bluuebunny/crossref_metadata_2025_split huggingface dataset:
Acidobacteria form a coherent but highly diverse group within the bacterial domain: evidence from environmental genomics
Authors: Quaiser Achim , Ochsenreiter Torsten , Lanz Christa , Schuster Stephan C. , Treusch Alexander H. , Eck Jürgen , Schleper Christa 
Published: October 2003

SummaryAcidobacteria have been established as a novel phylum of Bacteria that is consistently detected in many different habitats around the globe by 16S rDNA‐based molecular surveys. The phylogenetic diversity, ubiquity and abundance of this group, particularly in soil habitats, suggest an important ecological role and extensive metabolic versatility. However, the genetic and physiological information about Acidobacteria is scarce. In order to gain insight into genome structure, evolution and diversity of these microorganisms we have initiated an environmental genomic approach by constructing large insert libraries directly from DNA of a calcerous grassland soil. Genomic fragments of Acidobacteria were identified with specific 16S rDNA probes and sequence analyses of six independently identified clones were performed, representing in total more than 210 000 bp. The 16S rRNA genes of the genomic fragments differed between 2.3% and 19.9% and were placed into two different subgroups of Acidobacteria (groups III and V). Although partial co‐linearity was found between genomic fragments, the gene content around the rRNA operons was generally not conserved. Phylogenetic reconstructions with orthologues that were encoded on two of the six genomic fragments (PurF, PurL, PurB and formamidopyrimidine‐DNA glycosylase) confirmed the coherence of the acidobacterial phylum. One genomic fragment harboured a cluster of eight genes which was syntenic and highly homologous to genomic regions in Rhodopseudomonas palustris and Bradyrhizobium japonicum, including a conserved two‐component system. Phylogenetic analysis of the putative response regulator confirmed that this similarity between Rhizobiales and Acidobacteria might be due to a horizontal gene transfer. In total, our data give first insight into the genome content and diversity of the ubiquitously distributed but poorly characterized phylum of Acidobacteria. Furthermore they support the phylogenetic inferences made from 16S rRNA gene libraries, suggesting that Acidobacteria form a broad group in the same sense and with a similar diversity as that of many well‐studied bacterial phyla.
σ28 RNA polymerase regulates hctB, a late developmental gene in Chlamydia
Authors: Hiu Yin Yu Hilda , Tan Ming 
Published: October 2003

Summary Chlamydia is predicted to encode two alternative sigma factors that could provide a mechanism for the regulation of gene expression via alternative forms of RNA polymerase. We have demonstrated that σ28, one of these alternative sigma factors, is transcriptionally active. Chlamydial σ28 RNA polymerase was reconstituted from recombinant σ28 protein and core enzyme that was biochemically isolated from chlamydiae. In an in vitro transcription assay, σ28 RNA polymerase transcribed the hctB promoter in a σ28‐dependent manner. Transcription by σ28 RNA polymerase was salt tolerant compared with transcription by σ66 RNA polymerase, the major form of chlamydial RNA polymerase. As hctB encodes a histone‐like protein that is only expressed late in the developmental cycle, our results suggest that σ28 RNA polymerase has a role in the regulation of late gene expression in Chlamydia.
Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation
Authors: Mogk Axel , Deuerling Elke , Vorderwülbecke Sonja , Vierling Elizabeth , Bukau Bernd 
Published: October 2003

SummarySmall heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co‐operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co‐operatively the DnaK system in reversing protein aggregation. A ΔibpABΔclpB double mutant exhibits strongly increased protein aggregation at 42°C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37°C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for ΔibpAB, ΔclpB, and the double ΔibpABΔclpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.
Heat and DNA damage induction of the LexA‐like regulator HdiR from Lactococcus lactis is mediated by RecA and ClpP
Authors: Savijoki Kirsi , Ingmer Hanne , Frees Dorte , Vogensen Finn K. , Palva Airi , Varmanen Pekka 
Published: October 2003

