bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic and functional analysis of the phosphorylcholine moiety of commensal Neisseria lipopolysaccharide
Authors: Serino Laura , Virji Mumtaz
Published: March 2003

Phosphorylcholine (ChoP) is a common surface feature of many mucosal organisms, including Neisseria spp., in which it is present exclusively on pili of pathogenic Neisseria and on the lipopolysaccharide (LPS) of commensal Neisseria (Cn). Its presence in Cn has been confirmed by nuclear magnetic resonance. It appears that choline is the main source for the production of ChoP by Cn. We have sequenced a locus, containing four genes (licA–D) with 47–73% identity to the lic1 locus of Haemophilus influenzae (Hi) and 21–40% identity to lic genes in Streptococcus pneumoniae, involved in the production and incorporation of ChoP. The arrangement of the Cn genes and the presence of CAAT repeats, responsible for phase variation of ChoP expression, resemble Hi and differ from S. pneumoniae. Cn DNA flanking the lic locus contains genes ilvE and NMA2149 with >85% identity to the pathogenic Neisseria genes. However, there are no lic genes in the corresponding location or elsewhere in pathogenic Neisseria. This suggests either the loss of the locus from pathogenic Neisseria or a horizontal transfer of genes to Cn, perhaps from H. influenzae spp. As in Hi, ChoP enhances adherence to and invasion of human epithelial cells via the receptor for platelet‐activating factor. However, ChoP expression also increases susceptibility to serum killing mediated by complement and C‐reactive protein. Taken together, these observations support the hypothesis that the ability of many organisms to switch off ChoP expression rapidly represents an important adaptation to dif‐ferent environments encountered during the colonization/infection process and that the ChoP moiety apparently synthesized by distinct means in pathogenic and commensal Neisseria represents an advantage in the colonization properties of these bacteria.
Osmotic stress‐coupled maintenance of polar growth in Aspergillus nidulans
Authors: Han Kap‐Hoon , Prade Rolf A.
Published: March 2003

Free‐living cells monitor extracellular ‘osmotic strength’ and respond metabolically to offset unfavourable osmotic intracellular solute concentrations. Here, we report the reconstruction of the Aspergillus nidulans salt stress‐controlling
MAP kinase pathway, based on homology analysis with known yeast genes. In A. nidulans,
salt stress HOG genes, such as pbsA, hogA, ptpA and msnA,
are upregulated when exposed to high concentrations of salt and, in a hogA
deletion mutant (SIK1), the accumulation of pbsA is strongly reduced, suggesting a salt‐specific feedback induction mechanism. Growth of SIK1 appears to be unchanged in unstressed cells, but hyphal extension rates are reduced by as much as 60% in the presence of salt. Microscopic observation revealed abnormal hyperbranched hyphal tips, disproportionate accumulation of nuclei and absence of septa. Thus, the inability to maintain turgor pressure depresses cell expansion and results in slower volume increases. In addition, SIK1 fails to partition the apical cell; thus, nuclei are not likely to arrest mitosis in interphase as in normal cells, but continue to divide, accumulating to high levels.
Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two‐component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli
Authors: Sperandio Vanessa , Torres Alfredo G. , Kaper James B.
Published: March 2003

Quorum sensing is a cell‐to‐cell signalling mechanism in which bacteria secrete hormone‐like compounds called autoinducers. When these auto‐inducers reach a certain threshold concentration, they interact with bacterial transcriptional regulators, thereby regulating gene expression. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 as well as E. coli K‐12 produces the autoinducer‐2 (AI‐2), which is synthesized by the product of the luxS gene, and previous work from our laboratory has shown that genes encoding the EHEC type III secretion system were activated by quorum sensing. Recently, by hybridizing an E. coli K‐12 gene array with cDNA synthesized from RNA extracted from EHEC strain 86‐24 and its isogenic luxS mutant, we observed that other potential virulence‐associated factors, such as genes encoding the expression and assembly of flagella, motility and chemotaxis, were also activated by quorum sensing. The array data also indicated that several genes encoding putative E. coli regulators were controlled by quorum sensing. In this report, we describe a two‐component system regulated by quorum sensing that shares homology with Salmonella typhimurium PmrAB, which we have named quorum sensing E. coli regulator B and C (QseBC). The qseBC genes, previously identified only as open reading frames b3025 and b3026, are organized in an operon in the E. coli chromosome, with qseB encoding the response regulator and qseC the sensor kinase. We confirmed the regulation of qseBC by quorum sensing using qseB::lacZ transcriptional fusions and characterized the phenotypes of an isogenic qseC mutation in EHEC. This mutant expressed less flagellin and had reduced motility compared with the wild‐type and complemented strains. Transcription of flhD, fliA, motA and fliC::lacZ fusions was decreased in the qseC mutant, suggesting that qseBC is a transcriptional regulator of flagella genes. A qseC mutant was also generated in E. coli K‐12 strain MC1000 that showed the same phenotypes as the EHEC mutant, indicating that qseBC regulates flagella and motility by quorum sensing in both EHEC and K‐12. QseBC activates transcription of flhDC, which is the master regulator for the flagella and motility genes and, in the absence of flhD, QseBC failed to activate the transcription of fliA. Motility of a luxS, but not of a qseC, mutant can be restored by providing AI‐2 exogenously as preconditioned media, suggesting that the qseC mutant is unable to respond to AI‐2. However, QseC has no effect on the expression of other quorum sensing‐controlled genes such as those encoding for the type III secretion system. These data indicate that QseBC is one component of the quorum‐sensing regulatory cascade in both EHEC and K‐12 that is involved in the regulation of flagella and motility genes, but that additional regulators in this cascade remain to be characterized.
The WD40‐repeat protein CreC interacts with and stabilizes the deubiquitinating enzyme CreB in vivo in Aspergillus nidulans
Authors: Lockington Robin A. , Kelly Joan M.
Published: March 2003

Genetic dissection of carbon catabolite repression in Aspergillus nidulans

has identified two genes, creB and creC, which, when mutated, affect

expression of many genes in both carbon catabolite repressing and derepressing conditions.

