasmodium species and appears to be unique to this genus. It encodes a predicted secreted and soluble protein with a modular structure composed of two unique cysteine‐rich domains. Using the rodent malaria parasite Plasmodium berghei we show that SOAP is targeted to the micronemes and forms high molecular mass complexes via disulphide bonds. Moreover, SOAP interacts strongly with mosquito laminin in yeast‐two‐hybrid assays. Targeted disruption of the SOAP gene gives rise to ookinetes that are markedly impaired in their ability to invade the mosquito midgut and form oocysts. These results identify SOAP as a key molecule for ookinete‐to‐oocyst differentiation in mosquitoes.
An effective sporulation of Myxococcus xanthus requires glycogen consumption via Pkn4‐activated 6‐phosphofructokinase
Authors: Nariya Hirofumi , Inouye Sumiko
Published: December 2004

6‐Phosphofructokinase (PFK) is a key enzyme for glycolysis in both prokaryotes and eukaryotes. Previously, it was found that the activity of Myxococcus xanthus PFK increased 2.7‐fold upon phosphorylation at Thr‐226 by the Ser/Thr kinase Pkn4. The pkn4 gene is located 18 bp downstream of the pfk gene forming an operon, and both genes are expressed during vegetative growth and development. Here, we show that glycogen, which accumulates during stationary phase and early in development, is consumed during sporulation. A pfk–pkn4 deletion strain accumulated glycogen at a higher level than the wild‐type strain, was unable to consume glycogen during developmental progression and exhibited a poor spore yield. From genetic complementation analysis of the pfk–pkn4 deletion strain with the pfk and pkn4 genes, it was found that glycogen consumption and a high spore yield require not only the pfk gene but also the pkn4 gene. Furthermore, phosphorylation is critical for glycogen consumption because the pfk gene engineered to express the mutant PFK (Thr‐226‐Ala) did not complement a pfk mutant. We propose that glycogen metabolism in M. xanthus is regulated in a similar manner to that in eukaryotes requiring a protein Ser/Thr kinase.
Surface anchoring of bacterial subtilisin important for maturation function
Authors: Coutte Loic , Willery Eve , Antoine Rudy , Drobecq Hervé , Locht Camille , Jacob‐Dubuisson Françoise
Published: December 2004

Many extracytoplasmic proteins undergo proteolytic processing during secretion, which is essential to their maturation. These post‐translational modifications are carried out by specific enzymes whose subcellular localization is important for function. We have described a maturation subtilisin in Gram‐negative Bordetella pertussis, the autotransporter SphB1. SphB1 catalyses the maturation of the precursor of the adhesin filamentous haemagglutinin (FHA) at the bacterial surface, in addition to the processing of its own precursor. Here, we show that the outer membrane anchor of SphB1 is crucial to its function, as evidenced by the lack of FHA maturation in a strain releasing a variant of SphB1 into the milieu. In contrast, surface association is not required for automaturation of SphB1. The surface retention of mature SphB1 is mediated by lipidation of the protein. The tethered protease appears to be stabilized by unusual Gly‐ and Pro‐rich motifs at the N‐terminus of the protein. This represents a new mode of localization for a protease involved in protein secretion.
Identification of genes required for adventurous gliding motility in Myxococcus xanthus with the transposable element mariner
Authors: Youderian Philip , Burke Neal , White David J. , Hartzell Patricia L.
Published: December 2004

Myxococcus xanthus glides over solid surfaces without the use of flagella, dependent upon two large sets of adventurous (A) and social (S) genes, using two different mechanisms of gliding motility. Myxococcus xanthus A–S– double mutants form non‐motile colonies lacking migratory cells at their edges. We have isolated 115 independent mutants of M. xanthus with insertions of transposon magellan‐4 in potential A genes by screening for insertions that reduce the motility of a mutant S– parental strain. These insertions are found not only in the three loci known to be required for A motility, mglBA, cglB, and aglU, but also in 30 new genes. Six of these new genes encode different homologues of the TolR, TolB, and TolQ transport proteins, suggesting that adventurous motility is dependent on biopolymer transport. Other insertions which affect both A and S motility suggest that both systems share common energy and cell wall determinants. Because the spectrum of magellan‐4 insertions in M. xanthus is extraordinarily broad, transposon mutagenesis with this eukaryotic genetic element permits the rapid genetic analysis of large sets of genes that contribute to a complex microbial behaviors such as A motility.
Invasive M1T1 group A Streptococcus undergoes a phase‐shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB
Authors: Aziz Ramy K. , Pabst Michael J. , Jeng Arthur , Kansal Rita , Low Donald. E. , Nizet Victor , Kotb Malak
Published: December 2004