SummaryThe SOS response is a paradigm for bacterial cells response to DNA damage. Yet some bacteria lack a homologue of the SOS regulator, LexA, including the Gram‐positive, Lactococcus lactis. In this organism we have identified a negative transcriptional regulator, HdiR that induces target gene expression both upon DNA damage and heat shock. Gel mobility shift assays revealed that the binding site for HdiR is located within an inverted repeat structure. HdiR is able to carry out a self‐cleavage reaction in vitro at high pHs, while in vivo it undergoes RecA‐dependent self‐cleavage in the presence of a DNA‐damaging agent. Intriguingly, the N‐terminal cleavage product of HdiR retains DNA binding activity, and only when degraded by the Clp protease, is gene expression induced. Thus, the activity of HdiR in response to DNA damage is controlled by sequential proteolysis, involving self‐cleavage and Clp‐dependent degradation of HdiR. During heat‐stress, limited self‐cleavage occurs; however, recA and clpP are still required for full induction of target gene expression. Thus, our data show that common elements are involved in both the DNA damage and the heat‐mediated induction of the HdiR regulon.
Two different mechanisms for ribosome/mRNA interaction in archaeal translation initiation
Authors: Benelli Dario , Maone Enzo , Londei Paola 
Published: October 2003

SummaryIn this study, we have analysed the features of mRNA/ribosome interaction in the thermophilic archeon Sulfolobus solfataricus. Leadered mRNAs endowed with Shine–Dalgarno (SD) motifs formed stable binary complexes with 30S subunits, optimally at high temperature (65–70°C) and without the aid of initiator tRNA (tRNAi) or any factor. ‘Toeprinting’ assays revealed that the SD motifs were necessary and sufficient to direct the 30S subunit to the translation initiation region. Leaderless mRNAs, i.e. mRNAs entirely lacking a 5′‐untranslated region (UTR), did not interact directly with 30S subunits but required the presence of tRNAi, indicating that codon–anticodon pairing was required for positioning the ribosome on the initiation codon. The data suggest that archaea such as Sulfolobus routinely use two distinct mechanisms for translational initiation. SD‐dependent initiation, resembling the pathway prevalent in present‐day bacteria, would operate on distal cistrons of polycistronic mRNAs, whereas ‘leaderless’ initiation, reminiscent of the eukaryotic pathway, would operate on monocistronic mRNAs and on opening cistrons of polycistronic mRNAs.
Genomic analysis in Escherichia coli demonstrates differential roles for polynucleotide phosphorylase and RNase II in mRNA abundance and decay
Authors: Mohanty Bijoy K. , Kushner Sidney R. 
Published: October 2003

SummaryPrevious work has shown that simultaneous inactivation of polynucleotide phosphorylase (PNPase) and RNase II (both 3′→ 5′ exonucleases) in Escherichia coli leads to the loss of cell viability and the accumulation of partially degraded mRNA species. In order to help to distinguish how these two enzymes globally affect the abundance and decay of mRNAs, we have carried out a genome‐wide analysis of the steady‐state levels of E. coli transcripts using deletion mutations in either rnb or pnp. The data show that, in exponentially growing cells, inactivation of PNPase leads to an increase in the steady‐state level of more expressed mRNAs (17.3%) than inactivation of RNase II (7.3%). In contrast, the steady‐state levels of a large number of E. coli mRNAs (31%) are decreased in the absence of RNase II, including almost all the ribosomal protein genes, suggesting that a major function of this enzyme is to protect specific mRNAs from the activity of other ribonucleases. Array data were confirmed by Northern analysis of 12 individual mRNAs. A comparison between the steady‐state levels and the half‐lives of individual mRNAs indicates that there may be a direct interaction between transcription and mRNA decay for some of the transcripts. In addition, results are presented to show significant phenotypic differences between the pnp‐7 point mutant and the pnpΔ683 deletion allele.
amrA encodes a putative membrane protein necessary for maximal exponential phase expression of the Mga virulence regulon in Streptococcus pyogenes
Authors: Ribardo Deborah A. , McIver Kevin S. 
Published: October 2003