The creB gene encodes a functional deubiquitinating enzyme and the creC

gene encodes a protein that contains five WD40 repeat motifs, and a proline‐rich

region . These findings have allowed the in vivo molecular analysis of a cellular

switch involving deubiquitination. We demonstrate that overexpression of the CreB

deubiquitinating enzyme can partially compensate for a lack of the CreC WD40‐repeat

protein in the cell, but not vice versa and, thus, the CreB deubiquitinating

enzyme acts downstream of the CreC WD40‐repeat protein. We demonstrate using co‐immunoprecipitation

ex‐periments that the CreB deubiquitinating enzyme and the CreC WD40‐repeat protein

interact in vivo in both carbon catabolite repressing and carbon catabolite derepressing conditions. Further, we show that the CreC WD40‐repeat protein is required to prevent the proteolysis of the CreB deubiquitinating enzyme in the absence of carbon catabolite repression. This is the first case in which a regulatory deubiquitinating enzyme has been shown to interact with another protein that is required for the stability of the deubiquitinating enzyme.
An rRNA mutation identifies the apicoplast as the target for clindamycin in Toxoplasma gondii
Authors: Camps Manel , Arrizabalaga Gustavo , Boothroyd John
Published: March 2003

Toxoplasma gondii is a protozoan sensitive to several inhibitors of prokaryotic

translation (e.g. clindamycin, macrolides and tetracyclines). A priori, two

prokaryotic‐like organelles, the ‘apicoplast’ (a non‐photosynthetic

plastid) and the mitochondrion, are likely targets for these drugs. Without using

overt mutagenesis, we selected two independent clones (ClnR‐4 and ClnR‐21)

with strong and stable clindamycin resistance. Several lines with substantial but

lower levels of resistance were also isolated with (XR‐46) or without (ClnR‐23)

overt mutagenesis. The ClnR‐4 and ClnR‐21 mutants uniquely

possess a G→U point mutation at position 1857 of the apicoplast large‐subunit

rRNA, whereas no mutation was identified in this region for ClnR‐23 or

XR‐46. Position 1857 corresponds to position 2061 in Escherichia coli where

it is predicted to bind clindamycin. The mutation is present in all the apicoplast

rDNA copies (an estimated 12 per organelle), indicative of a strong selective advantage

in the presence of clindamycin. In the absence of drug, however, such a mutation

is unlikely to be neutral, as the G is a critical contributor to the transpeptidation

reaction and absolutely conserved in all kingdoms. This may explain why ClnR‐4

shows a slight growth defect in vitro. These mutants provide direct genetic

evidence that apicoplast translation is the target for clindamycin in Toxoplasma. Further, their sensitivity profiles to other antibiotics specific for the large ribosomal subunit (macrolides and chloramphenicol) and, intriguingly, the small subunit (doxycycline) argue that these drugs also target the apicoplast ribosome.
Overexpression of the putative thiol conjugate transporter TbMRPA causes melarsoprol resistance in Trypanosoma brucei
Authors: Shahi Sanjay K. , Krauth‐Siegel R. Luise , Clayton Christine E.
Published: March 2003

Melaminophenyl arsenical drugs are a mainstay of chemotherapy against late‐stage African sleeping sickness, but drug resistance is increasingly prevalent. We describe here the characterization of two genes encoding putative metal–thiol conjugate transporters from Trypanosoma brucei. The two proteins, TbMRPA and
TbMRPE, were each overexpressed in trypanosomes, with or without co‐expression
of two key enzymes in trypanothione biosynthesis, ornithine decarboxylase and gamma‐glutamyl‐cysteine
synthetase. Overexpression of gamma‐glutamyl‐cysteine synthetase resulted in a twofold
increase in cellular trypanothione, whereas overexpression of ornithine decarboxylase
had no effect on the trypanothione level. The overexpression of TbMRPA resulted
in a 10‐fold increase in the IC50 of melarsoprol. The overexpression of
the trypanothione biosynthetic enzymes alone gave two‐ to fourfold melarsoprol resistance,
but did not enhance resistance caused by MRPA. Overexpression of TbMRPE had little effect on susceptibility to melarsoprol but did give two‐ to threefold resistance to suramin.
A genome‐wide strategy for the identification of essential genes in Staphylococcus aureus
Authors: Forsyth R. Allyn , Haselbeck Robert J. , Ohlsen Kari L. , Yamamoto Robert T. , Xu Howard , Trawick John D. , Wall Daniel , Wang Liangsu , Brown‐Driver Vickie , Froelich Jamie M. , C. Kedar G. , King Paula , McCarthy Melissa , Malone Cheryl , Misiner Brian , Robbins David , Tan Zehui , Zhu Zhan‐yang , Carr Grant , Mosca Deborah A. , Zamudio Carlos , Foulkes J. Gordon , Zyskind Judith W.
Published: March 2003