A globally disseminated strain of M1T1 group A Streptococcus (GAS) has been associated with severe infections in humans including necrotizing fasciitis and toxic shock syndrome. Recent clinicoepidemiologic data showed a striking inverse relationship between disease severity and the degree to which M1T1 GAS express the streptococcal cysteine protease, SpeB. Electrophoretic 2‐D gel analysis of the secreted M1T1 proteome, coupled with MALDI‐TOF mass spectroscopy, revealed that expression of active SpeB caused the degradation of the vast majority of secreted GAS proteins, including several known virulence factors. Injection of a SpeB+/SpeA– M1T1 GAS strain into a murine subcutanous chamber model of infection selected for a stable phase‐shift to a SpeB–/SpeA+ phenotype that expressed a full repertoire of secreted proteins and possessed enhanced lymphocyte‐stimulating capacity. The proteome of the SpeB–in vivo phase‐shift form closely matched the proteome of an isogenic speB gene deletion mutant of the original M1T1 isolate. The absence or the inactivation of SpeB allowed proteomic identification of proteins in this M1T1 clone that are not present in the previously sequenced M1 genome including SpeA and another bacteriophage‐encoded novel streptodornase allele. Further proteomic analysis of the M1T1 SpeB+ and SpeB– phase‐shift forms in the presence of a cysteine protease inhibitor demonstrated differences in the expression of several proteins, including the in vivo upregulation of SpeA, which occurred independently of SpeB inactivation.
Characterization of components of the mismatch repair machinery in Trypanosoma brucei
Authors: Bell Joanna S. , Harvey Timothy I. , Sims Anne‐Marie , McCulloch Richard
Published: December 2004

Mismatch repair is one of a number of DNA repair pathways that cells possess to deal with damage to their genome. Mismatch repair is concerned with the recognition and correction of incorrectly paired bases, which can be base–base mismatches or insertions or deletions of a few bases, and appears to have been conserved throughout evolution. Primarily, this is concerned with increasing the fidelity of DNA replication, but also has important roles in the regulation of homologous recombination and the correction of chemical damage. In this study, we describe five genes in the protistan parasite Trypanosoma brucei that are likely to be involved in nuclear mismatch repair. The predicted T. brucei mismatch repair genes are diverged compared with their likely counterparts in the other eukaryotes examined to date. To demonstrate that these do indeed encode a functional nuclear mismatch repair system, we made T. brucei null mutants in two of the genes, MSH2 and MLH1, that are likely to be central to the functioning of the mismatch repair machinery. These mutations resulted in increased rates of sequence variation at a number of microsatellite loci in the parasite genome, and led to increased tolerance to the alkylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, both phenotypes consistent with mismatch repair impairment.
Authentic precursors to ribosomal subunits accumulate in Escherichia coli in the absence of functional DnaK chaperone
Authors: Hage Aziz El , Alix Jean‐Hervé
Published: December 2004

Escherichia coli dnaK‐ts mutants are defective in the late stages of ribosome biogenesis at high temperature. Here, we show that the 21S, 32S and 45S ribosomal particles that accumulate in the dnaK756‐ts mutant at 44°C contain unprocessed forms of their 16S and 23S rRNAs (partially processed in the case of 45S particles). Their 5S rRNA stoichiometry and ribosomal protein composition are typical of the genuine ribosomal precursors found in a wild‐type (dnaK+) strain. Despite the lack of a functional DnaK, a very slow maturation of these 21S, 32S and 45S particles to structurally and functionally normal 30S and 50S ribosomal subunits still occurs at high temperature. This conversion is accompanied by the processing of p16S and p23S rRNAs to their mature forms. We conclude that: (i) 21S, 32S and 45S particles are not dead‐end particles, but true precursors to active ribosomes (21S particles are converted to 30S subunits, and 32S and 45S to 50S subunits); (ii) DnaK is not absolutely necessary for ribosome biogenesis, but accelerates the late steps of this process considerably at high temperature; and (iii) 23S rRNA processing depends on the stage reached in the stepwise assembly of the 50S subunit, not directly on DnaK.
Evidence for dynamic clustering of carboxy‐terminal aromatic amino acids in TonB‐dependent energy transduction
Authors: Ghosh Joydeep , Postle Kathleen
Published: December 2004

