bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose
Authors: Solano Cristina , García Begoña , Valle Jaione , Berasain Carmen , Ghigo Jean‐Marc , Gamazo Carlos , Lasa Iñigo 
Published: March 2003

SummaryWe report here a new screening method based on the fluorescence of colonies on calcofluor agar plates to identify transposon insertion mutants of Salmonella enteritidis that are defective in biofilm development. The results not only confirmed the requirement of genes already described for the modulation of multicellular behaviour in Salmonella typhimurium and other species, but also revealed new aspects of the biofilm formation process, such as two new genetic elements, named as bcsABZC and bcsEFG operons, required for the synthesis of an exopolysaccharide, digestible with cellulase. Non‐polar mutations of bcsC and bcsE genes and complementation experiments demonstrated that both operons are respon‐sible for cellulose biosynthesis in both S. enteritidis and S. typhimurium. Using two different growth media, ATM and LB, we showed that the biofilm produced by S. enteritidis is made of different constituents, suggesting that biofilm composition and regulation depends on environmental conditions. Bacterial adherence and invasion assays of eukaryotic cells and in vivo virulence studies of cellulose‐deficient mutants indicated that, at least under our experimental conditions, the production of cellulose is not involved in the virulence of S. enteritidis. However, cellulose‐deficient mutants were more sensitive to chlorine treatments, suggesting that cellulose production and biofilm formation may be an important factor for the survival of S. enteritidis on surface environments.
An intrinsic control element for translational initiation in class 1 integrons
Authors: Hanau‐Berçot Béatrice , Podglajen Isabelle , Casin Isabelle , Collatz Ekkehard 
Published: March 2003

SummaryIntegrons are genetic elements able to capture anti‐biotic resistance and other genes and to promote their transcription. Here, we have investigated integron‐dependent translation of an aminoglycoside 6′‐N‐acetyltransferase gene (aac(6′)‐Ib7)
inserted at the attI1 site. N‐terminal sequencing revealed that translation
of this gene was initiated at a GTG codon, which is not part of a plausible translation
initiation region (TIR). A short open reading frame (called ORF‐11) overlapping the
attI1 site was probed by site‐directed mutagenesis for its contribution to
aac(6′)‐Ib7 translation. When ORF‐11 and its
TIR were deleted en bloc, translational efficiency dropped by over 80%,
as determined with an acetyltransferase– luciferase fusion product. Invalidation
of the ATG start codon of ORF‐11 or its putative Shine–Dalgarno sequence resulted
in a decrease of over 60%, whereas the decrease was much less pronounced
when the amino acid sequence of the putative ORF‐11‐encoded peptide was altered or
when the distance between ORF‐11 and aac(6′)‐Ib7
was doubled. This demonstrates that aac(6′)‐Ib7
translation is dependent upon the translation of ORF‐11, but almost certainly not
upon the corresponding peptide. These results lead us to conclude that an intrinsic
short ORF present in the 5′‐conserved segment of many class 1 integrons may substantially enhance expression at the translational level of captured TIR‐deficient anti‐biotic resistance genes.
Methylation at nucleotide G745 or G748 in 23S rRNA distinguishes Gram‐negative from Gram‐positive bacteria
Authors: Liu Mingfu , Douthwaite Stephen 
Published: March 2003

SummaryBacteria tune the function of their ribosomes by methylating specific rRNA nucleotides. Nucleotide G745 in Escherichia coli 23S rRNA is methylated by the methyltransferase
enzyme RrmA, whereas in Streptomyces fradiae, the neighbouring nucleotide
G748 is methylated by the enzyme TlrB. Both nucleotides line the peptide exit channel
of the ribosome at the binding site of macrolide, lincosamide and streptogramin B
antibiotics. Resistance to the macrolide tylosin, which is produced by S. fradiae,
is conferred by methylation of G748. RrmA and TlrB are homologues (29% identical),
and a database search against all presently available sequences revealed a further
two dozen homologues from a wide variety of Bacteria. No homologues were found among
the Archaea or Eukarya. The bacterial sequences adhere to the species phylogeny and
segregate into two groups, in which the Gramnegative sequences align with RrmA and
the Gram‐positives with TlrB. Consistently, in more than 20 species tested, the distribution
of methylation in the Gram‐negative rRNAs (methylated at G745) and the Gram‐positives
(methylated at G748) perfectly matches the bacterial phylogeny. Cloning and ex‐pression
of representative methyltransferase genes showed that this specificity of methylation
is determined solely by the methyltransferase enzyme and is independent of the origin
of the rRNA substrate. This is the first case in which the position of an RNA methylation
defines a sharp division between the Gram‐negative and Gram‐positive bacteria. Given
the specificities and distribution of these methyltransferases, we propose a change
in the nomenclature of RrmA to RlmAI (rRNA large subunit
methyltransferase) and of TlrB to RlmAII.
Candida albicans Als1p: an adhesin that is a downstream effector of the EFG1 filamentation pathway
Authors: Fu Yue , Ibrahim Ashraf S. , Sheppard Donald C. , Chen Yee‐Chun , French Samuel W. , Cutler Jim E. , Filler Scott G. , Edwards John E. 
Published: March 2003