To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus

aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus

chromosomal DNA fragments were cloned into a xylose‐inducible expression plasmid

and transformed into S. aureus. Homology comparisons between 658 S. aureus

genes identified in this particular antisense screen and the Mycoplasma genitalium

genome, which contains 517 genes in total, yielded 168 conserved genes, many of which

appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell‐based, drug‐screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.
Studies on the role of the metK gene product of Escherichia coli K‐12
Authors: Wei Yuhong , Newman E. B.
Published: March 2003

We show here that the metK gene is essential to the growth of Escherichia

coli K‐12 and can be deleted only in the presence of a rescue plasmid carrying

a functional metK gene. When metK expression was limited, genomic DNA

methylation decreased and cell division was hampered. Through primer extension, the

transcription start site of metK was located at 140 bp upstream of the translation

start site. The frequently used metK84 mutant has been shown to

carry an A(r)G transition in the −10 region of the metK promoter. This accounts for its low level of S‐adenosylmethionine (SAM) synthetase and SAM deficiency.
The luxS gene is involved in cell–cell signalling for toxin production in Clostridium perfringens
Authors: Ohtani Kaori , Hayashi Hideo , Shimizu Tohru
Published: March 2003

A Gram‐positive anaerobic pathogen, Clostridium perfringens, causes clostridial
myonecrosis or gas gangrene in humans by producing numerous extracellular toxins
and enzymes that act in concert to degrade host tissues. C. perfringens possesses
a homologue of the luxS gene that is reported to be responsible for the production
of autoinducer 2 (AI‐2), which participates in quorum sensing in bacteria. The luxS
mutant was constructed using C. perfringens strain 13, and the role of the
luxS gene in toxin production was examined. The cell‐free culture supernatant
from wild‐type strain 13 greatly stimulated the luminescence of Vibrio harveyi
BB170, whereas that from the luxS mutant caused no significant stimulation,
indicating that the luxS gene is necessary for AI‐2 production in C. perfringens.
The luxS mutant showed a reduced level of production of alpha‐, kappa‐ and
theta‐toxins. In the luxS mutant, the transcription of the theta‐toxin gene
(pfoA) was lower at mid‐exponential growth phase, whereas alpha‐ and kappa‐toxin
gene transcription was not significantly affected. The production of toxins in the
luxS mutant was stimulated by the addition of the culture supernatant from
the wild‐type cells, possibly because of the presence of AI‐2. Moreover, the expression
of the pfoA gene in the luxS mutant was apparently activated when the
mutant cells were cultured in the presence of culture supernatants from the wild‐type
C. perfringens, Escherichia coli DH5α carrying the luxS
gene of C. perfringens. A deletion analysis of the luxS operon showed
that the luxS gene alone is responsible for cell–cell signalling, and
that the metB or cysK genes located upstream of luxS are not
involved in regulating toxin production. Our results indicate that cell–cell
signalling by AI‐2 plays an important role in the regulation of toxin production
in C. perfringens.
The FtsH protease is involved in development, stress response and heat shock control in Caulobacter crescentus
Authors: Fischer B. , Rummel G. , Aldridge P. , Jenal Urs
Published: March 2003

The ftsH gene of Caulobacter crescentus has been isolated and identified as a component of the general stress response of this organism. In C. crescentus, ftsH expression is transiently induced after temperature upshift and in stationary phase. Consistent with this, mutants deprived of the FtsH protease are viable at normal growth conditions, but are highly sensitive to elevated temperature, increased salt concentration or the presence of antibiotics. Overexpression of ftsH resulted in an increased salt but not thermotolerance, emphasizing the importance of the FtsH protease in stress response. Mutants lacking FtsH were unable to undergo morphological and physiological adaptation in stationary phase and, upon starvation, experienced a more pronounced loss of viability than cells containing FtsH. In addition, cells lacking FtsH had an increased cellular concentration of the heat shock sigma factor σ32, indicating that, as in Escherichia coli, the FtsH protease is involved in the control of the C. crescentus heat shock response. In agreement with this, transcription of the heat‐induced σ32‐dependent gene dnaK was derepressed at normal temperature when FtsH was absent. In contrast, the groEL gene, which is controlled in response to heat stress by both σ32 and a HcrA/CIRCE mechanism, was not derepressed in an ftsH mutant. Finally, FtsH is involved in C. crescentus development and cell cycle control. ftsH mutants were unable to synthesize stalks efficiently and had a severe cell division phenotype. In the absence of FtsH, swarmer cells differentiated into stalked cells faster than when FtsH was present, even though the entire cell cycle was longer under these conditions. Thus, directly or indirectly, the FtsH protease is involved in the inherent biological clock mechanism, which controls the timing of cell differentiation in C. crescentus.
Competitive interaction of the OxyR DNA‐binding protein and the Dam methylase at the antigen 43 gene regulatory region in Escherichia coli
Authors: Waldron Denise E. , Owen Peter , Dorman Charles J.
Published: March 2003