SummaryThe transcriptional regulator Mga activates a regulon of virulence genes important for colonization and immune evasion in GAS. Using transposon mutagenesis of a serotype M6 group A streptococcus (GAS) reporter strain KSM148, we have identified an open reading frame (ORF) designated amrA that is required for maximal activation of the Mga regulon during exponential phase. A deletion in amrA, but not in the downstream transcriptionally linked ORF Spy0798, was able to reproduce the phenotype seen in the transposon mutants. Northern analysis for mga and emm transcripts, as well as Western analysis of Mga, confirmed a reduction in mga expression leading to a decrease in transcription of the Mga‐regulated emm in the amrA deletion and transposon mutants. Furthermore, both the amrA deletion mutant and an original transposon mutant could be complemented using amrA expressed from a nisin‐inducible expression system. As amrA is strongly conserved across the sequenced streptococcal M types, and inactivation of amrA in an M3 serotype also resulted in reduction of emm transcripts, the role of amrA does not appear to be serotype specific. Although the specific function of AmrA is unknown, its putative membrane localization and homology to transporters involved in cell wall synthesis suggest a link between growth and virulence gene expression in GAS.
Xanthomonas type III effector XopD targets SUMO‐conjugated proteins in planta
Authors: Hotson Andrew , Chosed Renee , Shu Hongjun , Orth Kim , Mudgett Mary Beth 
Published: October 2003

Summary Xanthomonas campestris pathovar vesicatoria (Xcv) uses the type III secretion system (TTSS) to inject effector proteins into cells of Solanaceous plants during pathogenesis. A number of Xcv TTSS effectors have been identified; however, their function in planta remains elusive. Here, we provide direct evidence for a functional role for a phytopathogenic bacterial TTSS effector in planta by demonstrating that the Xcv effector XopD encodes an active cysteine protease with plant‐specific SUMO substrate specificity. XopD is injected into plant cells by the TTSS during Xcv pathogenesis, translocated to subnuclear foci and hydrolyses SUMO‐conjugated proteins in vivo. Our studies suggest that XopD mimics endogenous plant SUMO isopeptidases to interfere with the regulation of host proteins during Xcv infection.
Transcription of σ54‐dependent but not σ28‐dependent flagellar genes in Campylobacter jejuni is associated with formation of the flagellar secretory apparatus
Authors: Hendrixson David R. , DiRita Victor J. 
Published: October 2003

SummaryWe performed a genetic analysis of flagellar regulation in Campylobacter jejuni, from which we elucidated key portions of the flagellar transcriptional cascade in this bacterium. For this study, we developed a reporter gene system for C. jejuni involving astA, encoding arylsulphatase, and placed astA under control of the σ54‐regulated flgDE2 promoter in C. jejuni strain 81‐176. The astA reporter fusion combined with transposon mutagenesis allowed us to identify genes in which insertions abolished flgDE2 expression; genes identified were on both the chromosome and the plasmid pVir. Included among the chromosomal genes were genes encoding a putative sensor kinase and the σ54‐dependent transcriptional activator, FlgR. In addition, we identified specific flagellar genes, including flhA, flhB, fliP, fliR and flhF, that are also required for transcription of flgDE2 and are presumably at the beginning of the C. jejuni flagellar transcriptional cascade. Deletion of any of these genes reduced transcription of both flgDE2 and another σ54‐dependent flagellar gene, flaB, encoding a minor flagellin. Transcription of the σ28‐dependent gene flaA, encoding the major flagellin, was largely unaffected in the mutants. Further examination of flaA transcription revealed significant σ28‐independent transcription and only weak repressive activity of the putative anti‐σ28 factor FlgM. Our study suggests that σ54‐dependent transcription of flagellar genes in C. jejuni is linked to the formation of the flagellar secretory apparatus. A key difference in the C. jejuni flagellar transcriptional cascade compared with other bacteria that use σ28 for transcription of flagellar genes is that a mechanism to repress significantly σ28‐dependent transcription of flaA in flagellar assembly mutants is absent in C. jejuni.
Tag‐mediated fractionation of yeast ribosome populations proves the monomeric organization of the eukaryotic ribosomal stalk structure
Authors: Guarinos Esther , Santos Cruz , Sánchez Arancha , Qiu De‐Yi , Remacha Miguel , Ballesta Juan P. G. 
Published: October 2003