SummaryFilamentation and adherence to host cells are critical virulence factors of Candida

albicans. Multiple filamentation regulatory pathways have been discovered in

C. albicans using Saccharomyces cerevisiae as a model. In S. cerevisiae,

these pathways converge on Flo11p, which functions as a downstream effector of filamentation

and also mediates cell–cell adherence (flocculation). In C. albicans,

such effector(s) have not yet been identified. Here, we demonstrate that the cell

surface protein Als1p is an effector of filamentation in C. albicans. We show

that Als1p expression is controlled by the transcription factor Efg1p, which is known

to be a key regulator of filamentation in C. albicans. Further, disruption

of ALS1 inhibited filamentation, and autonomous expression of Als1p restored

filamentation in an efg1 homozygous null mutant. Thus, Als1p functions as

a downstream effector of the EFG1 filamentation pathway. In addition, we found

that Als1p mediates both flocculation and adherence of C. albicans to endothelial

cells in vitro. As a cell surface glycoprotein that mediates filamentation

and adherence, Als1p has both structural and functional similarity to S. cerevisiae

Flo11p. Consistent with our in vitro results, Als1p was required for both normal filamentation and virulence in the mouse model of haematogenously disseminated candidiasis.
RusA proteins from the extreme thermophile Aquifex aeolicus and lactococcal phage r1t resolve Holliday junctions
Authors: Sharples Gary J. , Bolt Edward L. , Lloyd Robert G. 
Published: March 2003

SummaryThe RusA protein of Escherichia coli is a DNA structure‐specific endonuclease that resolves Holliday junction intermediates formed during DNA replication, recombination and repair by introducing symmetrically paired incisions 5′ to CC dinucleotides. It is encoded by the defective prophage DLP12, which raises the possibility that it may be of bacteriophage origin. We show that rusA‐like sequences are indeed often associated with prophage sequences in the genomes of several bacterial species. They are also found in many bacteriophages, including Lactococcus lactis phage r1t. However, rusA is also present in the chromosome of the hyperthermophilic bacterium Aquifex aeolicus. In this case, there is no obvious association of rusA with prophage‐like sequences. Given the ancient lineage of Aquifex aeolicus, this observation provides the first indication that RusA may be of bacterial origin. The RusA proteins of A. aeolicus and bacteriophage r1t were purified and shown to resolve Holliday junctions. The r1t enzyme also promotes DNA repair in strains lacking the RuvABC resolvase. Both enzymes cleave junctions in a sequence‐dependent manner, but the A. aeolicus RusA shows a different sequence preference (3′ to TG) from the E. coli protein (5′ to CC), and the r1t RusA has relaxed sequence dependence, requiring only a single cytosine.
Genetic dissection of the Ralstonia solanacearum hrp gene cluster reveals that the HrpV and HrpX proteins are required for Hrp pilus assembly
Authors: Gijsegem Frédérique Van , Vasse Jacques , Rycke Riet De , Castello Philippe , Boucher Christian 
Published: March 2003

SummaryIn both plant and mammalian Gram‐negative pathogenic bacteria, type III secretion systems (TTSSs) play a crucial role in interactions with the host. All these systems share conserved proteins (called Hrc in plant pathogens), but each bacterium also produces a variable number of additional type III proteins either unique or with counterparts only in a limited number of related systems. In order to investigate the role of the different proteins encoded by the hrp gene cluster of the
phytopathogenic bacterium Ralstonia solanacearum, non‐polar mutants in all
hrp genes (except for hrcQ) were analysed for their interactions with
plants, their ability to secrete the PopA protein and their production of the Hrp
pilus. In addition to Hrc proteins and the HrpY major component of the Hrp pilus,
four additional Hrp proteins are indispensable for type III secretion and for interactions
with plants. We also provide evidence that hrpV and hrpX mutants can still target the HrpY pilin outside the bacterial cell but are impaired in the production of Hrp pili, indicating that HrpV and HrpX proteins are involved in the assembly of this appendage.
Host versus in vitro signals and intrastrain allelic differences in the expression of a Candida albicans virulence gene
Authors: Staib Peter , Kretschmar Marianne , Nichterlein Thomas , Hof Herbert , Morschhäuser Joachim 
Published: March 2003