The antigen 43 surface protein of Escherichia coli is expressed in a phase‐variable manner by a mechanism involving alternative activation and repression of transcription of the agn43 gene. The repressor is the OxyR DNA‐binding protein, and its binding site was found to be located downstream of the agn43 transcription start site in a region of DNA that encompasses three 5′‐GATC‐3′ sequences that are subject to Dam‐mediated DNA methylation. It has been suggested previously that the phase‐variable expression of antigen 43 results from a competition between Dam methylase and the OxyR repressor for these sites. The 5′‐GATC‐3′ sequences were inactivated for methylation by site‐directed mutagenesis, and all possible combinations of inactive and active sites were assessed for effects on phase‐variable expression of the agn43 gene. Inactivation of any 5′‐GATC‐3′ site individually had no effect; at least two sites had to be inactivated to disrupt the normal pattern of expression. Studies of OxyR interaction with agn43 DNA showed that methylation of any two 5′‐GATC‐3′ sites was necessary and sufficient to block binding of the repressor. It was also found that the adenines of the second and third 5′‐GATC‐3′ sites are required for OxyR binding, demonstrating that the sites for Dam methylation and for repressor binding are intimately associated. This is consistent with a competition model in which Dam and OxyR share a preference for specific DNA sequences in the regulatory region of the agn43 gene.
Bacteriophage control of Shiga toxin 1 production and release by Escherichia coli
Authors: Wagner Patrick L. , Livny Jonathan , Neely Melody N. , Acheson David W. K. , Friedman David I. , Waldor Matthew K.
Published: March 2003

The stx genes of many Shiga toxin‐encoding Escherichia coli (STEC) strains are encoded by prophages of the λ bacteriophage family. In the genome of the Stx1‐encoding phage H‐19B, the stx1AB genes are found ≈ 1 kb downstream of the late phage promoter, pR′, but are known to be regulated by the associated iron‐regulated promoter, pStx1. Growth of H‐19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production. Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized. The studies reported here identify the factors that contribute to Stx1 production after induction of the H‐19B prophage. We found that replication of the phage genome, with the associated increase in stx1AB copy number, is the most quantitatively important mechanism by which H‐19B induction increases Stx1 production. Three promoters are shown to be involved in stx1AB transcription after phage induction, the iron‐regulated pStx1 and the phage‐regulated pR and pR′ promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the pR′ promoter, contrary to previous findings in the related Stx2‐encoding phage φ361, was found to be unnecessary for high‐level Stx1 production after phage induction. Finally, we present evidence that phage‐mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release. By amplifying stx1AB copy number, regulating stx1AB transcription and allowing for Stx1 release, the biology of the Stx‐encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC.
Specific activation of the Bacillus quorum‐sensing systems by isoprenylated pheromone variants
Authors: Ansaldi Mireille , Marolt Darja , Stebe Tina , Mandic‐Mulec Ines , Dubnau David
Published: March 2003

Natural genetic competence in Bacillus subtilis is controlled by quorum‐sensing
(QS). The ComP– ComA two‐component system detects the signalling molecule
ComX, and this signal is transduced by a conserved phosphotransfer mechanism. ComX
is synthesized as an inactive precursor and is then cleaved and modified by ComQ
before export to the extracellular environment. The comQXP′ loci
of a set of natural Bacillus isolates have been sequenced and shown to possess a striking polymorphism that determines specific patterns of both activation and inhibition of the quorum‐sensing response. We have developed a simple purification method for the modified peptide signalling pheromones allowing the characterization of four distinct ComX molecules representing different pherotypes. Genetic and biochemical evidence demonstrate that all the ComX variants are isoprenylated by the post‐translational modification of a conserved tryptophan residue and that the modifications on the ComX peptide backbones vary in mass among the various pherotypes. These results give new insights into peptidemediated quorum‐sensing signalling in Gram‐positive bacteria and emphasize the role of isoprenylation in bacterial signal transduction.
Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface
Authors: Claessen Dennis , Wösten Han A. B. , Keulen Geertje van , Faber Onno G. , Alves Alexandra M. C. R. , Meijer Wim G. , Dijkhuizen Lubbert
Published: March 2003

The filamentous bacteria Streptomyces coelicolor and Streptomyces lividans exhibit a complex life cycle. After a branched submerged mycelium has been established, aerial hyphae are formed that may septate to form chains of spores. The aerial structures possess several surface layers of unknown nature that make them hydrophobic, one of which is the rodlet layer. We have identified two homologous proteins, RdlA and RdlB, that are involved in the formation of the rodlet layer in both streptomycetes. The rdl genes are expressed in growing aerial hyphae but not in spores. Immunolocalization showed that RdlA and RdlB are present at surfaces of aerial structures, where they form a highly insoluble layer. Disruption of both rdlA and rdlB in S. coelicolor and S. lividans (ΔrdlAB strains) did not affect the formation and differentiation of aerial hyphae. However, the characteristic rodlet layer was absent. Genes rdlA and rdlB were also expressed in submerged hyphae that were in contact with a hydrophobic solid. Attachment to this substratum was greatly reduced in the ΔrdlAB strains. Sequences homologous to rdlA and rdlB occur in a number of streptomycetes representing the phylogenetic diversity of this group of bacteria, indicating a general role for these proteins in rodlet formation and attachment.
Two separate DNA sequences within oriC participate in accurate chromosome segregation in Bacillus subtilis
Authors: Kadoya Ryosuke , Hassan Anwarul K. M. , Kasahara Yasuhiro , Ogasawara Naotake , Moriya Shigeki
Published: March 2003