Escherichia coli uses the proton motive force of the cytoplasmic membrane and TonB protein to energize the active transport of iron‐siderophores and vitamin B12 across the outer membrane. TonB shuttles between the cytoplasmic and outer membranes, presumably during the course of energy transduction. Previous results indicated that the carboxy‐terminal 65 amino acids of TonB are essential for both its outer membrane association and activity. A highly conserved region (residues 199–216) within this domain, predicted to be an amphipathic α‐helix, was the initial focus of this study. Scanning mutagenesis indicated that only the aromatic residues F202, W213 and Y215 were individually important for activity. When the crystal structure of a dimeric TonB carboxy‐terminus subsequently became available, we observed that two additional aromatic residues outside that region, F180 and F230, were potentially engaged in end‐on hydrophobic interactions with the three residues identified previously. Changing these five aromatic residues individually to alanine reduced TonB activity. Surprisingly, however, each substitution exhibited a unique phenotypic profile with respect to ability to support [55Fe]‐ferrichrome transport, sensitivity to colicins B, D, Ia and M or sensitivity to bacteriophage φ80. The phenotypic results suggested that the carboxy‐terminus of TonB was a flexible and dynamic domain that could interact specifically with different ligands or transporters, perhaps through the aromatic residues. The possibility of interactions among all the aromatic residues was tested using double‐mutant cycle analysis. All possible combinations of alanine substitutions were constructed, with the result that TonB containing any double‐alanine substitution was inactive in the phenotypic assays, while retaining the ability to associate with the outer membrane. This synergistic, rather than additive, effect of the double mutants suggested that, consistent with the flexibility suggested by analysis of the single substitutions, all the aromatic residues might be capable of interacting with one another. A means of reconciling these results with the crystal structure is presented.
Characterization of a non‐mitochondrial type I phosphatidylserine decarboxylase in Plasmodium falciparum
Authors: Baunaure Françoise , Eldin Patrick , Cathiard Anne‐Marie , Vial Henri
Published: December 2004

In search of key enzymes in Plasmodium phospholipid metabolism, we demonstrate the presence of a parasite‐encoded phosphatidylserine decarboxylase (PSD) in the membrane fraction of Plasmodium falciparum‐infected erythrocytes. PSD cDNA, encoding phosphatidylserine decarboxylase (PfPSD), was cloned by screening a directional cDNA library derived from the trophozoite erythrocytic stage. The corresponding PfPSD gene is located on chromosome 9 of P. falciparum, contains one intron of 938 nucleotides and is transcribed into a 3.7 kb mRNA. PfPSD cDNA encodes a putative protein of 362 amino acids, with a predicted molecular mass of 42.6 kDa, which clearly belongs to the type I PSD family. Only a 35 kDa polypeptide was detected in the parasite using a specific rabbit antiserum. PfPSD has a 314VGSS317 sequence near its carboxyl‐terminus that is related to the Escherichia coli, yeast and human LGST motif, which is the site of proenzyme processing. PSD enzyme was expressed in E. coli with a KM of 63 ± 19 µM and a VMAX of 680 ± 49 nmol of phosphatidylethanolamine formed h−1 mg−1 protein. Site‐directed mutagenesis of the VGSS active site demonstrated that the PfPSD proenzyme was processed into two non‐identical subunits (α and β) and revealed the crucial role played by each residue in enzyme processing and activity. Using indirect immunofluorescence, PfPSD labelling was co‐localized with an endoplasmic reticulum marker, but not with a mitochondrial vital dye. This P. falciparum PSD is the first type I PSD identified in the endoplasmic reticulum compartment.
The antioxidant systems in Toxoplasma gondii and the role of cytosolic catalase in defence against oxidative injury
Authors: Kwok Lai Yu , Schlüter Dirk , Clayton Christine , Soldati Dominique
Published: December 2004