SummaryThe yeast Candida albicans is a harmless colonizer of mucosal surfaces in healthy people but can become a serious pathogen in immunocompromised patients, causing superficial as well as systemic infections. The evolution of gene families encoding pathogenicity‐related functions, like adhesins and secreted aspartic proteinases (Saps), which are differentially induced by host signals at various stages of colonization and infection, may have allowed C. albicans an optimal adaptation to many different host niches. We found that even the two alleles of a single gene can be differentially regulated in the diploid C. albicans. In the model strain SC5314, the in vitro expression of one of the two SAP2 alleles, SAP2‐1, depended on the presence of a functional SAP2‐2 allele. In contrast, inactivation of SAP2‐1 did not in‐fluence the expression of SAP2‐2. The proteinase encoded by the SAP2‐2 allele serves as a signal sensor and amplifier to enhance its own expression as well as to induce the SAP2‐1 allele to achieve maximal proteolytic activity under appropriate conditions. Using in vivo expression technology, we could demonstrate that the SAP2‐1 allele is significantly activated only in the late stages of systemic candidiasis in mice, whereas the SAP2‐2 allele is induced much earlier. The differential regulation of the two SAP2 alleles was due to differences in their pro‐moters, which contained a variable number of two pentameric nucleotide repeats. Mutations that re‐duced or increased the copy number of these repeats diminished the inducibility of the SAP2 promoter during infection but not in vitro, suggesting that the mutations affected interactions of regulatory factors that are necessary for SAP2 activation in vivo but dispensable for its induction in vitro. Therefore, the signals and signal transduction pathways that mediate SAP2 expression within certain host niches may differ from those that activate the gene in vitro. In addition to the generation of gene families whose members exhibit functional and regulatory diversification, C. albicans seems to use its diploid genome to create further variability and host adaptation by differential evolution of even the two alleles of a single gene.
The role of co‐transcriptional translation and protein translocation (transertion) in bacterial chromosome segregation
Authors: Woldringh Conrad L. 
Published: March 2003

SummaryMany recent reviews in the field of bacterial chromosome segregation propose that newly replicated DNA is actively separated by the functioning of specific proteins. This view is primarily based on an interpretation of the position of fluorescently labelled DNA regions and proteins in analogy to the active segregation mechanism in eukaryotic cells, i.e. to mitosis. So far, physical aspects of DNA organization such as the diffusional movement of DNA supercoil segments and their interaction with soluble proteins, leading to a phase separation between cytoplasm and nucleoid, have received relatively little attention. Here, a quite different view is described taking into account DNA–protein interactions, the large variation in the cellular position of fluorescent foci and the compaction and fusion of segregated nucleoids upon inhibition of RNA or protein synthesis. It is proposed that the random diffusion of DNA supercoil segments is transiently constrained by the process of co‐ transcriptional translation and translocation (transertion) of membrane proteins. After initiation of DNA replication, a bias in the positioning of transertion areas creates a bidirectionality in chromosome segre‐gation that becomes self‐enhanced when neigh‐bouring genes on the same daughter chromosome are expressed. This transertion‐mediated segregation model is applicable to multifork replication during rapid growth and to multiple chromosomes and plasmids that occur in many bacteria.
Diethylmaleate activates the transcription factor Pap1 by covalent modification of critical cysteine residues
Authors: Castillo Esther A. , Ayté José , Chiva Cristina , Moldón Alberto , Carrascal Montse , Abián Joaquín , Jones Nic , Hidalgo Elena 
Published: March 2003

SummaryDuring the last decade, much has been learnt about the mechanisms by which oxidative stress is perceived by aerobic organisms. The Schizosaccharomyces pombe Pap1 protein is a transcription factor localized at the cytoplasm, which accumulates in the nucleus in response to different inducers, such as the pro‐oxidant hydrogen peroxide (H2O2) or the glutathione‐depleting agent diethylmaleate (DEM). As described for other H2O2 sensors, our genetic data indicates that H2O2 reversibly oxidizes two cysteine residues in Pap1 (Cys278 and Cys501). Surprisingly, our studies demonstrate that DEM generates a non‐reversible modification of at least two cysteine residues located in or close to the nuclear export signal of Pap1 (Cys523 and Cys532). This modification impedes the interaction of the nuclear exporter Crm1 with the nuclear export signal located at the carboxy‐terminal domain of Pap1. Mass spectrometry data suggest that DEM binds to the thiol groups of the target cysteine residues through the formation of a thioether. Here we show that DEM triggers Pap1 nuclear accumulation by a novel molecular mechanism.
The plastid DNA of the malaria parasite Plasmodium falciparum is replicated by two mechanisms
Authors: Williamson D. H. , Preiser P. R. , Moore P. W. , McCready S. , Strath M. , Wilson R. J. M. (Iain) 
Published: March 2003