Current views of bacterial chromosome segregation vary in respect of the likely presence or absence of an active segregation mechanism involving a mitotic‐like apparatus. Furthermore, little is known about cis‐acting elements for chromosome segregation in bacteria. In this report, we show that two separate DNA regions, a 3′ coding region of dnaA and the AT‐rich sequence between dnaA and dnaN (the initial opening site of duplex DNA during replication), are necessary for efficient segregation of the chromosome in Bacillus subtilis. When a plasmid replicon was integrated into argG, far from oriC, on the chromosome and then the oriC function was disrupted, the oriC‐deleted mutant formed anucleate cells at 5% possibly because of defects in chromosome segregation. However, when the two DNA sequences were added near oriN, frequency of anucleate cells decreased to 1%. In these cells, the origin (argG) regions were localized near cell poles, whereas they were randomly distributed in cells without the two DNA sequences. These results suggest that the two DNA sequences in and downstream of the dnaA gene participate in correct positioning of the replication origin region within the cell and that this function is associated with accurate chromosome segregation in B. subtilis.
A bZIP protein from halophilic archaea: structural features and dimer formation of cGvpE from Halobacterium salinarum
Authors: Plößer Petra , Pfeifer Felicitas
Published: March 2003

The cGvpE protein of Halobacterium salinarum PHH4 has been identified as transcriptional activator for the promoter of the c‐gvpA gene encoding the major gas vesicle structural protein cGvpA. Molecular modelling of the carboxy‐terminal region of cGvpE suggests that this protein resembles a basic leucine‐zipper protein, and mutations in the putative DNA binding domain DNAB completely abolish the activator function in Haloferax volcanii transformants. Mutations in the key residues of the putative leucine‐zipper region AH6 of cGvpE confirmed that the three residues V159, L166 and L173 were essential for the activator function of cGvpE at the c‐gvpA promoter, whereas the cysteine residue C180 could be altered to a leucine or an aspartate residue without the loss of this function. Mutations in basic residues of helix AH4 demonstrated the importance of the lysine K104 for the activator function of cGvpE. A cGvpE protein containing a his‐tag at the C‐terminus was still able to activate the expression of c‐gvpA in vivo. The cGvpE his‐purified from Hf. volcanii formed a dimer in Blue‐native polyacrylamide gels that could be resolved into monomers by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Dimers of cGvpE were already seen using SDS–PAGE, but not with cGvpE mutant proteins containing the alterations L166E or L173E/C180L in the leucine zipper. These results imply that the hydrophobic surface of helix AH6 is indeed required for the establishment of cGvpE dimers.
Pseudomonas aeruginosa RoxR, a response regulator related to Rhodobacter sphaeroides PrrA, activates expression of the cyanide‐insensitive terminal oxidase
Authors: Comolli James C. , Donohue Timothy J.
Published: March 2003

The facultative anaerobe Pseudomonas aeruginosa has multiple aerobic electron transport pathways, one of which is terminated by a cyanide‐insensitive oxidase (CIO). This study characterizes a P. aeruginosa two‐component system that regulates CIO production. The response regulator of this system (RoxR) has significant amino acid sequence similarity to PrrA of Rhodobacter sphaeroides and related proteins in other α‐proteobacteria. In heterologous complementation analysis, R. sphaeroides PrrA rescued the growth defect of a P. aeruginosa mutant lacking RoxR, and RoxR enabled photosynthetic growth of an R. sphaeroides PrrA mutant. Also, RoxR could substitute for PrrA in activating transcription in vitro, demonstrating that these proteins are functional homologues. P. aeruginosa strains lacking RoxR or the sensor kinase (RoxS) were more sensitive than wild type to the respiratory inhibitors cyanide and azide. The phenotypes of these mutant strains correlated with reduced cyanide‐insensitive O2 utilization and less cyanide‐dependent expression of the locus encoding the CIO (cioAB). The ability of purified RoxR to bind to the cioAB promoter region also suggests that this protein acts directly to regulate cioAB transcription. Therefore, RoxR appears to play a role in regulating the transcription of loci for P. aeruginosa energy‐generating enzymes similar to that of its homologues in α‐proteobacteria.
Proteomic analysis of the Caulobacter crescentus stalk indicates competence for nutrient uptake
Authors: Ireland Marcia M. E. , Karty Jonathan A. , Quardokus Ellen M. , Reilly James P. , Brun Yves V.
Published: March 2003