Superoxide dismutase, catalase, glutathione peroxidase and peroxiredoxins form an antioxidant network protecting cells against reactive oxygen species (ROS). Catalase is a potent H2O2‐detoxifying enzyme, which is unexpectedly absent in some members of the Kinetoplastida and Apicomplexa, but present in Toxoplasma gondii. In T. gondii, catalase appears to be cytosolic. In addition, T. gondii also possesses genes coding for other types of peroxidases, including glutathione/thioredoxin‐like peroxidases and peroxiredoxins. This study presents a detailed analysis of the role of catalase in the parasite and reports the existence of antioxidant enzymes localized in the cytosol and the mitochondrion of T. gondii. The catalase gene was disrupted and, in addition, T. gondii cell lines overexpressing either catalase or a cytosolic 1‐cys peroxiredoxin, TgPrx2, under the control of a strong promoter were created. Analysis of these mutants confirmed that the catalase activity is cytosolic and is encoded by a unique gene in T. gondii. Furthermore, the catalase confers protection against H2O2 exposure and contributes to virulence in mice. The overexpression of Prx2 also increases protection against H2O2 treatment, suggesting that catalase and other peroxidases function as a defence mechanism against endogenously produced reactive oxygen intermediates and the oxidative stress imposed by the host.
Modulation of CRP‐dependent transcription at the Escherichia coli acsP2 promoter by nucleoprotein complexes: anti‐activation by the nucleoid proteins FIS and IHF
Authors: Browning Douglas F. , Beatty Christine M. , Sanstad Erik A. , Gunn Kathryn E. , Busby Stephen J. W. , Wolfe Alan J.
Published: December 2004

acs encodes acetyl‐coenzyme A synthetase, a high‐affinity enzyme that allows cells to scavenge for acetate during carbon starvation. CRP activates acs transcription by binding tandem DNA sites located upstream of the major promoter, acsP2. Here, we used electrophoretic mobility shift assays and DNase I footprint analyses to demonstrate that the nucleoid proteins FIS and IHF each bind multiple sites within the acs regulatory region, that FIS competes successfully with CRP for binding to their overlapping and neighbouring sites and that IHF binds independently of either FIS or CRP. Using in vitro transcription assays, we demonstrated that FIS and IHF independently reduce CRP‐dependent acs transcription. Using in vivo reporter assays, we showed that disruption of DNA sites for FIS or deletion of DNA sites for IHF increases acs transcription. We propose that FIS and IHF each function directly as anti‐activators of CRP, each working independently at different times during growth to set the levels of CRP‐dependent acs transcription.
Regulatory proteins with a sense of direction: cell cycle signalling network in Caulobacter
Authors: Jacobs‐Wagner Christine
Published: December 2004

Localization of kinases and other signalling molecules at discrete cellular locations is often an essential component of signal transduction in eukaryotes. Caulobacter crescentus is a small, single‐celled bacterium that presumably lacks intracellular organelles. Yet in Caulobacter, the subcellular distribution of several two‐component signal transduction proteins involved in the control of polar morphogenesis and cell cycle progression changes from a fairly dispersed distribution to a tight accumulation at one or both poles in a spatial and temporal pattern that is reproduced during each cell cycle. This cell cycle‐dependent choreography suggests that similarly to what happens in eukaryotes, protein localization provides a means of modulating signal transduction in bacteria. Recent studies have provided important insights into the biological role and the mechanisms for the differential localization of these bacterial signalling proteins during the Caulobacter cell cycle.
Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation
Authors: Burke Mary E. , Arnold Patricia H. , He Jiuya , Wenwieser Sandra V. C. T. , Rowland Sally‐J. , Boocock Martin R. , Stark W. Marshall
Published: December 2004

Catalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent ‘accessory’ binding sites II and III. ‘Hyperactive’ resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli. Some single mutants can resolve a res × site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I × site I plasmid. Site I × site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2–3′ interface that abolish activity of wild‐type resolvase. Activating mutations may allow regulatory mechanisms of the wild‐type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.
Characterization of cis‐acting prgQ mutants: evidence for two distinct repression mechanisms by Qa RNA and PrgX protein in pheromone‐inducible enterococcal plasmid pCF10
Authors: Bae Taeok , Kozlowicz Briana K. , Dunny Gary M.
Published: December 2004

The pCF10‐encoded negative regulators PrgX and Qa (prgQ antisense) RNA inhibit pCF10 transfer by blocking prgQ transcription extension past a potential transcription terminator sequence IRS1. To identify potential target sites for negative regulation, we isolated and analysed 13 cis‐acting mutations in the prgXQ region. Determination of the 3′ end of Qa RNA showed that eight mutations mapped in the region encoding Qa RNA. Four mutations were in the Qa promoter region and one was in IRS1. Three mutations in Qa greatly reduced the intracellular level of this RNA but did not affect that of PrgX. However, both Qa RNA and PrgX protein were reduced in three Qa promoter region mutants and the expression of prgQ transcripts extending 3′ from IRS1 became constitutive. Qa RNA could mediate its negative regulatory activity in the absence of PrgX, and this activity was not abolished by cCF10, the peptide pheromone that induces pCF10 transfer. RNA analysis showed that Qa RNA abolished transcription readthrough. Based on the experimental data as well as computer analysis of predicted secondary structures of prgQ mRNA in the presence or absence of Qa, we concluded that Qa RNA is a pheromone‐insensitive effector of prgQ mRNA termination or degradation at IRS1. In cells lacking a Qa target sequence, expression of PrgX repressed transcription from the prgQ promoter, and this repression was relieved by addition of exogenous cCF10. Thus, even though the synthesis of these negative regulators is coupled, they each act independently on separate targets to regulate expression of conjugation functions.
Structure‐function analysis of the self‐recognizing Antigen 43 autotransporter protein from Escherichia coli
Authors: Klemm Per , Hjerrild Louise , Gjermansen Morten , Schembri Mark A.
Published: December 2004

Antigen 43 (Ag43) is a self‐recognizing surface adhesin found in most Escherichia coli strains. Expression of Ag43 confers aggregation and fluffing of cells, promotes biofilm formation and is associated with enhanced resistance to antimicrobial agents. Ag43 is an autotransporter protein and consists of two moieties: a transporter, the β‐module, and a passenger domain, the α‐module. Here we have employed various molecular approaches to probe structure/function aspects of Ag43. An entire family of Ag43 variants was identified. The gene encoding Ag43 (flu) was cloned from a diverse range of E. coli subtypes and found to encode variant proteins with different properties. Several novel variants were identified and characterized that were unable to promote cell–cell aggregation. By employing a combination of linker insertion mutagenesis and domain swapping between clumping and non‐clumping variants, we have pinpointed the region of the protein responsible for autoaggregation to be located within the N‐terminal one‐third of the passenger domain. Our data suggest that ionic interactions between charged residues residing in interacting pairs of Ag43α domains may be important for the self‐recognition process. Based on its similarity to other related proteins, we predict the passenger, Ag43α, domain primarily to consist of an extended β‐helix structure in which numerous repeats or rungs are stacked in parallel orientation in an extended cylindrical formation. Finally, we found that in spite of their different aggregative pattern all Ag43 variants promoted biofilm formation to abiotic surfaces.
Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization – RING‐FISH
Authors: Zwirglmaier K. , Ludwig W. , Schleifer K. ‐H.
Published: December 2004

Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 104−105 copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (101−103 copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method – a halo‐like, ring‐shaped concentration of fluorescence in the cell periphery – we coined the term RING‐FISH (recognition of individual genes) to differentiate it from conventional FISH.
Site‐directed mutagenesis of a LuxR‐type quorum‐sensing transcription factor: alteration of autoinducer specificity
Authors: Chai Yunrong , Winans Stephen C.
Published: December 2004