SummaryIn common with other apicomplexan parasites, Plasmodium falciparum, a causative organism of human malaria, harbours a residual plastid derived from an ancient secondary endosymbiotic acquisition of an alga. The function of the 35 kb plastid genome is unknown, but its evolutionary origin and genetic content make it a likely target for chemotherapy. Pulsed field gel electrophoresis and ionizing radiation have shown that essentially all the plastid DNA comprises covalently closed circular monomers, together with a tiny minority of linear 35 kb molecules. Using two‐dimensional gels and electron microscopy, two replication mechanisms have been revealed. One, sensitive to the topoisomerase inhibitor ciprofloxacin, appears to initiate at twin D‐loops located in a large inverted repeat carrying duplicated rRNA and tRNA genes, whereas the second, less drug sensitive, probably involves rolling circles that initiate outside the inverted repeat.
The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition
Authors: Huston Wilhelmina M. , Jennings Michael P. , McEwan Alastair G. 
Published: March 2003

SummaryRecently it has been observed that multicopper oxidases are present in a number of microbial genomes, raising the question of their function in prokaryotes. Here we describe the analysis of an mco mutant from the opportunistic pathogen Pseudomonas aeruginosa. Unlike wild‐type Pseudomonas aeruginosa, the mco mutant was unable to grow aerobically on minimal media with Fe(II) as sole iron source. In contrast, both the wild‐type and mutant strain were able to grow either anaerobically via denitrification with Fe(II) or aerobically with Fe(III). Analysis of iron uptake showed that the mco mutant was impaired in Fe(II) uptake but unaffected in Fe(III) uptake. Purification and analysis of the MCO protein confirmed ferroxidase activity. Taken together, these data show that the mco gene encodes a multicopper oxidase that is involved in the oxidation of Fe(II) to Fe(III) subsequent to its acquisition by the cell. In view of the widespread distribution of the mco gene in bacteria, it is suggested that an iron acquisition mechanism involving multicopper oxidases may be an important and hitherto unrecognized feature of bacterial pathogenicity.
MotX and MotY, specific components of the sodium‐driven flagellar motor, colocalize to the outer membrane in Vibrio alginolyticus
Authors: Okabe Mayuko , Yakushi Toshiharu , Kojima Masaru , Homma Michio 
Published: March 2003

SummaryRotation of the sodium‐driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium‐driven motor of Vibrio, have been believed to be localized in the inner (cytoplasmic) membrane via their N‐terminal hydrophobic segments. Here we show that MotX and MotY colocalize to the outer membrane. Both proteins, when expressed together, were detected in the outer membrane fraction separated by sucrose density gradient centrifugation. As mature MotX and MotY proteins do not have N‐terminal hydrophobic segments, the N‐termini of the primary translation products must have signal sequences that are removed upon translocation across the inner membrane. Moreover, MotX and MotY require each other for efficient localization to the outer membrane. Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane. This is the first case that describes motor proteins function in the outer membrane for flagellar rotation.
A novel class of microbial phosphocholine‐specific phospholipases C
Authors: Stonehouse Martin J. , Cota‐Gomez Adela , Parker Sarah K. , Martin Wesley E. , Hankin Joseph A. , Murphy Robert C. , Chen Weibin , Lim Kheng B. , Hackett Murray , Vasil Adriana I. , Vasil Michael L. 
Published: March 2003

SummaryIn this report we describe the 1500‐fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17 117.2 Da (PlcR2) and 78 417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline‐containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the con‐clusion that the members of this PC‐PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.
The Salmonella enterica sv. Typhimurium smvA, yddG and ompD (porin) genes are required for the efficient efflux of methyl viologen
Authors: Santiviago Carlos A. , Fuentes Juan A. , Bueno Susan M. , Trombert A. Nicole , Hildago Alejandro A. , Socias L. Teresa , Youderian Philip , Mora Guido C. 
Published: March 2003

SummaryIn Gram‐negative bacteria, a subset of inner membrane proteins in the major facilitator superfamily (MFS) acts as efflux pumps to decrease the intracellular concentrations of multiple toxic substrates and confers multidrug resistance. The Salmonella enterica sv. Typhimurium smvA gene encodes a product predicted to be an MFS protein most similar to QacA of Staphylococcus aureus. Like mutations in qacA, mutations in smvA confer increased sensitivity to methyl viologen (MV). Mutations in the adjacent ompD (porin) and yddG (drug/metabolite transporter) genes also confer increased sensitivity to MV, and mutations in smvA are epistatic to mutations in ompD or yddG for this phenotype. YddG and OmpD probably comprise a second efflux pump in which the OmpD porin acts as an outer membrane channel (OMC) protein for the efflux of MV and functions independently of the SmvA pump. In support of this idea, the pump dependent on YddG and OmpD has a different substrate specificity from the pump dependent on SmvA. Mutations in tolC, which encodes an OMC protein, confer increased resistance to MV. TolC apparently facilitates the import of MV, and a subset of OMC proteins including the OmpD porin and TolC may facilitate both import and export of distinct subsets of toxic substrates.