Caulobacter crescentus
, a Gram‐negative
α
‐purple proteobacterium, is an oligotroph that lives in aquatic environments dilute in nutrients. This bacterium divides asymmetrically. Part of this asymmetric cell division involves the formation of a prosthecum at one pole, referred to as the stalk, which replaces the flagellum of the motile swarmer cell. Little is known about the synthesis or function of the stalk. The stalk is an extension of the cell membranes and peptidoglycan layer, and stalk elongation is stimulated by phosphate starvation. In this study, we have taken advantage of two‐dimensional gel (2D gel) electro‐phoresis as well as the fully sequenced genome of
Caulobacter
to study the proteome of the stalk. We modified a stalk‐shedding mutant strain of
Caulobacter crescentus
to increase the yield of stalk material shed and performed 2D gel electrophoresis of purified stalks and cellular fractions. Comparison of the stalk 2D gel with the 2D gels of cell membrane and soluble fractions showed that the stalk is mostly free of cytoplasmic proteins and has a profile very similar to that of the cell membrane. Of the 172 proteins on a stalk 2D gel, we report the identification of 64 spots, corresponding to 39 different proteins present in the stalk of
Caulobacter. The identifications include several TonB‐dependent receptors, two OmpA family proteins, a dipeptidase, GlpQ, two alkaline phosphatases, 3‐phytase, a putative TolC protein and 11 proteins of unknown function. These identifications are consistent with the hypothesis that the stalk plays a role in nutrient uptake.
Identification of a second Listeria secA gene associated with protein secretion and the rough phenotype
Authors: Lenz Laurel L. , Portnoy Daniel A.
Published: March 2003

We describe the identification and characterization of a second secA gene in Listeria monocytogenes. This gene, termed secA2, is involved in smooth–rough phenotypic variation and secA2 expression contributes to bacterial virulence. Spontaneous rough (R‐) variants of L. monocytogenes grow in chains and form rough colonies on solid media. A subset of R‐variants, classified here as type I, also shows reduced secretion of an autolysin, p60. We find that disruptions and in frame deletions in secA2 confer phenotypes identical to those of spontaneous type I R‐variants. Additionally, the secA2 genes from two spontaneous type I R‐variants encoded truncated SecA2 proteins. Mutations were not found in the secA2 genes from the remaining five independent R‐variants, four of which showed a distinct (type II) rough morphology and secreted wild‐type levels of p60. Expression of an epitope‐tagged SecA2 in the ΔsecA2 strain and a spontaneous R‐variant restored normal cell septation and smooth colony morphology. These data suggest that mutations in both secA2 and other genes contribute to smooth–rough phase variation in L. monocytogenes. Expression of the full‐length SecA2 also promotes secretion of p60 and a set of additional L. monocytogenes proteins. We hypothesize that SecA2‐dependent protein secretion plays a role in the colonization of environmental and host surfaces.
GeneChip® expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes
Authors: Ochsner Urs A. , Wilderman Paula J. , Vasil Adriana I. , Vasil Michael L.
Published: March 2003

Upon iron restriction, the opportunistic pathogen Pseudomonas aeruginosa produces various virulence factors, including siderophores, exotoxin, proteases and haemolysin. The ferric uptake regulator (Fur) plays a central role in this response and also controls other regulatory genes, such as pvdS, which encodes an alternative sigma factor. This circuit leads to a hierarchical cascade of direct and indirect iron regulation. We used the GeneChip® to analyse the global gene expression profiles in response to iron. In iron‐starved cells, the expression of 118 genes was increased at least fivefold compared with that in iron‐replete cells, whereas the expression of 87 genes was decreased at least fivefold. The GeneChip® data correlated well with results obtained using individual lacZ gene fusions. Strong iron regulation was observed for previously identified genes involved in biosynthesis or uptake of the siderophores pyoverdine and pyochelin, utilization of heterologous siderophores and haem and ferrous iron transport. A low‐iron milieu led to increased expression of the genes encoding TonB, alkaline protease, PrpL protease, exotoxin A, as well as fumarase C, Mn‐dependent superoxide dismutase SodA, a ferredoxin and ferredoxin reductase and several oxidoreductases and dehydrogenases. Iron‐controlled regulatory genes included seven alternative sigma factors and five other transcriptional regulators. Roughly 20% of the iron‐regulated genes encoded proteins of unknown function and lacked any conclusive homologies. Under low‐iron conditions, expression of 26 genes or operons was reduced in a ΔpvdS mutant compared with wild type, including numerous novel pyoverdine biosynthetic genes. The GeneChip® proved to be a very useful tool for rapid gene expression analysis and identification of novel genes controlled by Fur or PvdS.
A malaria scavenger receptor‐like protein essential for parasite development
Authors: Claudianos Charles , Dessens Johannes T. , Trueman Holly E. , Arai Meiji , Mendoza Jacqui , Butcher Geoff A. , Crompton Tessa , Sinden Robert E.
Published: March 2003

Malaria parasites suffer severe losses in the mosquito as they cross the midgut, haemolymph and salivary gland tissues, in part caused by immune responses of the insect. The parasite compensates for these losses by multiplying during the oocyst stage to form the infectious sporozoites. Upon human infection, malaria parasites are again attenuated by sustained immune attack. Here, we report a single copy gene that is highly conserved amongst Plasmodium species that encodes a secreted protein named PxSR. The predicted protein is composed of a unique combination of metazoan protein domains that have been previously associated with immune recognition/activation and lipid/protein adhesion interactions at the cell surface, namely: (i) scavenger receptor cysteine rich (SRCR); (ii) pentraxin (PTX); (iii) polycystine‐1, lipoxygenase, alpha toxin (LH2/PLAT); (iv) Limulus clotting factor C, Coch‐5b2 and Lgl1 (LCCL). In our assessment the PxSR molecule is completely novel in biology and is only found in Apicomplexa parasites. We show that PxSR is expressed in sporozoites of both human and rodent malaria species. Disruption of the PbSR gene in the rodent malaria parasite P. berghei results in parasites that form normal numbers of oocysts, but fail to produce any sporozoites. We suggest that, in addition to a role in sporogonic development, PxSR may have a multiplicity of functions.
Differential binding to and biofilm formation on, HEp‐2 cells by Salmonella enterica Serovar Typhimurium is dependent upon allelic variation in the fimH gene of the fim gene cluster
Authors: Boddicker Jennifer D. , Ledeboer Nathan A. , Jagnow Jennifer , Jones Bradley D. , Clegg Steven
Published: March 2003