TraR is a quorum‐sensing transcription factor from Agrobacterium tumefaciens that regulates replication and conjugation genes of the tumour‐inducing (Ti) plasmid. TraR activity requires the autoinducer pheromone N‐3‐oxooctanoyl‐l‐homoserine lactone (OOHL). Structural studies of TraR–OOHL–DNA complexes showed that one molecule of OOHL is completely engulfed within the N‐terminal domain of each TraR subunit. TraR is thought to bind OOHL via four hydrogen bonds, three of them direct and one water mediated, and by numerous hydrophobic interactions. Here, we show that all residues predicted to hydrogen bond with OOHL are essential for wild‐type protein function. Mutants that failed to detect OOHL in vivo invariably failed to sequester exogenous OOHL. We showed previously that TraR is protected from cellular proteases by OOHL, and now show that mutants that failed to detect OOHL were also not protected from proteolysis by OOHL. We also describe several mutants with altered autoinducer specificity. Three mutants (T129V, T129A and T115I) detected 3‐oxo‐AHLs and 3‐unsubstituted AHLs with equal sensitivity, indicating that these mutations perturb the water‐mediated hydrogen bond to the 3‐oxo moiety of OOHL. Three other mutants (A49I, A49M and Q58L) preferentially detected AHLs containing six or seven carbon atoms rather than eight. The bulkier residues in these mutations appear to have occupied a portion of the OOHL binding site, interfering with binding of the acyl chain of AHLs.
A novel biotin protein required for reductive carboxylation of 2‐oxoglutarate by isocitrate dehydrogenase in Hydrogenobacter thermophilus TK‐6
Authors: Aoshima Miho , Ishii Masaharu , Igarashi Yasuo
Published: December 2004

Isocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters. Notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2‐oxoglutarate. The carboxylation reaction required the addition of cell extract and ATP‐Mg, suggesting the existence of additional carboxylation factor(s). Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides. N‐terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation. Pyruvate carboxylase was also purified, but was not active in stimulating isocitrate dehydrogenase. Isocitrate dehydrogenase, the novel biotin protein, ATP‐Mg and NADH were essential for the reductive carboxylation of 2‐oxoglutarate. These observations indicate that the novel biotin protein is an ATP‐dependent factor, which is involved in the reverse (carboxylating) reaction of isocitrate dehydrogenase.
The tetraspanin BcPls1 is required for appressorium‐mediated penetration ofBotrytis cinereainto host plant leaves
Authors: Gourgues M. , Brunet‐Simon A. , Lebrun M‐H. , Levis C.
Published: December 2004

Animal tetraspanins are membrane proteins controlling cell adhesion, morphology and motility. In fungi, the tetraspanin MgPls1 controls an appressorial function required for the penetration ofMagnaporthe griseainto host plants. An orthologue ofMgPLS1,BcPLS1, was identified in the necrotrophic fungal plant pathogenBotrytis cinerea. We constructed aBcpls1::barnull mutant by targeted gene replacement.Bcpls1::baris not pathogenic on intact plant tissues of bean, tomato or rose, but it infects wounded plant tissues. Both wild type andBcpls1::bardifferentiate appressoria on plant and artificial surfaces, a process involving an arrest of polarized growth, apex swelling and its cell wall reinforcement. Although wild‐type appressoria allowed the penetration of the fungus into the host plant within 6–12 h, no successful penetration events were observed withBcpls1::bar, suggesting that its appressoria are not functional. AneGFPtranscriptional fusion showed thatBcPLS1was specifically expressed in conidia, germ tubes and appressoria during host penetration. Our results indicate thatBcPLS1is required for the penetration ofB. cinereainto intact host plants. The defect in pathogenicity ofBcpls1::baralso demonstrates that functionalB. cinereaappressoria are required for a successful penetration process. AsBcpls1::barandMgpls1Δ::hphpenetration defects are similar, fungal tetraspanins are likely to be required for an essential appressorial function widespread among fungi.
Initiation of intracellular offspring in Epulopiscium
Authors: Angert Esther R. , Clements Kendall D.
Published: December 2004