SummaryThe analysis of the not well understood composition of the stalk, a key ribosomal structure, in eukaryotes having multiple 12 kDa P1/P2 acidic protein components has been approached using these proteins tagged with a histidine tail at the C‐terminus. Tagged Saccharomyces cerevisiae ribosomes, which contain two P1 proteins (P1α and P1β) and two P2 proteins (P2α and P2β), were fractionated by affinity chromatography and their stalk composition was determined. Different yeast strains expressing one or two tagged proteins and containing either a complete or a defective stalk were used. No indication of protein dimers was found in the tested strains. The results are only compatible with a stalk structure containing a single copy of each one of the four 12 kDa proteins per ribosome. Ribosomes having an incomplete stalk are found in wild‐type cells. When one of the four proteins is missing, the ribosomes do not carry the three remaining proteins simultaneously, containing only two of them distributed in pairs made of one P1 and one P2. Ribosomes can carry two, one or no acidic protein pairs. The P1α/P2β and P1β/P2α pairs are preferentially found in the ribosome, but they are not essential either for stalk assembly or function.
Cell cycle regulator Cdc14 is expressed during sporulation but not hyphal growth in the fungus‐like oomycete Phytophthora infestans
Authors: Ah Fong Audrey M. V. , Judelson Howard S. 
Published: October 2003

SummaryCdc14 proteins are important regulators of mitosis and the cell cycle. These phosphatases have been studied previously only in yeasts and metazoans, which grow by fission or budding. Here we describe a homologue (piCdc14) from the oomycete Phytophthora infestans, a primitive eukaryote lacking a classical cell cycle. PiCdc14 complements a cdc14ts mutant of Saccharomyces cerevisiae and may function like other Cdc14 proteins, but displays a strikingly different pattern of expression. Whereas previously studied Cdc14 genes are constitutively transcribed, piCdc14 is not expressed during normal growth but instead only during asexual sporulation. In transformants of P. infestans expressing a fusion between the piCdc14 promoter and the β‐glucuronidase reporter, expression was first detected in sporangiophore initials, persisted in sporangiophores bearing immature sporangia, and later became restricted to mature sporangia. After germination, expression ended a few hours before the resumption of mitosis in hyphae emerged from the spores. Homology‐dependent silencing experiments supported an essential role of piCdc14 in sporulation. It is proposed that the function of piCdc14 may be to synchronise nuclear behaviour during sporulation and maintain dormancy in spores until germination. These results help illuminate the process of sporulation in oomycetes and the evolution of the cell cycle in eukaryotes.
SetB: an integral membrane protein that affects chromosome segregation in Escherichia coli
Authors: Espeli Olivier , Nurse Pearl , Levine Cindy , Lee Chong , Marians Kenneth J. 
Published: October 2003

Summary setB was identified as a high‐copy suppressor of the partition defect of a mutation in parC, encoding one of the subunits of topoisomerase IV. Deletion of this integral inner membrane protein causes a delay in chromosome segregation, whereas its overproduction causes nucleoid disintegration and stretching, leading to a cell division defect. setB deletion mutants also exhibit a synthetic phenotype when combined with mutations that delete the C‐terminal motor domain of the septal ring protein FtsK. SetB localizes in the cell as a helix and interacts with MreB, the bacterial actin homologue, which also forms a helix. These observations suggest that there may be a link between chromosome segregation and cellular infrastructure.
Partition operon expression in the linear plasmid prophage N15 is controlled by both Sop proteins and protelomerase
Authors: Dorokhov Boris D. , Lane David , Ravin Nikolai V. 
Published: October 2003

SummaryThe temperate coliphage N15, unlike most low copy‐number prokaryotic replicons, is maintained as a linear DNA molecule with covalently closed ends. Accurate partitioning of the plasmid prophage is assured by a close homologue of the sop locus of the F plasmid. However, the region upstream of the N15 sopAB genes contains multiple putative promoters, in contrast to F sop whose expression is driven by one negatively autoregulated promoter. In addition, the centromere of N15 is represented by four inverted repeats located at widely separated sites within the region essential for replication and control of lytic functions. We have analysed expression of N15 sop genes. We find that transcription of N15 sop is driven by two major promoters. The first, P1, is similar in sequence and function to the F sop promoter; it is repressed by Sop proteins. The second promoter, P2, is upstream of P1 and is several times stronger. It is insensitive to regulation by Sop proteins but is tightly repressed by protelomerase, the N15 enzyme that completes prophage replication by generating hairpin telomeres. These results establish a regulatory link between the partition system and other processes of N15 maintenance.
New effects of type III effectors
Authors: Innes Roger 
Published: October 2003