Type 1 fimbria‐mediated adherence to HEp‐2 cells by two strains of Salmonella enterica serovar Typhimurium was found to be different. Although both strains exhibited a strong mannose‐sensitive haemagglutination reaction with guinea pig erythrocytes, characteristic of the expression of type 1 fimbriae, only one of the strains adhered in large numbers to HEp‐2 cells. Characterization of the fimH genes, encoding the fimbrial adhesins, indicated two allelic variants. Using fimH mutants of the two strains it was possible to demonstrate that binding to HEp‐2 cells was associated with the presence of one of the alleles regardless of the host strain. Therefore, this differential binding was not a function of the type I fimbrial shaft or the presence of other types of fimbriae produced by one strain but not the other. These observations may explain the differences in HEp‐2 binding by type 1 fimbriate strains of Salmonella previously reported by several groups. Also, our studies demonstrate that the FimH adhesin can influence the efficiency of biofilm formation on HEp‐2 cells using once‐flow‐through continuous culture conditions. The formation of biofilms on eukaryotic cells using this procedure is more likely to represent those conditions found in the intestinal tract than conditions using batch culture techniques to investigate adherence and biofilm formation. Indeed, the increased efficiency of biofilm formation in the murine intestinal tract confirmed the role of one of the fimH alleles in this process.
Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier
Authors: Purdy Des , O'Keeffe Triona A. T. , Elmore Michael , Herbert Mike , McLeod Anne , Bokori‐Brown Monika , Ostrowski Anna , Minton Nigel P.
Published: March 2003

Progress towards understanding the molecular basis of virulence in Clostridium difficile has been hindered by the lack of effective gene transfer systems. We have now, for the first time, developed procedures that may be used to introduce autonomously replicating vectors into this organism through their conjugative, oriT‐based mobilization from Escherichia coli donors. Successful transfer was achieved through the use of a plasmid replicon isolated from an indigenous C. difficile plasmid, pCD6, and through the characterization and subsequent circumvention of host restriction/modification (RM) systems. The characterized replicon is the first C. difficile plasmid replicon to be sequenced and encodes a large replication protein (RepA) and a repetitive region composed of a 35 bp iteron sequence repeated seven times. Strain CD6 has two RM systems, CdiCD6I/M.CdiCD6I and CdiCD6II/M. CdiCD6II, with equivalent specificities to Sau96I/M. Sau96I (5′‐GGNMCC‐3′) and MboI/M. MboI (5′‐GMATC‐3′) respectively. A second strain (CD3) possesses a type IIs restriction enzyme, Cdi I, which cleaves the sequence 5′‐CATCG‐3′ between the fourth and fifth nucleotide to give a blunt‐ended fragment. This is the first time that an enzyme with this specificity has been reported. The sequential addition of this site to vectors showed that each site caused between a five‐ and 16‐fold reduction in transfer efficiency. The transfer efficiencies achieved with both strains equated to between 1.0 × 10−6 and 5.5 × 10−5 transconjugants per donor.
Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL‐37
Authors: Schmidtchen Artur , Frick Inga‐Maria , Andersson Emma , Tapper Hans , Björck Lars
Published: March 2003

Effectors of the innate immune system, the anti‐bacterial peptides, have pivotal roles in preventing infection at epithelial surfaces. Here we show that proteinases of the significant human pathogens Pseudomonas aeruginosa, Enterococcus faecalis, Proteus mirabilis and Streptococcus pyogenes, degrade the antibacterial peptide LL‐37. Analysis by mass spectrometry of fragments generated by P. aeruginosa elastase in vitro revealed that the initial cleavages occurred at Asn‐Leu and Asp‐Phe, followed by two breaks at Arg‐Ile, thus inactivating the peptide. Proteinases of the other pathogens also degraded LL‐37 as determined by SDS‐PAGE. Ex vivo, P. aeruginosa elastase induced LL‐37 degradation in human wound fluid, leading to enhanced bacterial survival. The degradation was blocked by the metalloproteinase inhibitors GM6001 and 1, 10‐phenantroline (both of which inhibited P. aeruginosa elastase, P. mirabilis proteinase, and E. faecalis gelatinase), or the inhibitor E64 (which inhibited S. pyogenes cysteine proteinase). Additional experiments demonstrated that dermatan sulphate and disaccharides of the structure [ΔUA(2S)‐GalNAc(4,6S)], or sucroseoctasulphate, in‐hibited the degradation of LL‐37. The results indicate that proteolytic degradation of LL‐37 is a common virulence mechanism and that molecules which block this degradation could have therapeutic potential.
A nuclear protein in Schizosaccharomyces pombe with homology to the human tumour suppressor Fhit has decapping activity
Authors: Salehi Zivar , Geffers Lars , Vilela Cristina , Birkenhäger Ralf , Ptushkina Marina , Berthelot Karine , Ferro Myriam , Gaskell Simon , Hagan Iain , Stapley Ben , McCarthy John E. G.
Published: March 2003