Epulopiscium spp. are the largest heterotrophic bacteria yet described. A distinguishing feature of the Epulopiscium group is their viviparous production of multiple, internal offspring as a means of cellular reproduction. Based on their phylogenetic position, among low G + C Gram‐positive endospore‐forming bacteria, and the remarkable morphological similarity between developing endospores and Epulopiscium offspring, we hypothesized that intracellular offspring production in Epulopiscium evolved from endospore formation. These observations also raise the possibility that a cell with the capacity to form multiple intracellular offspring was the ancestor of all contemporary endospore‐forming bacteria. In an effort to characterize mechanisms common to both processes, we describe the earliest stages of offspring formation in Epulopiscium. First, in anticipation of polar division, some of the mother cell DNA coalesces at the cell poles. FtsZ then localizes in a bipolar pattern and the cell divides. A portion of the pole‐associated DNA is trapped within the small cells formed by division at both poles. As development progresses, more pole‐associated DNA is apparently packaged into the offspring primordia. These results illustrate three mechanisms, the reorganization of cellular DNA, asymmetric division and DNA packaging, that are common to both endospore formation in Bacillus subtilis and the production of active, intracellular offspring in Epulopiscium. Unlike most endospore formers, Epulopiscium partitions only a small proportion of mother cell DNA into the developing offspring.
Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK‐DnaJ‐GrpE system in the management of protein misfolding under severe stress conditions
Authors: Mujacic Mirna , Bader Martin W. , Baneyx François
Published: December 2004

Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48°C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50°C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB‐DnaK‐DnaJ‐GrpE system to clear thermally aggregated proteins at 30°C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, ΔclpB or ΔibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK‐DnaJ‐GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding.
Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms
Authors: Friedman Lisa , Kolter Roberto
Published: December 2004

Pseudomonas aeruginosa forms diverse matrix‐enclosed surface‐associated multicellular assemblages (biofilms) that aid in its survival in a variety of environments. One such biofilm is the pellicle that forms at the air–liquid interface in standing cultures. We screened for transposon insertion mutants of P. aeruginosa PA14 that were unable to form pellicles. Analysis of these mutants led to the identification of seven adjacent genes, named pel genes, the products of which appear to be involved in the formation of the pellicle's extracellular matrix. In addition to being required for pellicle formation, the pel genes are also required for the formation of solid surface‐associated biofilms. Sequence analyses predicted that three pel genes encode transmembrane proteins and that five pel genes have functional homologues involved in carbohydrate processing. Microscopic and macroscopic observations revealed that wild‐type P. aeruginosa PA14 produces a cellulase‐sensitive extracellular matrix able to bind Congo red; no extracellular matrix was produced by the pel mutants. A comparison of the carbohydrates produced by the wild‐type strain and pel mutants suggested that glucose was a principal component of the matrix material. Together, these results suggest that the pel genes are responsible for the production of a glucose‐rich matrix material required for the formation of biofilms by P. aeruginosa PA14.
A bacterial cell–cell communication signal with cross‐kingdom structural analogues
Authors: Wang Lian‐Hui , He Yawen , Gao Yunfeng , Wu Ji En , Dong Yi‐Hu , He Chaozu , Wang Su Xing , Weng Li‐Xing , Xu Jin‐Ling , Tay Leng , Fang Rong Xiang , Zhang Lian‐Hui
Published: December 2004

Extracellular signals are the key components of microbial cell–cell communication systems. This report identified a diffusible signal factor (DSF), which regulates virulence in Xanthomonas campestris pv. campestris, as cis‐11‐methyl‐2‐dodecenoic acid, an α,β unsaturated fatty acid. Analysis of DSF derivatives established the double bond at the α,β positions as the most important structural feature for DSF biological activity. A range of bacterial pathogens, including several Mycobacterium species, also displayed DSF‐like activity. Furthermore, DSF is structurally and functionally related to farnesoic acid (FA), which regulates morphological transition and virulence by Candida albicans, a fungal pathogen. Similar to FA, which is also an α,β unsaturated fatty acid, DSF inhibits the dimorphic transition of C. albicans at a physiologically relevant concentration. We conclude that α,β unsaturated fatty acids represent a new class of extracellular signals for bacterial and fungal cell–cell communications. As prokaryote–eukaryote interactions are ubiquitous, such cross‐kingdom conservation in cell–cell communication systems might have significant ecological and economic importance.