SummaryThe enzymatic activities and/or targets of four type III effector proteins from plant pathogens have been reported in a flurry of new papers. In this issue, XopD is shown to remove SUMO groups from host cell proteins, while in previous issues of Molecular Microbiology, HopPtoD2 was shown to function as a tyrosine phosphatase and AvrRpt2 as probably a cysteine protease that targets the host RIN4 protein. Finally, AvrPphB is revealed in a recent Science paper to function as a cysteine protease that targets the host PBS1 kinase. This work is providing some of the first insights into how plant pathogens subvert host cell signalling machinery to cause disease.
The LKA gene is a BRASSINOSTEROID INSENSITIVE 1 homolog of pea
Authors: Nomura Takahito , Bishop Gerard J. , Kaneta Tsuyoshi , Reid James B. , Chory Joanne , Yokota Takao 
Published: October 2003

SummaryBrassinosteroids (BRs) are growth‐promoting plant steroid hormones, and in garden pea (Pisum sativum L.), the lka mutant is defective in BR perception. Here, we show that LKA encodes P. sativum BRI1 (PsBRI1), a homolog of BRI1, which is the Arabidopsis leucine‐rich repeat receptor‐like kinase/BR receptor. PsBRI1 was isolated by screening a pea cDNA library using Arabidopsis BRI1 cDNA as the probe. PsBRI1 is predicted to encode a 1188‐amino‐acid protein that has 78% similarity with Arabidopsis BRI1. Sequence analysis of PsBRI1 in the lka mutant led to the identification of a missense mutation that converts the highly conserved aspartic acid residue to asparagine, which is located in the leucine‐rich repeat, just before the island domain that may bind BR or a BR–protein complex. The mutation identified in PsBRI1 co‐segregated with the semi‐erectoide lka phenotype. Transcript analysis of LKA/PsBRI1 indicates that it is ubiquitously expressed in pea and that the expression was downregulated by exogenous BR. The lka mutant was then utilized in further studies to analyze the independent actions of BR and gibberellin (GA) through the characterization of BR response on GA mutants and GA response on BR mutants.
Sequence database of 1172 T‐DNA insertion sites in Arabidopsis activation‐tagging lines that showed phenotypes in T1 generation
Authors: Ichikawa Takanari , Nakazawa Miki , Kawashima Mika , Muto Shu , Gohda Kazushi , Suzuki Kumiko , Ishikawa Akie , Kobayashi Hiroko , Yoshizumi Takeshi , Tsumoto Yuko , Tsuhara Yumi , Iizumi Haruko , Goto Yukiko , Matsui Minami 
Published: October 2003

SummaryPlant genomic resources harbouring gain‐of‐function mutations remain rare, even though this type of mutation is believed to be one of the most useful for elucidating the function of unknown genes that have redundant partners in the genome. An activation‐tagging T‐DNA was introduced into the genome of Arabidopsis creating 55 431 independent transformed lines. Of these T1 lines, 1262 showed phenotypes different from those of wild‐type plants. We called these lines ‘AT1Ps’ (activation T1 putants). The phenotypes observed include abnormalities in morphology, growth rate, plant colour, flowering time and fertility. Similar phenotypes re‐appeared either in dominant or semi‐dominant fashion in 17% of 177 AT2P plants tested. Plasmid rescue or an adaptor‐PCR method was used to identify 1172 independent genomic loci of T‐DNA integration sites in the AT1P plants. Mapping of the integration sites revealed that the chromosomal distribution of these sites is similar to that observed in conventional T‐DNA knock‐out lines, except that the intragenic type of integration is slightly lower (27%) in the AT1P plants compared to that observed in other random knock‐out populations (30–35%). Ten AT2P lines that showed dominant phenotypes were chosen to monitor expression levels of genes adjacent to the T‐DNA integration sites by RT‐PCR. Activation was observed in 7 out of 17 of the adjacent genes detected. Genes located up to 8.2 kb away from the enhancer sequence were activated. One of the seven activated genes was located close to the left‐border sequence of the T‐DNA, having an estimated distance of 5.7 kb from the enhancer. Surprisingly, one gene, the first ATG of which is located 12 kb away from the enhancer, showed reduced mRNA accumulation in the tagged line. Application of the database generated to Arabidopsis functional genomics research is discussed. The sequence database of the 1172 loci from the AT1P plants is available (http://pfgweb.gsc.riken.go.jp/index.html).