A number of eukaryotic proteins are already known to orchestrate key steps of mRNA metabolism and translation via interactions with the 5′ m7GpppN cap. We have characterized a new type of histidine triad (HIT) motif protein (Nhm1) that co‐purifies with the cap‐binding complex eIF4F of Schizosaccharomyces pombe. Nhm1 is an RNA‐binding protein that binds to m7GTP‐Sepharose, albeit with lower specificity and affinity for methylated GTP than is typical for the cap‐binding protein known as eukaryotic initiation factor 4E. Sequence searches have revealed that proteins with strong sequence similarity over all regions of the new protein exist in a wide range of eukaryotes, yet none has been characterized up to now. However, other proteins that share specific motifs with Nhm1 include the human Fhit tumour suppressor protein and the diadenosine 5′, 5′′′‐P1, P4‐tetraphosphate asymmetrical hydrolase of S. pombe. Our experimental work also reveals that Nhm1 inhibits translation in a cell‐free extract prepared from S. pombe, and that it is therefore a putative translational modulator. On the other hand, purified Nhm1 manifests mRNA decapping activity, yet is physically distinct from the Saccharomyces cerevisiae decapping enzyme Dcp1. Moreover, fluorescence and immunofluorescence microscopy show that Nhm1 is predominantly, although not exclusively, nuclear. We conclude that Nhm1 has evolved as a special branch of the HIT motif superfamily that has the potential to influence both the metabolism and the translation of mRNA, and that its presence in S. pombe suggests the utilization of a novel decapping pathway.
Molecular and functional analysis of the type III secretion signal of the Salmonella enterica InvJ protein
Authors: Rüssmann Holger , Kubori Tomoko , Sauer Jeannette , Galán Jorge E.
Published: March 2003

Central to the pathogenicity of Salmonella enterica is the function of a type III secretion system (TTSS) encoded within a pathogenicity island at centisome 63 (SPI‐1). An essential component of this system is a supramolecular structure termed the needle complex. Proteins to be delivered into host cells possess specific signals that route them to the type III secretion pathway. In addition, some bacterial proteins have signals that deliver them to the secretion complex to either become their structural components or exert their function at that location. One of these proteins is InvJ, which controls the length of the needle substructure of the needle complex. In this study, we have analysed the signal that targets InvJ to the TTSS. We found that amino acid residues 4 to 7 of InvJ are necessary and sufficient to mediate secretion of InvJ or a reporter protein in a TTSS‐dependent manner. InvJ secretion was found to be essential for its function in needle length determination, effector protein secretion and bacterial invasion of epithelial cells. Frameshift mutagenesis analysis indicated that the InvJ type III secretion signal sequence tolerates significant alterations in its amino acid sequence without affecting InvJ secretion. Introduction of silent mutations in the secretion signal coding sequence that result in drastically different predicted mRNA folds had no effect on InvJ secretion or expression.
Requirement of spermidine for developmental transitions in Aspergillus nidulans
Authors: Jin Yuan , Bok Jin Woo , Guzman‐de‐Peña Doralinda , Keller Nancy P.
Published: March 2003

Deletion of the spermidine synthase gene in the fungus Aspergillus nidulans results in a strain, ΔspdA, which requires spermidine for growth and accumulates putrescine as the sole polyamine. Vegetative growth but not sporulation or sterigmatocystin production is observed when ΔspdA is grown on media supplemented with 0.05–0.10 mM exogenous spermidine. Supplementation of ΔspdA with ≥ 0.10 mM spermidine restores sterigmatocystin production and ≥ 0.50 mM spermidine produces a phenotype with denser asexual spore production and decreased radial hyphal growth compared with the wild type. ΔspdA spores germinate in unsupplemented media but germ tube growth ceases after 8 h upon which time the spores swell to approximately three times their normal diameter. Hyphal growth is resumed upon addition of 1.0 mM spermidine. Suppression of a G protein signalling pathway could not force asexual sporulation and sterigmatocystin production in ΔspdA strains grown in media lacking spermidine but could force both processes in ΔspdA strains supplemented with 0.05 mM spermidine. These results show that increasing levels of spermidine are required for the transitions from (i) germ tube to hyphal growth and (ii) hyphal growth to tissue differentiation and secondary metabolism. Suppression of G protein signalling can over‐ride the spermidine requirement for the latter but not the former transition.
The histidine kinase Hik33 perceives osmotic stress and cold stress in Synechocystis sp. PCC 6803
Authors: Mikami Koji , Kanesaki Yu , Suzuki Iwane , Murata Norio
Published: March 2003

The stress imposed on living organisms by hyperosmotic conditions and low temperature appears to be perceived via changes in the physical state of membrane lipids. We compared genome‐wide patterns of transcription between wild‐type Synechocystis sp. PCC 6803 and cells with a mutation in the histidine kinase Hik33 using a DNA microarray. Our results indicated that Hik33 regulated the expression of both osmostress‐inducible and cold‐inducible genes. The respective genes that were regulated by Hik33 under hyperosmotic and low‐temperature conditions were, for the most part, different from one another. However, Hik33 also regulated the expression of a set of genes whose expression was induced both by osmotic stress and by cold stress. These results indicate that Hik33 is involved in responses to osmotic stress and low‐temperature stress but that the mechanisms of the responses differ.